Information

Where can I find an at least Semi-Comprehensive list of what biochemical reactions Acetyl-CoA participates in?


Acetyl-CoA has a number of biochemical roles in the body and I'm curious as to whether anybody knows where I can find at least a semi-comprehensive list (i.e. comprising all the major roles in the human body it serves) of the biochemical reactions in which acetyl-CoA partakes in the human body.


I may be missing some fantastic resource, but my first thought is to send you to KEGG. Is that crazy? Maybe, but a simple search for Acetyl-CoA in humans can give you tons of useful information, including GO terms, enzymes, genes, and, yes, biochemical reactions; just use the KEGG Pathways tool and specify humans (hsa). You can click on each individual pathway for more information, such as the cholinergic synapse.


Metacyc is an excelent methabolic pathways database that includes all kind of organisms. Of course you can search for methabolic compound.


You should look at Lehninger's Principles of Biochemistry. Just search for Acetyl-coa in the index, and you will find it's roles in different aspects of the metabolism.


Regulation of glycolysis

Metabolic flow through glycolysis can be regulated at three key points:

  • hexokinase: is inhibited by glucose-6-P (product inhibition)
  • phosphofructokinase: is inhibited by ATP and citrate (which signals the abundance of citric acid cycle intermediates). It is also inhibited by H + , which becomes important under anaerobiosis (lactic fermentation produces lactic acid, resulting on a lowering of the pH ). Probably this mechanism prevents the cell from using all its ATP stock in the phosphofrutokinase reaction, which would prevent glucose activation by hexokinase. It is stimulated by its substrate (fructose-6-phosphate), AMP and ADP (which signal the lack of available energy), etc.
  • pyruvate kinase: inhibited by ATP, alanine, free fatty acids and acetyl-CoA. Activated by fructose-1,6-bisphosphate and AMP

Regulation of gluconeogenesis

Flow is regulated in the gluconeogenesis-specific reactions. Pyruvate carboxilase is activated by acetyl-CoA, which signals the abundance of citric acid cycle intermediates, i.e., a decreased need of glucose.

Regulation of the citric acid cycle

The citric acid cycle is regulated mostly by substrate availability, product inhibition and by some cycle intermediates.

  • pyruvate dehydrogenase: is inhibited by its products, acetyl-CoA and NADH
  • citrate synthase: is inhibited by its product, citrate. It is also inhibited by NADH and succinyl-CoA (which signal the abundance of citric acid cycle intermediates).
  • isocitrate dehydrogenase and a -ketoglutarate dehydrogenase: like citrate synthase, these are inhibited by NADH and succinyl-CoA. Isocitrate dehydrogenase is also inhibited by ATP and stimulated by ADP. All aforementioned dehydrogenases are stimulated by Ca 2+ . This makes sense in the muscle, since Ca 2+ release from the sarcoplasmic reticulum triggers muscle contraction, which requires a lot of energy. This way, the same "second messenger" activates an energy-demanding task and the means to produce that energy.

Regulation of the urea cycle

Carbamoyl-phosphate sinthetase is stimulated by N-acetylglutamine, which signals the presence of high amounts of nitrogen in the body.

Regulation of glycogen metabolism

Liver contains a hexokinase (hexokinase D or glucokinase)with low affinity for glucose which (unlike "regular" hexokinase) is not subject to product inhibition. Therefore, glucose is only phosphrylated in the liver when it is present in very high concentrations (i.e. after a meal). In this way, the liver will not compete with other tissues for glucose when this sugar is scarce, but will accumulate high levels of glucose for glycogen synthesis right after a meal.

Regulation of fatty acids metabolism

Acyl-CoA movement into the mitochondrion is a crucial factor in regulation. Malonyl-CoA (which is present in the cytoplasm in high amounts when metabolic fuels are abundant) inhibits carnitine acyltransferase, thereby preventing acyl-CoA from entering the mitochondrion. Furthermore, 3-hydroxyacyl-CoA dehydrogenase is inhibited by NADH and thiolase is inhibited by acetyl-CoA, so that fatty acids wil not be oxidized when there are plenty of energy-yielding substrates in the cell.

Regulation of the pentose phosphate pathway

Metabolic flow through the pentose phosphate pathway is controled by the activity of glucose-6-phosphate dehydrogenase, which is controlled by NADP + availability.


Contents

There are two major factors that lead to deficiency of CoQ10 in humans: reduced biosynthesis, and increased use by the body. Biosynthesis is the major source of CoQ10. Biosynthesis requires at least 12 genes, and mutations in many of them cause CoQ deficiency. CoQ10 levels also may be affected by other genetic defects (such as mutations of mitochondrial DNA, ETFDH, APTX, FXN, and BRAF, genes that are not directly related to the CoQ10 biosynthetic process). Some of these, such as mutations in COQ6, can lead to serious diseases such as steroid-resistant nephrotic syndrome with sensorineural deafness.

Some adverse effects, largely gastrointestinal, are reported with very high intakes. The observed safe level (OSL) risk assessment method indicated that the evidence of safety is strong at intakes up to 1200 mg/day, and this level is identified as the OSL. [9]

Assessment Edit

Although CoQ10 may be measured in blood plasma, these measurements reflect dietary intake rather than tissue status. Currently, most clinical centers measure CoQ10 levels in cultured skin fibroblasts, muscle biopsies, and blood mononuclear cells. [10] Culture fibroblasts can be used also to evaluate the rate of endogenous CoQ10 biosynthesis, by measuring the uptake of 14 C-labelled p-hydroxybenzoate. [11]

Statins Edit

It has been suggested that the myotoxicity of statins is due to impairment of CoQ biosynthesis, but the evidence supporting this was deemed controversial in 2011. [10] [ needs update ]

While statins may reduce coenzyme Q10 in the blood it is unclear if they reduce coenzyme Q10 in muscle. [12] Evidence does not support that supplementation improves side effects from statins. [12]

Regulation and composition Edit

CoQ10 is sold in many jurisdictions as a dietary supplement in the name of UbiQ 300 & UbiQ 100, not subject to the same regulations as medicinal drugs, but not approved for the treatment of any medical condition. [1] [13] The manufacture of CoQ10 is not regulated, and different batches and brands may vary significantly: [1] a 2004 laboratory analysis by ConsumerLab.com of CoQ10 supplements on sale in the US found that some did not contain the quantity identified on the product label. Amounts ranged from "no detectable CoQ10", through 75% of stated dose, up to a 75% excess. [14]

Generally, CoQ10 is well tolerated. The most common side effects are gastrointestinal symptoms (nausea, vomiting, appetite suppression, and abdominal pain), rashes, and headaches. [15]

While there is no established ideal dosage of CoQ10, a typical daily dose is 100–200 milligrams. Different formulations have varying declared amounts of CoQ10 and other ingredients.

Heart disease Edit

A 2014 Cochrane review found "no convincing evidence to support or refute" the use of CoQ10 for the treatment of heart failure. [16] Another 2014 Cochrane review found insufficient evidence to make a conclusion about its use for the prevention of heart disease. [17] A 2016 Cochrane review concluded that CoQ10 had no effect on blood pressure. [18] In a 2017 meta-analysis of people with heart failure 30–100 mg/d of CoQ10 resulted in 31% lower mortality. Exercise capacity was also increased. No significant difference was found in the endpoints of left heart ejection fraction and New York Heart Association (NYHA) classification. [19]

Migraine headaches Edit

The Canadian Headache Society guideline for migraine prophylaxis recommends, based on low-quality evidence, that 300 mg of CoQ10 be offered as a choice for prophylaxis. [20]

Statin myopathy Edit

CoQ10 has been routinely used to treat muscle breakdown associated as a side effect of use of statin medications. A 2015 meta-analysis of randomized controlled trials found that CoQ10 had no effect on statin myopathy. [21] A 2018 meta-analysis concluded that there was preliminary evidence for oral CoQ10 reducing statin-associated muscle symptoms, including muscle pain, muscle weakness, muscle cramps and muscle tiredness. [22]

Cancer Edit

As of 2014 [update] no large clinical trials of CoQ10 in cancer treatment had been conducted. [1] The US' National Cancer Institute identified issues with the few, small studies that had been carried out, stating, "the way the studies were done and the amount of information reported made it unclear if benefits were caused by the CoQ10 or by something else". [1] The American Cancer Society concluded, "CoQ10 may reduce the effectiveness of chemo and radiation therapy, so most oncologists would recommend avoiding it during cancer treatment." [23]

Dental disease Edit

A 1995 review study found that there is no clinical benefit to the use of CoQ10 in the treatment of periodontal disease. [24] Most of the studies suggesting otherwise were outdated, focused on in vitro tests, [25] [26] [27] had too few test subjects and/or erroneous statistical methodology and trial setup, [28] [29] or were sponsored by a manufacturer of the product. [30]

Chronic kidney disease Edit

A review of the effects of CoQ10 supplementation in people with CKD was proposed in 2019. [31]

Additional uses Edit

Coenzyme Q10 has also been used to treat Alzheimer's disease, high cholesterol, or amyotrophic lateral sclerosis (Lou Gehrig's disease). However, research has shown that this medicine may not be effective in treating these conditions [32]

Coenzyme Q10 has potential to inhibit the effects of theophylline as well as the anticoagulant warfarin coenzyme Q10 may interfere with warfarin's actions by interacting with cytochrome p450 enzymes thereby reducing the INR, a measure of blood clotting. [33] The structure of coenzyme Q10 is very similar to that of vitamin K, which competes with and counteracts warfarin's anticoagulation effects. Coenzyme Q10 should be avoided in patients currently taking warfarin due to the increased risk of clotting. [15]

The oxidized structure of CoQ10 is shown on the top-right. The various kinds of Coenzyme Q may be distinguished by the number of isoprenoid subunits in their side-chains. The most common coenzyme Q in human mitochondria is CoQ10. Q refers to the quinone head and 10 refers to the number of isoprene repeats in the tail. The molecule below has three isoprenoid units and would be called Q3.

In its pure state, it is an orange-coloured lipophile powder, and has no taste nor odour. [34] : 230

Biosynthesis occurs in most human tissue. There are three major steps:

  1. Creation of the benzoquinone structure (using phenylalanine or tyrosine, via 4-hydroxybenzoate)
  2. Creation of the isoprene side chain (using acetyl-CoA)
  3. The joining or condensation of the above two structures

The initial two reactions occur in mitochondria, the endoplasmic reticulum, and peroxisomes, indicating multiple sites of synthesis in animal cells. [35]

An important enzyme in this pathway is HMG-CoA reductase, usually a target for intervention in cardiovascular complications. The "statin" family of cholesterol-reducing medications inhibits HMG-CoA reductase. One possible side effect of statins is decreased production of CoQ10, which may be connected to the development of myopathy and rhabdomyolysis. However, the role statin plays in CoQ deficiency is controversial. Although these drug reduce blood levels of CoQ, studies on the effects of muscle levels of CoQ are yet to come. CoQ supplementation also does not reduce side effects of statin medications. [10] [12]

Genes involved include PDSS1, PDSS2, COQ2, and ADCK3 (COQ8, CABC1). [36]

Organisms other than human use somewhat different source chemicals to produce the benzoquinone structure and the isoprene structure. For example, the bacteria E. coli produces the former from chorismate and the latter from a non-mevalonate source. The common yeast S. cerevisiae, however, derives the former from either chorismate or tyrosine and the latter from mevalonate. Most organisms share the common 4-hydroxybenzoate intermediate, yet again uses different steps to arrive at the "Q" structure. [37]

Absorption Edit

CoQ10 is a crystalline powder insoluble in water. Absorption follows the same process as that of lipids the uptake mechanism appears to be similar to that of vitamin E, another lipid-soluble nutrient. This process in the human body involves secretion into the small intestine of pancreatic enzymes and bile, which facilitates emulsification and micelle formation required for absorption of lipophilic substances. [38] Food intake (and the presence of lipids) stimulates bodily biliary excretion of bile acids and greatly enhances absorption of CoQ10. Exogenous CoQ10 is absorbed from the small intestine and is best absorbed if taken with a meal. Serum concentration of CoQ10 in fed condition is higher than in fasting conditions. [39] [40]

Metabolism Edit

Data on the metabolism of CoQ10 in animals and humans are limited. [41] A study with 14 C-labeled CoQ10 in rats showed most of the radioactivity in the liver two hours after oral administration when the peak plasma radioactivity was observed, but CoQ9 (with only 9 isoprenyl units) is the predominant form of coenzyme Q in rats. [42] It appears that CoQ10 is metabolised in all tissues, while a major route for its elimination is biliary and fecal excretion. After the withdrawal of CoQ10 supplementation, the levels return to normal within a few days, irrespective of the type of formulation used. [43]

Some reports have been published on the pharmacokinetics of CoQ10. The plasma peak can be observed 2–6 hours after oral administration, depending mainly on the design of the study. In some studies, a second plasma peak also was observed at approximately 24 hours after administration, probably due to both enterohepatic recycling and redistribution from the liver to circulation. [38] Tomono et al. used deuterium-labeled crystalline CoQ10 to investigate pharmacokinetics in humans and determined an elimination half-time of 33 hours. [44]

Improving the bioavailability of CoQ10 Edit

The importance of how drugs are formulated for bioavailability is well known. In order to find a principle to boost the bioavailability of CoQ10 after oral administration, several new approaches have been taken different formulations and forms have been developed and tested on animals and humans. [41]

Reduction of particle size Edit

Nanoparticles have been explored as a delivery system for various drugs, such as improving the oral bioavailability of drugs with poor absorption characteristics. [45] However, this has not proved successful with CoQ10, although reports have differed widely. [46] [47] The use of aqueous suspension of finely powdered CoQ10 in pure water also reveals only a minor effect. [43]

Soft-gel capsules with CoQ10 in oil suspension Edit

A successful approach is to use the emulsion system to facilitate absorption from the gastrointestinal tract and to improve bioavailability. Emulsions of soybean oil (lipid microspheres) could be stabilised very effectively by lecithin and were used in the preparation of softgel capsules. In one of the first such attempts, Ozawa et al. performed a pharmacokinetic study on beagles in which the emulsion of CoQ10 in soybean oil was investigated about twice the plasma CoQ10 level than that of the control tablet preparation was determined during administration of a lipid microsphere. [43] Although an almost negligible improvement of bioavailability was observed by Kommuru et al. with oil-based softgel ucapsules in a later study on dogs, [48] the significantly increased bioavailability of CoQ10 was confirmed for several oil-based formulations in most other studies. [49]

Novel forms of CoQ10 with increased water-solubility Edit

Facilitating drug absorption by increasing its solubility in water is a common pharmaceutical strategy and also has been shown to be successful for CoQ10. Various approaches have been developed to achieve this goal, with many of them producing significantly better results over oil-based softgel capsules in spite of the many attempts to optimize their composition. [41] Examples of such approaches are use of the aqueous dispersion of solid CoQ10 with the polymer tyloxapol, [50] formulations based on various solubilising agents, such as hydrogenated lecithin, [51] and complexation with cyclodextrins among the latter, the complex with β-cyclodextrin has been found to have highly increased bioavailability [52] [53] and also is used in pharmaceutical and food industries for CoQ10-fortification. [41]

In 1950, G. N. Festenstein was the first to isolate a small amount of CoQ10 from the lining of a horse's gut at Liverpool, England. In subsequent studies the compound was briefly called substance SA, it was deemed to be quinone and it was noted that it could be found from many tissues of a number of animals. [54]

In 1957, Frederick L. Crane and colleagues at the University of Wisconsin–Madison Enzyme Institute isolated the same compound from mitochondrial membranes of beef heart and noted that it transported electrons within mitochondria. They called it Q-275 for short as it was a quinone. [55] [54] Soon they noted that Q-275 and substance SA studied in England may be the same compound. This was confirmed later that year and Q-275/substance SA was renamed ubiquinone as it was a ubiquitous quinone that could be found from all animal tissues. [54] [34]

In 1958, its full chemical structure was reported by D. E. Wolf and colleagues working under Karl Folkers at Merck in Rahway. [56] [54] [34] Later that year D. E. Green and colleagues belonging to the Wisconsin research group suggested that ubiquinone should be called either mitoquinone or coenzyme Q due to its participation to the mitochondrial electron transport chain. [54] [34]

In 1966, A. Mellors and A. L. Tappel at the University of California were to first to show that reduced CoQ6 was an effective antioxidant in cells. [57] [34]

In 1960s Peter D. Mitchell enlarged upon the understanding of mitochondrial function via his theory of electrochemical gradient, which involves CoQ10, and in late 1970s studies of Lars Ernster enlargened upon the importance of CoQ10 as an antioxidant. The 1980s witnessed a steep rise in the number of clinical trials involving CoQ10. [34]

Detailed reviews on occurrence of CoQ10 and dietary intake were published in 2010. [58] Besides the endogenous synthesis within organisms, CoQ10 also is supplied to the organism by various foods. Despite the scientific community's great interest in this compound, however, a very limited number of studies have been performed to determine the contents of CoQ10 in dietary components. The first reports on this aspect were published in 1959, but the sensitivity and selectivity of the analytical methods at that time did not allow reliable analyses, especially for products with low concentrations. [58] Since then, developments in analytical chemistry have enabled a more reliable determination of CoQ10 concentrations in various foods:

CoQ10 levels in selected foods [58]
Food CoQ10 concentration (mg/kg)
Beef heart 113
liver 39–50
muscle 26–40
Pork heart 12–128
liver 23–54
muscle 14–45
Chicken breast 8–17
thigh 24–25
wing 11
Fish sardine 5–64
mackerel:
– red flesh 43–67
– white flesh 11–16
salmon 4–8
tuna 5
Oils soybean 54–280
olive 4–160
grapeseed 64–73
sunflower 4–15
canola 64–73
Nuts peanut 27
walnut 19
sesame seed 18–23
pistachio 20
hazelnut 17
almond 5–14
Vegetables parsley 8–26
broccoli 6–9
cauliflower 2–7
spinach up to 10
Chinese cabbage 2–5
Fruit avocado 10
blackcurrant 3
grape 6–7
strawberry 1
orange 1–2
grapefruit 1
apple 1
banana 1

Meat and fish are the richest sources of dietary CoQ10 levels over 50 mg/kg may be found in beef, pork, and chicken heart and liver. Dairy products are much poorer sources of CoQ10 than animal tissues. Vegetable oils also are quite rich in CoQ10. Within vegetables, parsley and perilla are the richest CoQ10 sources, but significant differences in their CoQ10 levels may be found in the literature. Broccoli, grapes, and cauliflower are modest sources of CoQ10. Most fruit and berries represent a poor to very poor source of CoQ10, with the exception of avocados, which have a relatively high CoQ10 content. [58]

Intake Edit

In the developed world, the estimated daily intake of CoQ10 has been determined at 3–6 mg per day, derived primarily from meat. [58]


Contents

Glycosidic bonds of the form discussed above are known as O-glycosidic bonds, in reference to the glycosidic oxygen that links the glycoside to the aglycone or reducing end sugar. In analogy, one also considers S-glycosidic bonds (which form thioglycosides), where the oxygen of the glycosidic bond is replaced with a sulfur atom. In the same way, N-glycosidic bonds, have the glycosidic bond oxygen replaced with nitrogen. Substances containing N-glycosidic bonds are also known as glycosylamines. C-glycosyl bonds have the glycosidic oxygen replaced by a carbon the term "C-glycoside" is considered a misnomer by IUPAC and is discouraged. [1] All of these modified glycosidic bonds have different susceptibility to hydrolysis, and in the case of C-glycosyl structures, they are typically more resistant to hydrolysis.

One distinguishes between α- and β-glycosidic bonds by the relative stereochemistry of the anomeric position and the stereocenter furthest from C1 in the saccharide. [2] An α-glycosidic bond is formed when both carbons have the same stereochemistry, whereas a β-glycosidic bond occurs when the two carbons have different stereochemistry. One complicating issue is that the alpha and beta conformations were originally defined based on the relative orientation of the major constituents in a Haworth projection. In this case, for D-sugars, a beta conformation would see the major constituent at each carbon drawn above the plane of the ring (nominally the same conformation), while alpha would see the anomeric constituent below the ring (nominally opposite conformations). For L-sugars, the definitions would then, necessarily, reverse. This is worth noting as these older definitions still permeate the literature and can lead to confusion.

Pharmacologists often join substances to glucuronic acid via glycosidic bonds in order to increase their water solubility this is known as glucuronidation. Many other glycosides have important physiological functions.

Nüchter et al. (2001) have shown a new approach to Fischer glycosidation. [3] [4] [5] Employing a microwave oven equipped with refluxing apparatus in a rotor reactor with pressure bombs, Nüchter et al. (2001) were able to achieve 100% yield of α- and β-D-glucosides. This method can be performed on a multi-kilogram scale.

Joshi et al. (2006) [6] propose the Koenigs-Knorr method in the stereoselective synthesis of alkyl D-glucopyranosides via glycosylation, with the exception of using lithium carbonate which is less expensive and toxic than the conventional method of using silver or mercury salts. D-glucose is first protected by forming the peracetate by addition of acetic anhydride in acetic acid, and then addition of hydrogen bromide which brominates at the 5-position. On addition of the alcohol ROH and lithium carbonate, the OR replaces the bromine and on deprotecting the acetylated hydroxyls the product is synthesized in relatively high purity. It was suggested by Joshi et al. (2001) that lithium acts as the nucleophile that attacks the carbon at the 5-position and through a transition state the alcohol is substituted for the bromine group. Advantages of this method as well as its stereoselectivity and low cost of the lithium salt include that it can be done at room temperature and its yield compares relatively well with the conventional Koenigs-Knorr method. [7]

Glycoside hydrolases (or glycosidases), are enzymes that break glycosidic bonds. Glycoside hydrolases typically can act either on α- or on β-glycosidic bonds, but not on both. This specificity allows researchers to obtain glycosides in high epimeric excess, one example being Wen-Ya Lu's conversion of D-Glucose to Ethyl β-D-glucopyranoside using naturally-derived glucosidase. It is worth noting that Wen-Ya Lu utilized glucosidase in a reverse manner opposite to the enzyme's biological functionality: [8]

Before monosaccharide units are incorporated into glycoproteins, polysaccharides, or lipids in living organisms, they are typically first "activated" by being joined via a glycosidic bond to the phosphate group of a nucleotide such as uridine diphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), or cytidine monophosphate (CMP). These activated biochemical intermediates are known as sugar nucleotides or sugar donors. Many biosynthetic pathways use mono- or oligosaccharides activated by a diphosphate linkage to lipids, such as dolichol. These activated donors are then substrates for enzymes known as glycosyltransferases, which transfer the sugar unit from the activated donor to an accepting nucleophile (the acceptor substrate).

Different biocatalytic approaches have been developed toward the synthesis of glycosides in the past decades, which using “glycosyltransferases” and “glycoside hydrolases” are among the most common catalysis. The former often needs expensive materials and the later often shows low yields, De Winter et al. [9] investigated use of cellobiose phosphorylase (CP) toward synthesis of alpha-glycosides in ionic liquids. The best condition for use of CP was found to be in the presence of IL AMMOENG 101 and ethyl acetate.

Multiple chemical approaches exist to encourage selectivity of α- and β-glycosidic bonds. The highly substrate specific nature of the selectivity and the overall activity of the pyranoside can provide major synthetic difficulties. The overall specificity of the glycosylation can be improved by utilizing approaches which take into account the relative transition states that the anomeric carbon can undergo during a typical glycosylation. Most notably, recognition and incorporation of Felkin-Ahn-Eisenstein models into rationale chemical design can generally provide reliable results provided the transformation can undergo this type of conformational control in the transition state.

Fluorine directed glycosylations represent an encouraging handle for both B selectivity and introduction of a non-natural biomimetic C2 functionality on the carbohydrate. One innovative example provided by Bucher et al. provides a way to utilize a fluoro oxonium ion and the trichloroacetimidate to encourage B stereoselectivity through the gauche effect. This reasonable stereoselectivity is clear through visualization of the Felkin-Ahn models of the possible chair forms.

This method represents an encouraging way to selectivity incorporate B-ethyl, isopropyl and other glycosides with typical trichloroacetimidate chemistry.

O-linked glycopeptides recently have been shown to exhibit excellent CNS permeability and efficacy in multiple animal models with disease states. In addition one of the most intriguing aspects thereof is the capability of O-glycosylation to extend half life, decrease clearance, and improve PK/PD thereof the active peptide beyond increasing CNS penetration. The innate utilization of sugars as solubilizing moieties in Phase II and III metabolism (glucuronic acids) has remarkably allowed an evolutionary advantage in that mammalian enzymes are not directly evolved to degrade O glycosylated products on larger moieties.

The peculiar nature of O-linked glycopeptides is that there are numerous examples which are CNS penetrant. The fundamental basis of this effect is thought to involve "membrane hopping" or "hop diffusion". The non-brownian motion driven "hop diffusion" process is thought to occur due to discontinuity of the plasma membrane. "Hop diffusion" notably combines free diffusion and intercomparmental transitions. Recent examples notably include high permeability of met-enkephalin analogs amongst other peptides. The full mOR agonist pentapeptide DAMGO is also CNS penetrant upon introduction of glycosylation. [10] [11] [12] [13]


Contents

Pantothenic acid is a water-soluble vitamin, one of the B vitamins. It is synthesized from the amino acid β-alanine and pantoic acid (see biosynthesis and structure of coenzyme A figures). Unlike vitamin E, which occurs in eight chemically related forms known as vitamers, pantothenic acid is only one chemical compound. It is a starting compound in the synthesis of coenzyme A (CoA), a cofactor for many enzyme processes. [8] [10] [11]

Pantothenic acid is a precursor to CoA via a five-step process. The biosynthesis requires pantothenic acid, cysteine, four equivalents of ATP (see figure). [12]

  1. Pantothenic acid is phosphorylated to 4′-phosphopantothenate by the enzyme pantothenate kinase. This is the committed step in CoA biosynthesis and requires ATP. [13]
  2. A cysteine is added to 4′-phosphopantothenate by the enzyme phosphopantothenoylcysteine synthetase to form 4'-phospho-N-pantothenoylcysteine (PPC). This step is coupled with ATP hydrolysis. [13]
  3. PPC is decarboxylated to 4′-phosphopantetheine by phosphopantothenoylcysteine decarboxylase
  4. 4′-Phosphopantetheine is adenylated (or more properly, AMPylated) to form dephospho-CoA by the enzyme phosphopantetheine adenylyl transferase
  5. Finally, dephospho-CoA is phosphorylated to coenzyme A by the enzyme dephosphocoenzyme A kinase. This final step also requires ATP. [13]

This pathway is suppressed by end-product inhibition, meaning that CoA is a competitive inhibitor of pantothenate kinase, the enzyme responsible for the first step. [13]

Coenzyme A is necessary in the reaction mechanism of the citric acid cycle. This process is the body's primary catabolic pathway and is essential in breaking down the building blocks of the cell such as carbohydrates, amino acids and lipids, for fuel. [14] CoA is important in energy metabolism for pyruvate to enter the tricarboxylic acid cycle (TCA cycle) as acetyl-CoA, and for α-ketoglutarate to be transformed to succinyl-CoA in the cycle. [15] CoA is also required for acylation and acetylation, which, for example, are involved in signal transduction, and various enzyme functions. [15] In addition to functioning as CoA, this compound can act as an acyl group carrier to form acetyl-CoA and other related compounds this is a way to transport carbon atoms within the cell. [10] CoA is also required in the formation of acyl carrier protein (ACP), [16] which is required for fatty acid synthesis. [10] [17] Its synthesis also connects with other vitamins such as thiamin and folic acid. [18]

Dietary Edit

Food sources of pantothenic acid include animal-sourced foods, including dairy foods and eggs. [7] [9] Potatoes, tomato products, oat-cereals, sunflower seeds, avocado and mushrooms are good plant sources. Whole grains are another source of the vitamin, but milling to make white rice or white flour removes much of the pantothenic acid, as it is found in the outer layers of whole grains. [7] [11] In animal feeds, the most important sources are alfalfa, cereal, fish meal, peanut meal, molasses, rice bran, wheat bran, and yeasts. [19]

Supplements Edit

Dietary supplements of pantothenic acid commonly use pantothenol (or panthenol), a shelf-stable analog, which is converted to pantothenic acid once consumed. [8] Calcium pantothenate – a salt – may be used in manufacturing because it is more resistant than pantothenic acid to factors that deteriorate stability, such as acid, alkali or heat. [10] [19] The amount of pantothenic acid in dietary supplement products may contain up to 1,000 mg (200 times the Adequate Intake level for adults), without evidence that such large amounts provide any benefit. [8] [7] According to WebMD, pantothenic acid supplements have a long list of claimed uses, but there is insufficient scientific evidence to support any of them. [20]

As a dietary supplement, pantothenic acid is not the same as pantethine, which is composed of two pantothenic acid molecules linked by a disulfide bridge. [8] Sold as a high-dose supplement (600 mg), pantethine may be effective for lowering blood levels of LDL cholesterol – a risk factor for cardiovascular diseases – but its long-term effects are unknown, requiring that its use be supervised by a physician. [8] Dietary supplementation with pantothenic acid does not have the same effect on LDL. [8]

Fortification Edit

According to the Global Fortification Data Exchange, pantothenic acid deficiency is so rare that no countries require that foods be fortified. [21]

The US Institute of Medicine (IOM) updated Estimated Average Requirements (EARs) and Recommended Dietary Allowances (RDAs) for B vitamins in 1998. At that time there was not sufficient information to establish EARs and RDAs for pantothenic acid. In instances such as this, the Board sets Adequate Intakes (AIs), with the understanding that at some later date, AIs may be replaced by more exact information. [11] [22]

The current AI for teens and adults ages 14 and up is 5 mg/day. This was based in part on the observation that for a typical diet, urinary excretion was approximately 2.6 mg/day, and that bioavailability of food-bound pantothenic acid was roughly 50%. [11] AI for pregnancy is 6 mg/day. AI for lactation is 7 mg/day. For infants up to 12 months the AI is 1.8 mg/day. For children ages 1–13 years the AI increases with age from 2 to 4 mg/day. Collectively the EARs, RDAs, AIs and ULs are referred to as Dietary Reference Intakes (DRIs). [11] [22]

Age group Age Adequate intake [11]
Infants 0–6 months 1.7 mg
Infants 7–12 months 1.8 mg
Children 1–3 years 2 mg
Children 4–8 years 3 mg
Children 9–13 years 4 mg
Adult men and women 14+ years 5 mg
Pregnant women (vs. 5) 6 mg
Breastfeeding women (vs. 5) 7 mg

While for many nutrients, the US Department of Agriculture uses food composition data combined with food consumption survey results to estimate average consumption, the surveys and reports do not include pantothenic acid in the analyses. [23] Less formal estimates of adult daily intakes report about 4 to 7 mg/day. [11]

The European Food Safety Authority (EFSA) refers to the collective set of information as Dietary Reference Values, with Population Reference Intake (PRI) instead of RDA, and Average Requirement instead of EAR. AI and UL are defined the same as in the US. For women and men over age 11 the Adequate Intake (AI) is set at 5 mg/day. AI for pregnancy is 5 mg/day, for lactation 7 mg/day. For children ages 1–10 years the AI is 4 mg/day. These AIs are similar to the US AIs. [24]

Safety Edit

As for safety, the IOM sets Tolerable upper intake levels (ULs) for vitamins and minerals when evidence is sufficient. In the case of pantothenic acid there is no UL, as there is no human data for adverse effects from high doses. [11] The EFSA also reviewed the safety question and reached the same conclusion as in United States – that there was not sufficient evidence to set a UL for pantothenic acid. [25]

Labeling requirements Edit

For US food and dietary supplement labeling purposes the amount in a serving is expressed as a percent of Daily Value (%DV). For pantothenic acid labeling purposes 100% of the Daily Value was 10 mg, but as of 27 May 2016 it was revised to 5 mg to bring it into agreement with the AI. [26] [27] Compliance with the updated labeling regulations was required by 1 January 2020 for manufacturers with US$10 million or more in annual food sales, and by 1 January 2021 for manufacturers with lower volume food sales. [28] [29] A table of the old and new adult daily values is provided at Reference Daily Intake.

When found in foods, most pantothenic acid is in the form of CoA or bound to acyl carrier protein (ACP). For the intestinal cells to absorb this vitamin, it must be converted into free pantothenic acid. Within the lumen of the intestine, CoA and ACP are hydrolyzed into 4'-phosphopantetheine. The 4'-phosphopantetheine is then dephosphorylated into pantetheine. Pantetheinase, an intestinal enzyme, then hydrolyzes pantetheine into free pantothenic acid. [30] Free pantothenic acid is absorbed into intestinal cells via a saturable, sodium-dependent active transport system. [15] At high levels of intake, when this mechanism is saturated, some pantothenic acid may also be additionally absorbed via passive diffusion. [19] As a whole, when intake increases 10-fold, absorption rate decreases to 10%. [15]

Pantothenic acid is excreted in urine. This occurs after its release from CoA. Urinary amounts are on the order of 2.6 mg/day, but decreased to negligible amounts when subjects in multi-week experimental situations were fed diets devoid of the vitamin. [11]

Pantothenic acid deficiency in humans is very rare and has not been thoroughly studied. In the few cases where deficiency has been seen (prisoners of war during World War II, victims of starvation, or limited volunteer trials), nearly all symptoms were reversed with orally administered pantothenic acid. [15] [10] Symptoms of deficiency are similar to other vitamin B deficiencies. There is impaired energy production, due to low CoA levels, which could cause symptoms of irritability, fatigue, and apathy. [15] Acetylcholine synthesis is also impaired therefore, neurological symptoms can also appear in deficiency [31] they include sensation of numbness in hands and feet, paresthesia and muscle cramps. Additional symptoms could include restlessness, malaise, sleep disturbances, nausea, vomiting and abdominal cramps. [31]

In animals, symptoms include disorders of the nervous, gastrointestinal, and immune systems, reduced growth rate, decreased food intake, skin lesions and changes in hair coat, and alterations in lipid and carbohydrate metabolism. [32] In rodents, there can be loss of hair color, which led to marketing of pantothenic acid as a dietary supplement which could prevent or treat graying of hair in humans (despite the lack of any human trial evidence). [10]

Pantothenic acid status can be assessed by measuring either whole blood concentration or 24-hour urinary excretion. In humans, whole blood values less than 1 μmol/L are considered low, as is urinary excretion of less than 4.56 mmol/day. [10]

Calcium pantothenate and dexpanthenol (D-panthenol) are European Food Safety Authority (EFSA) approved additives to animal feed. [1] Supplementation is on the order of 8–20 mg/kg for pigs, 10–15 mg/kg for poultry, 30–50 mg/kg for fish and 8–14 mg/kg feed for pets. These are recommended concentrations, designed to be higher than what are thought to be requirements. [1] There is some evidence that feed supplementation increases pantothenic acid concentration in tissues, i.e., meat, consumed by humans, and also for eggs, but this raises no concerns for consumer safety. [1]

No dietary requirement for pantothenic acid has been established in ruminant species. Synthesis of pantothenic acid by ruminal microorganisms appears to be 20 to 30 times more than dietary amounts. [33] Net microbial synthesis of pantothenic acid in the rumen of steer calves has been estimated to be 2.2 mg/kg of digestible organic matter consumed per day. Supplementation of pantothenic acid at 5 to 10 times theoretical requirements did not improve growth performance of feedlot cattle. [34]

Bacteria synthesize pantothenic acid from the amino acids aspartate and a precursor to the amino acid valine. Aspartate is converted to β-alanine. The amino group of valine is replaced by a keto-moiety to yield α-ketoisovalerate, which, in turn, forms α-ketopantoate following transfer of a methyl group, then D-pantoate (also known as pantoic acid) following reduction. β-alanine and pantoic acid are then condensed to form pantothenic acid (see figure). [13]

The term vitamin is derived from the word vitamine, which was coined in 1912 by Polish biochemist Casimir Funk, who isolated a complex of water-soluble micronutrients essential to life, all of which he presumed to be amines. [35] When this presumption was later determined not to be true, the "e" was dropped from the name, hence "vitamin". [19] Vitamin nomenclature was alphabetical, with Elmer McCollum calling these fat-soluble A and water soluble B. [19] Over time, eight chemically distinct, water-soluble B vitamins were isolated and numbered, with pantothenic acid as vitamin B5. [19]

The essential nature of pantothenic acid was discovered by Roger J. Williams in 1933 by showing it was required for the growth of yeast. [36] Three years later Elvehjem and Jukes demonstrated that it was a growth and anti-dermatitis factor in chickens. [10] Williams dubbed the compound "pantothenic acid," deriving the name from the Greek word pantothen, which translates as "from everywhere." His reason was that he found it to be present in almost every food he tested. [10] Williams went on to determine the chemical structure in 1940. [10] In 1953, Fritz Lipmann shared the Nobel Prize in Physiology or Medicine "for his discovery of co-enzyme A and its importance for intermediary metabolism", work he had published in 1946. [37]


Discussion

Obtaining information about the activity of individual members within a microbial community with respect to environmental perturbation, such as availability of different electron donors or acceptors, has long been stymied by the inability to link metatranscriptomic data to specific microorganisms. Metatranscriptomic data were generated for the community growing on hexadecane, butyric acid or caprylic acid using a low-input metatranscriptomics protocol optimized for the very low biomass of the community (Figure 1b). These metatranscriptomes were used in conjunction with the Smithella draft genome, obtained from single-cell sequencing and other representative genomes, to investigate both metabolic capabilities and microbial interactions of the community.

In Smithella, a number of pathways potentially related to alkane degradation were identified in the genome. Comparison of transcriptomic data obtained from all three carbon source additions explicated the activity of these genes during hexadecane degradation. Emulsification and membrane genes that are known to assist in the degradation of hydrophobic alkanes (Tzintzun-Camacho et al., 2012) were both present and active within Smithella. The gene encoding an apolipoprotein N-acyltransferase is of particular note, as previous studies have shown that deletion of lipoprotein synthesis genes can decrease membrane hydrophobicity in bacteria (Okugawa et al., 2012). Expression of this gene during growth with hexadecane may assist Smithella to adapt its membrane composition to the presence of the hydrophobic substrate. Transporters for long-chain fatty acids, such as palmitate, octadecanoate and tetradecanoate, were also encoded in the genome and actively expressed during growth with hexadecane only, suggesting that these genes may be assisting in the export of biosurfactants for hexadecane emulsification (Breuil and Kushner, 1980 Liu et al., 2012).

To date, very few enzymes involved in the activation of alkanes without molecular oxygen have been elucidated (So et al., 2003 Zedelius et al., 2011 Callaghan et al., 2012). Although the Smithella genome enodes a gene for methylmalonyl-CoA mutase, a protein known to have a role in fumarate-dependent anaerobic hexadecane degradation (Wilkes et al., 2002 Callaghan et al., 2012), this mode of degradation seems unlikely in this organism. Genes for alkyl-succinate synthase subunits, critical for the fumarate-dependent activation of alkanes, were not identified in the genome. This is consistent with previous studies that suggest a mechanism other than fumarate addition that drives alkane activation under methanogenic conditions (Aitken et al., 2013). Genes highly expressed during growth with hexadecane and not with fatty acids, particularly hypothetical proteins with no annotated function (Supplementary Tables S7 and S8), could provide potential candidates involved in this pathway.

Biological conversion of hexadecane to methane requires the interaction of syntrophic bacteria with methanogenic archaea (Zengler et al., 1999). A number of methanogens, notably the acetoclastic M. concilii and M. harundinacea as well as the hydrogenotrophic M. marisnigri and Methanocalculus were found to be highly abundant in our community. On the basis of 16S rDNA data, Methanosaeta species were well represented under all three different growth conditions, but dominated during fatty acid degradation (Figure 4b). While M. concilii was almost exclusively active during hexadecane degradation based on transcriptomic analysis, both M. concilii and M. harundinacea were active during oxidation of fatty acids (Supplementary Table S4). Although at least two different species of Methanosaeta appeared to be active within the community, they are fulfilling different metabolic roles under each condition. M. harundinacea is likely using formate as an electron donor in addition to acetate utilization during fatty acid degradation. The M. concilii genome does not encode a complete formate–dehydrogenase complex. Thus, if formate is being used as an electron carrier during hexadecane degradation as was revealed through de novo transcriptome analysis, it is not being utilized by a member of Methanosaeta.

M. concilii is known for its high affinity to acetate, and is unable to use hydrogen for methanogenesis (Smith and Ingram-Smith, 2007). Despite this, the M. concilii genome encodes genes required for CO2 reduction, which, based on our transcriptome analysis, were all found to be highly transcriptionally active under all three conditions with the majority of the enzymes being expressed within the upper 50th percentile. Although the entire CO2 fixation branch is active, different subunits of formyl-MF dehydrogenase, the first step in CO2 reduction (Figure 4a), were expressed at variable levels across conditions. FmdA was the most highly expressed during fatty acid degradation (97th percentile), but did not appear to be expressed during hexadecane degradation (13th percentile). From previous studies, FmdA has been shown to be the catalytic subunit exhibiting amidohydrolase activity (Holm and Sander, 1997), suggesting that M. concilii may be utilizing methylamines during fatty acid degradation. Alternatively, FmdC also exhibited higher levels of expression during fatty acid degradation and is located right after the FmdA gene, so these two genes may be co-regulated. FwdG was the second most highly expressed (82nd percentile) subunit during hexadecane degradation, whereas FwdG was expressed near the median during fatty acid degradation (42nd percentile). M. concilii has previously been shown to form conductive aggregates with species of Geobacter, suggesting that direct interspecies electron transfer (DIET) can occur between these species (Morita et al., 2011). It has recently been demonstrated by the Lovley group that Methanosaeta can reduce CO2 while accepting electrons by DIET using a transcriptomic and radiotracer approach (Rotaru et al., 2013). The higher expression of FwdG during hexadecane degradation potentially indicates that a source of electrons in addition to acetate is being utilized for methanogenesis, as FwdG is predicted to be a potential electron carrier. Although we cannot directly confirm that DIET is occurring in this consortium, it is possible that Smithella is transferring electrons to M. concilii for CO2 reduction in addition to acetate during hexadecane degradation.

Methanocalculus, a member of the family Methanocorpusculaceae that can reduce CO2 with hydrogen or formate as an electron donor (Zellner et al., 1989), was the predominant methanogen under hexadecane-degrading conditions. Members of this family were previously found in methanogenic alkane-degrading communities, composing around 20–30% of the archaeal community (Grabowski et al., 2005 Gray et al., 2011). Although we could not directly confirm the activity of Methanocalculus, both formate dehydrogenases and ech hydrogenases were actively transcribed during growth with hexadecane. It has been suggested that methanogenic degradation of long-chain alkanes requires the presence of both acetoclastic as well as hydrogenotrophic methanogens (Zengler et al., 1999 Jones et al., 2008). Thus, it is possible that Methanocorpusculaceae is fulfilling this role in concurrence with M. concilii in this community.

The hydrogenotrophic M. marisnigri, a member of the Methanomicrobiales, was most abundant during hexadecane degradation (Figure 4b), but only a small percentage of reads (11 762, representing 0.02%) mapped under this condition. This discrepancy likely arises from sequence dissimilarity between sequenced strains available in databases and the actual organism present in the community. Thus, in order to fully assess the activity of specific community members, draft genomes of targeted species must be obtained by a single-cell approach used here or alternatively by a computational approach that bins metagenomic sequences (Chatterji et al., 2008 Dick et al., 2009 Albertsen et al., 2013).

Smithella, a genus that to date has not been sequenced, was targeted for this study. Integration of single-cell genome sequencing and metatranscriptomics allowed us to identify potential genes involved in anaerobic hexadecane degradation. Extension of the metatranscriptomic data sets to additional representative genomes of community members yielded insight into the nature of potential syntrophic interactions Smithella participates in. Furthermore, applying species-specific genome-scale analysis of this community across multiple conditions provided insights into the mechanisms driving major shifts in abundance and activity of key community constituents. Moving beyond purely descriptive, meta-level analyses of communities will eventually allow an understanding of the plasticity of community composition and the capacity of individual members to accommodate and respond to environmental perturbation.


Organic Molecules Found in the Plant Cells | Plant Physiology

The following points highlight the seven main organic molecules found in the plant cells. The organic molecules are: 1. Nucleic Acids 2. Proteins 3. Organic Acids 4. Carbohydrates 5. Lipids 6. Fatty Acids 7. Secondary Plant Products.

Organic Molecule # 1. Nucleic Acids:

Nucleic acids are polymers of nucleotides. Each nucleotide is made up of a base and a sugar esterified with a molecule of phosphoric acid.

Nucleoside is a base-sugar combination but without phosphoric acid (Fig. 4-3). Nucleotides are either ribonucleotides or deoxyribonucleotides depending upon the presence of a specific sugar, which is ribose in the former and deoxyribose (lacking an oxygen atom) in the latter.

Ribonucleic acid (RNA) is a polymer of ribonucleotides whereas deoxyribonucleic acid (DNA) is a polymer of deoxyribonucleotides.

RNA consists of four organic bases e.g. adenine, uracil, cytosine and guanine whereas DNA has adenine, thymine, cytosine and guanine bases (Fig. 4-2).

Easter bonds link the monomers with the phosphate group from the C-5 of one sugar to the C-3 of the next (Fig. 4-1, 3).

The ribonucleotides are highly important in the major energy transfer reactions during cellular metabolism. In fact, large amount of energy is needed for the synthesis of phosphate esters. When these bonds are hydrolysed large amount of energy is released.

Adenosine series ribonucleotides are the most important ones and some of these include adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP).

It may be added that other ribonucleotides and deoxyribonucleotides also form di-and tri-phosphate esters. The triple esters of the nucleotides combine to form RNA and DNA. Further two phosphate groups are eliminated in the condensation of each nucleotide.

Deoxyribonucleotide combination or deoxyribonucleic acid (DNA) carries most of the genetic information in the cell. It comprises two parallel strands which are tightly linked together through hydrogen bonds between amino and carbonyl groups (C = O) group of adjacent bases (Fig. 4-1). The linking is very specific so that a purine always links with a pyrimidine.

Thus, adenine always bonds to thymine and cytosine to guanine. These pairs of bases are referred to as complementary since exact fit is permitted between complementary bases because of their molecular size and structure. The two strands then coil into a double helix because of hydrogen bonding as depicted in Fig. 4-1.

The double helix is self-duplicating because of similar base pairing and because only complementary bases can pair. Replication of DNA usually occurs by semiconservative process where each half of an original double helix is used as a template. Thus, two exact copies of the original double helix are formed during cell division. Replication may proceed in one or both directions from a given point.

DNA model as proposed by Watson and Crick envisages conservation of genetic information from generation to generation and also synthesis of identical replicate molecules. The general view is that at least three DNA polymerases (I, II, III) are involved in DNA replication of these DNA polymerase I fill the gaps between Okazaki fragments whereas II and III polymerases remove 3′ → 5′ pieces.

DNA appears to have two main functions: storage and replication of genetic information specific to an organism. It acts as a template for its own replication and also stores coded information which is transcribed into RNA. Tables 4-2, 4-2A show different structural forms of DNA (Fig. 4-3).

The information stored in the DNA is read by the formation of RNA strand which is complementary to that part of the DNA strand forming its template. The RNA formed is called messenger RNA (mRNA) mRNA carries a specific codon which consists of a triplet of nucleotides, and codes for a specific amino acid. There are two additional RNAs present in the cell and these are soluble RNA (sRNA) and transfer RNA (tRNA).

All the three RNAs are essentially involved in accomplishing translation process. Table 4-4 gives a comparison of three RNA types. Table 4-3 Compares DNA and RNA.

Figure 4-4 depicts diagrammatic representation of a set of processes leading from the transcription to final protein synthesis. Each of the tRNA comprises a triplet of bases which is precisely complementary to a codon.

Clearly, each of the specific tRNA has a specific codon which is able to combine with a specific amino acid required by the codon. Through hybridization between single strands of DNA or RNA or DNA-DNA DNA-RNA RNA-RNA it is possible to detect the complementary nucleotide sequences.

The required amino acids are lined up in a sequence determined by the mRNA where they are successively linked together chemically and then released from the tRNAs.

Nirenberg and Matthei (1961) artificially synthesized mRNA which comprised poly U (UUU) and was specific for phenylalanine amino acid. Subsequent studies indicated that poly C (CCC) RNA directed formation of poly-proline only. When AAA was used as mRNA only poly-lysine was formed.

It may be mentioned that UUU was the first triplet code word or codon identified. There are 20 different amino acids as units of translation and triplets of nucleotides only serve as codons for amino acids. Thus nucleotides triplets could form 64(43) different combinations.

Using artificially prepared messengers made up of nucleotides in diverse combinations, as many as 50 codons were recognised. Soon after it became evident that genetic code was degenerative i.e. more than one codon could be concerned with one amino acid.

Further studies revealed that except for tryptophan and methionine, all other amino acids were specified by 2 to 6 different codons. The sequence of nucleotides on the tRNA is called anticodon. For instance, for leucine the codon is CUG and the anticodon is GAC.

The code letter is a nucleotide in DNA or RNA. The sequence of 3 nucleotides in a codon is called code word. Some are nonsense codons and do not code for any amino acid. There are some ambiguous codons which code for more than one amino acids e.g. CCA codes for glycine and glutamic acid. In bacteria the starting amino acid is N-formyl methionine whereas in eucaryotes it is methionine.

The anticodon for both is UAC while the codon is AUG. AUG is also referred to as the initiating codon and protein synthesis is initiated at this site. On the contrary some of the codons like UAA, UAG and UGA are called termination codons. The genetic code is universal and the sequence of nucleotides on a mRNA molecule is called reading frame.

The multicellular plant develops from a zygote following growth and differentiation. Growth is defined variously either as an increase in dry or fresh weight, or even increase in DNA or proteins.

It may also be considered as non-reversible increase in volume. Growth also involves cell division and cell elongation.

Cell division usually implies mitotic cell comprising several phases (prophase, metaphase, anaphase, telophase, etc.).

In a division cycle the chromosome is split longitudinally to produce two homologous chromatids.

Thus, from one nucleus, two daughter nuclei are produced. The two daughter nuclei are genetically similar.

In the meristematic tissues, cells undergo cycle of DNA synthesis or undergo mitotic cycle. These are G1 (DNA synthesis or post-mitotic phase), G2 (pre­mitotic phase without DNA synthesis) separated by S phase. The mitotic phase begins after G2 and comprises several subphases like prophase, metaphase, anaphase and telophase.

Chromosomes are made up of DNA which has autocatalytic function e.g., replication.

Similarly enzymes (DNA polymerases) have been isolated which could successfully support DNA synthesis in a cell-free system. These enzymes need DNA building blocks as well as some cofactors.

Active cell division is restricted to meristematic cells. In the fully differentiated cells, cell division is rare or absent. Sometimes process (s) of cell differentiation could be disturbed and thus cell division could continue. In a tissue culture medium, a part of plant could be divided repeatedly. Sometimes tumors are produced due to uncontrolled cell division.

Much of the work has been done in Nicotiana especially in hybrids involving N. Langsdorffii. The tumors may also be produced by viruses. Agrobacterium is known to induce crown galls. These galls may be produced on any part of the plant following an inflicting of wound.

The entry of bacteria specifically through the wounds is a must for gall formation. Initially a callus is formed after injury and it grows into a tumor. Cell divisions in a gall are not oriented periclinally.

Introduction of 5-FUD in the wounds inhibits gall tumor formation. There is thus synthesis of DNA in a gall and also synthesis of tumor-inducing principle (TIP) released by bacterium. This TIP appears to be bacterial DNA. The ability of cells to divide is under the regulation of the organism. The fact that the cells of plants differentiate in different ways even though they have the same genetic material, is quite intriguing.

In recent years it has become increasingly evident that cells are omnipotent or are bestowed with the genetic totipotency. Isolated single cells from carrot stem or root when grown in tissue culture have been shown to differentiate into callus or even carrot plantlets.

Similarly enzyme-treated cells, where cell wall is removed, produce protoplasts and the latter are grown in full-fledged plants. The protoplasts may be raised from mesophyll cells, fruit wall or even microspores.

All the diploid cells possess genetic information for variable characters. Interestingly not all of these genes are active or functional at the same time. Seemingly specific gene (s) or gene blocks become active at one developmental phase whereas in another tissue yet another set of genes becomes functional. This would explain differentiation at a specific time.

Thus differences in gene activity causing variable differentiation could be attributed to number and position of genes and also their activity. In summary, gene activity may be visualized in terms of transcription or translation. Differential gene activity may affect these two levels and hence variable differentiation may be brought about.

Giant and lambrush chromosomes provide interesting system to demonstrate differential gene activity. Like the salivary glands of diptera insects, giant chromosomes have been reported in endosperm, antipodals, and suspensor cells. These giant chromosomes comprise hundreds of parallel aligned homologous chromosomes.

In fact, each chromatid is divided longitudinally. There is also discernability of transverse bands connected by faintly staining portions of chromatids. Genes are located on these deeply- staining transverse bands. Under the spell of primary activity of genes these bands undergo structural changes.

Consequently their DNA unfolds into a puff like structure or Balbiani ring. The unfolded DNA transcribes mRNA. As will be noticed, these puffs are areas of high transcription or primary gene activity. During the different phases of development, puffs disappear and new ones are formed. In summary, specific “puffing” patterns indicate microscopically visible expression of differential gene activity.

Using biochemical methods it has been possible to demonstrate structural differences in mRNA produced during different phases of growth and differentiation of an organ, seedling, etc. It may be inferred that differential gene activity is manifested at the level of mRNA.

The proteins represent the secondary gene products. Similarly, enzymatic proteins also point towards the same. Using electrophoresis technique with polyacrylamide gels, phase and tissue specific patterns of proteins, different isozymes have been shown. Some of the enzymes where isozyme patterns have been worked out are those of peroxidase, dehydrogenases and amylases, etc.

This is an interesting demonstration of differential gene activity at the proteins levels. For instance, pattern of peroxidase and protein has been shown to vary with the tissue. Obviously differential gene activity controls differentiation. Coupled with their behaviour we need to provide answers on the basis and/or factors causing their activation or inactivation.

In a way we need to understand how gene activity is “switched on” and “switched off’? Else we need to know the type of stimulus which affects the different states of activity of the genes. In general, either internal and external factors or conditions affect regulation of gene activity.

Regulation of gene activity is best explained in terms of transcription (RNA) and translation. As early as 1961, Jacob and Monad proposed a model to explain the regulation of gene activity (Fig. 4-5). As is evident from Fig. 4-5, there are regulator, operator and structural genes. The group of functionally related structural genes, along with operator gene is referred to as an Operon.

In multicellular systems a modification of this model (Fig. 4-6) has been proposed by Britten and Davidson (1969).

The genes could be possibly activated because of the interaction of the 3 types of genes and possibly through substrate induction. Similarly genes could be inactivated through end product repression. It is assumed that repressors are presumably inactivated by low molecular weight metabolites (effectors).

Once the repressor is in activated, the blockage of the operon is released and hence it is activated and thereafter its structural genes code for specific proteins. Obviously, repressor which is produced by the regulator gene, blocks the operator gene and thus the activity of the latter is stopped.

The structural genes transcribe mRNA. The effector has been shown to be a substrate of an enzyme also which is coded by the operon. Sometimes the effector has been shown to be an end product of a reaction or a series of reactions.

In eukaryotes, DNA of chromosomes is surrounded by proteins. Some of the proteins are basic histones. In addition a little amount of acidic and neutral nuclear proteins is also associated with DNA. Histones abound in arginine and lysine. Thus histones may be rich in either of the two amino acids.

Histones have been suggested to participate in the regulation of gene activity. Available evidences have shown that inhibition of transcription is caused by histones and that histones cause specific inhibitory effect. Some workers dispute this suggestive role of histones. Majority of the evidences do support the role of histones in the repression of gene activity or transcription.

In general, histones may be causing contraction of chromosomes and thereby regulating the heterochromatic areas. Such an effect appears to be unspecific. According to second viewpoint specific histones inhibit particular gene locus. Yet another thinking is that histones complex with RNA.

Phytochrome may be playing a vital role in intercellular regulation. A reference was made to the model of Britten and Davidson (1969). The varied developmental processes were shown to be mediated by different phytohormones.

Elsewhere, the recent hypothesis concerning the role of cyclic AMP or even ethylene as second messenger. Accordingly phytohormones would be primary messengers and they possibly induce second messenger.

From amongst the external factors, temperature and light contribute significantly in the regulation of gene (s) activity. Temperature affects RNA production in plant giant chromosomes. Possibly temperature affects metabolic processes.

The role of light of different wavelengths in regulating metabolic and developmental processes. In fact photomorphogensis has received considerable attention in recent years. It seems well established that phytochrome system activates gene activity. In fact, a parallelism has been shown to exist between the external factor light and phytohormone (s) (GA) action.

The possibility of red light in promoting ethylene production has also been suggested. There is a good justification to believe that external factors and phytohormones may be affecting the state of activity of the genes through a second messenger.

Organic Molecule # 2. Proteins:

Proteins are polymers of twenty different amino acids. Structurally and functionally, no two proteins of an organism are similar. The differences exist at the sequence of the amino acids. Differences among proteins cause variations in organs or developmental stages. The structure of protein is best visualized at four levels of organization (primary, secondary, tertiary and quaternary).

Primary structure is the simplest level of organization of protein which is constituted by the polymers of amino acids (Fig. 4-7). Some correlation between the primary structure of nucleic acids and proteins is assumed. The secondary structure is obtained by the folding and intra-molecular hydrogen bonding in the polypeptide while the tertiary structure refers to the three dimensional structure of protein.

The tertiary structure is stabilized by the side chains of amino acids. Sometimes in a protein, several polypeptides may be associated to constitute a complex macromolecule. This is the quaternary structure of the protein and each one of its polypeptide units has its own primary, secondary or tertiary structures. The mechanism of the synthesis of polypeptides i.e. primary structure is well understood.

Figure 4-8 shows basic amino acid structure having an amino group near a carboxylic acid group. To the latter are attached different side groups (R). These side groups are lipophilic or hydrophilic. Proteins are formed from amino acid monomers by the splitting of a molecule of water from the two adjacent amino acids rather than the chemical structure and based on this following types are recognized:

One of the classifications takes into account solubility rather than the chemical structure and based on these following types are recognised:

These proteins are made up of amino acids only.

These are of six types and described below:

These are water solube and also dissolve in dilute salt solutions. Most of the enzymatic proteins are included in it.

They are insoluble or very slightly soluble in water. They are important enzymatic or storage proteins.

These proteins are rich in arginine and are of low molecular weight. They may be associated with nuclear proteins.

They are rich in basic amino acids and soluble in water. They are in the nuclei and associated with nucleoproteins.

They are rich in proline and insoluble in water. Some enzymes are prolamines.

They are major storage proteins which are soluble in dilute acid or base. There are also conjugated proteins which besides their polypeptides contain prosthetic group. Their further classification is based on the prosthetic group.

Some enzymes have metal prosthetic group closely associated with the protein moiety e.g., molybdenum in nitrate reductase.

Here it is a combination of protein and a carbohydrate. They may be components of membranes.

Here protein is associated with different types of lipids.

These are histones associated with nucleic acids.

Here pigments are associated with proteins. For example, chlorophyll-protein complexes, flavoproteins are included in this.

Organic Molecule # 3. Organic Acids:

Plants also contain organic acids which are highly significant in several different kinds of metabolic processes. Some of the important organic acids which participate in the TCA cycle are malic, citric, α-keto-glutaric and succinic acids. In some systems malic and citric acids accumulate in large quantities in the vacuoles of the plants and offer a sour taste to the tissue.

Some organic acids impart flavour to the fruits. Within the cell several metabolic intermediates are also present and some of these serve as substrates for reactions leading to biosynthesis of plant constituents.

For instance, from pyruvic acid, acetyl-CoA is formed which is an important intermediate. This enters the TCA cycle and is metabolized into carbon dioxide and water under aerobic conditions.

Several other plant constituents like gibberellins, carotenoids, steroids, rubber, etc. are formed from acetyl-CoA. The processes of catabolism and anabolism are not haphazard but controlled through several processes which include structure and organization of an organ competition of metabolites enzymes, etc.

Organic Molecule # 4. Carbohydrates:

Simple carbohydrates, the first product formed during Calvin cycle are converted into simple carbohydrates through series of reactions.

Carbohydrates not only serve as a source of chemical energy but also furnish the basic carbon skeleton for all other organic substances found in plants and animals.

Originally, carbohydrates were thought to be the hydrates of carbon with empirical formula Cx (H2 O)x or organic substances containing elements, carbon, hydrogen and oxygen, generally in the ratio of 1:2: 1.

Now we know that they are not truly hydrates of carbon because there are several exceptions to this generalised formula, lactic acid has the formula C3 H6 O3 whereas myoinositol has C6 H12 O6 and they are not carbohydrates.

Deoxyribose, a sugar of common biological importance, has the formula of <>3 H10 O4 rather than C3 H1 0 O5. Moreover, some compounds have general properties of carbohydrates but contain nitrogen and sulphur in addition to C, H and O. Such compounds do not stick to the rigorous definition based on carbon, hydrogen and oxygen composition.

A more appropriate definition of carbohydrates or saccharides is, the polyhydroxyaldehydes or ketones or their derivatives. They can be classified roughly into three main groups: monosaccharides, oligosaccharides and polysaccharides.

Monosaccharides:

These are the least complex carbohydrates or simple sugars which cannot be hydrolysed into smaller units under reasonably mild conditions.

Oligosaccharides:

(Oligo=”few”. These comprise two to ten molecules of simple sugars joined by glycosidic linkage.

Polysaccharides:

These are polymers of monosaccharide units which are joined in a chained fashion or in a branched structure.

If polysaccharides contain recurring units of similar kind of monosaccharides, they are termed as homopolysaccharides when two or more different monosaccharide units are bound together, then they are called as heteropolysaccharides.

Carbohydrates can also be classified as reducing and non-reducing sugars. The reducing sugars function as reducing agents due to the presence of free aldehyde or ketone groups in a molecule.

They reduce the metal ions like copper and silver in the alkaline solution and ‘Benedicts’ solution is used to detect these reducing sugars.

Optical Isomerism:

This type of isomerism is mostly found in carbohydrates, like the amino acids, they are also optically active and are able to rotate the plane of polarized light due to the presence of asymmetric carbon atom.

We shall consider D-glyceraldehyde, a triose as an initial example of carbohydrate because it has a single asymmetric carbon atom and it can exist in the form of two optical isomer which are mirror images of each other and are known as enantiomers.

Depending upon the ability to rotate the plane of polarized light to left or right they are classifies as Levorotatory (l or L) and dextrorotatory (d or D), respectively. All naturally occurring sugars are D- form (Fig. 4-16, 18).

Sugars may exist in two forms:

(i) Six membered ring form called pyranose (derivatives of heterocyclic compound pyran) (Fig. 4-17), and

(ii) Five membered form called as furanose (derivatives of furon) (Fig. 4-17).

Some common examples of hexose and pentose with pyranose and furanose forms are given elsewhere.

Monosaccharides:

These are the simplest sugars forming basic building blocks of all the complex carbohydrates. The empirical formula for monosaccharide is (CH2O)n where n=3 or larger. They can be aldehyde derivative (aldose) or ketone derivatives (ketose). Further they can also be classified as trioses (3 carbon), tetroses (4 carbon), pentoses (5 carbon), hexoses (6 carbon), heptoses (7 carbon) and octoses (8 carbons) depending upon the number of carbon atoms (Table 4-5).

Of all these, hexoses and pentoses are most abundant sugars found in plants whereas heptoses are rare. Each of these sugars exists in two series, aldo and keto.

Amongst the pentoses D-xylose and L-arabinose are particularly important constituents of cell walls. D-ribose and 2- deoxyribose are components of ribonucleic acid and deoxyribonucleic acid, respectively. Most common hexose sugar found in an uncombined state is D-glucose (or dextrose). D-glucose is the result of the hydrolysis of starch or cellulose and mostly found in fruits.

D-galactose and D-mannose are the important constituents of cell wall polysaccharides. D-galactose also occurs as a component of oligosaccharide like raffinose, stachyose and lactose.

Another two branched chain monosaccharides, apinose (5 carbon) and hamamelose (6 carbon), have been shown to occur in plants like oleander and bark of witch hazeel. Hamamelose has been found to occur in combination with tannin in Primula species.

All the monosaccharides are soluble in water and sweet in taste but are insoluble in non-polar solvents.

Derivatives of Monosaccharides:

Several different types of derivatives of monosaccharides exist in a plant. We shall be dealing with a few of them.

Sugar Alcohols, Sugar acids and amino sugars:

In sugar alcohols, carboxyl group of the monosaccharide is reduced by H2 gas in the presence of a catalyst or by sodium amalgam in H2O. Sugar alcohols like mannitol, glycerol, sorbitol, etc. are found in nature.

Among sugar acids, three main types are more common aldonic acid, aldaric acid and uronic acid. Aldonic acids are formed when aldehyde group of the aldose sugars are oxidized by some oxidizing agent or by specific enzymes e.g. D-glucose gives rise to D-gluconic acid.

Aldaric acids have both aldehyde-carbon atom and carbon of hydroxyl group oxidized by strong oxidizing agents like nitric acid. Uronic acids are biologically very important. Here, only carbon atom of primary hydroxyl group is oxidized. In plants, D-glucuronic acid and D-galactouronic acid are commonly found as components of cell wall.

Amino sugars which occur as components of glycoproteins have one carbon at 2-position of the hydroxyl group replaced by amino group. Commonest amino sugars are D-glucosamine and D- galactosamine (Fig. 4-19).

Oligosaccharides:

Oligosaccharides are the connecting links between disaccharides and polysaccharides (Table 4-6). Disaccharides are most abundantly found in plant kingdom.

Since the monosaccharides have free and reactive hydroxyl groups, therefore two units or molecules may join by glycosidic linkage to form a disaccharide. The most common disaccharides are sucrose, maltose, gentianose and melibiose. Sucrose is the principal disaccharide present in higher plants and is known as cane sugar.

It is produced either directly as a product of photosynthesis or indirectly from simple sugars in many non-photosynthetic tissues. Carbohydrates are transported mainly in the form of sucrose.

Latter comprises glucose and fructose moieties (O-β-D-fructo furanosyl (2→ 1) α -D-glucopyranoside). Unlike most disaccharides, sucrose has no free anomieric carbon atom and free aldehyde group (Fig. 4-20). That is why it is a non-reducing sugar and does not reduce cupric ions.

It is a disaccharide, product of CO2 fixation during photosynthesis and economically important agricultural commodity, as a nutrient to living organisms. It is a non-reducing, sweet, highly soluble molecule chemically inert when in contact with proteins since it cannot form covalent adducts with free amino groups.

Sucrose molecules retain a high free energy of hydrolysis-highest known for glycosidic bond. A-glucosyl residue in sucrose is very efficient mode of energy conservation by the plant. Major portion of CO2 fixed photo-synthetically is captured in sucrose. Via sucrose organic carbon is trans-located to different non-photosynthetic organs from green tissues.

It is also used in the synthesis of starch and fructans-storage glycosides. Sucrose which is stored in the seeds is utilized during seed germination and seedling growth. Sucrose is usually stored in the vacuoles and it is estimated that leaf vacuole at mid-day has sucrose nearly as 11 mM whereas cytosol has 55 mM. In the metabolism of sucrose several regulatory factors and enzymes play a vital role.

In the following a simplified scheme of its synthesis is given:

Synthesis of Sucrose:

Sucrose is synthesized in the cytosol and the chloroplast where the Calvin cycle operates. Free glucose and fructose are not the precursors of sucrose.

On the other hand their phosphorylated forms are the precursors. Triose phosphates extruded from the chloroplasts serve as precursor of hexose phosphates and then sucrose.

More details are given below:

The above equation also indicates that only one ATP is required to form the glycosidic bond that connects glucose to fructose in sucrose:

Glucose – 1- phosphate + fructose – 6 – phosphate + 2H2 O + ATP → sucrose + 3Pi + ADP

Since three ATP molecules are needed during the Calvin cycle for each carbon in each of the hexose of sucrose (36 of total ATP), the one additional ATP required to form the glycosidic bond in sucrose is only a minor additional requirement.

F-1, 6-P2 regulates formation of F-6-P and hence sucrose synthesis.

Sucrose phosphate synthase is regulated by several factors e., light, Pi, G-6-P/Pi ratio F-2, 6-P2. The last factor is a modulatory molecule which regulates sucrose synthesis.

It may be observed that combined action of sucrose-6-phosphate phosphatase and sucrose phosphate synthase (SPS) causes sucrose synthesis.

Recently maize SPS gene promoter has been expressed in transgenic tomato resulting in high sucrose synthesis and more biomass production. It is an excellent example of biotechnology in the application of crop improvement.

Sucrose synthase is a cytoplasmic enzyme and is present in photosynthetic and non-photosynthetic tissues, and helps in sucrose translocation.

Sucrose mobilization:

It may be stated that cleavage of sucrose molecule is pivotal for entering the metabolic pathways of cell.

Following diagram provides a schematic representation of the process:

Invertase enzyme plays a very significant role and the enzyme occurs in cytoplasm, vacuole as well as apoplast. However, it is absent in plastids. The enzyme occurs in multiple forms viz. alkaline (cytosol), acidic (vacuole, apoplast) and neutral.

Transgenic studies have revealed the role of invertase in starch accumulation, organ shape and size and grain filling, in leaves high invertase in vacuole prevents sucrose accumulation in vacuole. Sucrose is shown to control the expression of several enzymes and other proteins. It represses α- amylase, is synergistic and antagonistic for the input of other signals (Pi concentration, auxins, GA, O2 levels).

Other disaccharides like trehalose, maltose, are uncommon (Fig. 4.20). Trehalose is another example of non-reducing sugar mainly observed in fungi, blue green algae and red algae. Now presence of this sugar has also been observed in leaves of fern Botrychium lunaria. It has 2 glucose units joined by α (1 → 1) bond.

On the other hand, maltose, cellobiose sugars are the result of partial degradation of starch (by amylases) and cellulose or lignin (by cellulases) respectively. Two glucose moieties in pyranose form are joined by (1 → 4) in maltose, (1 → 4) in cellulose and (1 → 6) in gentiabiose sugar.

Trehalose protects cells invariably during stress. Its presence in food indicates fungal infection.

It also stabilizes protein against stress denaturation. Lastly disaccharide lactose (O – β – D – Galactopyranosyl- (1 → 4) – β – D Glucopyranose) is found only as milk sugar.

Tri-, Tetra- and Penta- saccharides:

Among trisaccharides raffinose, gentianose and tetrasaccharide stachyose are commonly found in plants (Fig. 4-21).

Raffinose on hydrolysis yields glucose, fructose and galactose and occurs mainly in leaves and storage organs, whereas gentianose on hydrolysis yields two molecules of glucose and one of fructose and found mainly in Gentiana.

A tetrasaccharide, stachyose has been found in several tree species and comprises glucose, fructose and two molecules of galactose (Fig. 4- 21).

This sugar has been found to replace sucrose as transport metabolite in Verbascum thapsus, Fraxinus americana. Large amount of this sugar has been detected in leguminous seeds. Verbacose is only pentasaccharide found in plants.

Polysaccharides:

Simple sugars produced during photosynthesis are not utilized immediately but are converted to high molecular weight compounds, the polysaccharides. Latter are widely distributed in plants (Table 4-7). Being insoluble, polysaccharides can function as reserve substances without disturbing the osmotic equilibrium of the cell and as components of cell wall polysaccharides mainly in leaves, seeds and fruits.

On hydrolysis with specific enzymes or acids, they yield monosaccharides or monosaccharide derivatives.

Depending upon the type of monomeric units, they are divided into two main classes e.g., homopolysaccharides (one type of monomeric unit) and heteropolysaccharides (different types of monomeric units).

Homopolysaccharides are given names according to the type of building blocks e.g. polysaccharides with glucose units (Glucan) with fructose (Fructosan) with mannose units (Mannans). We shall be dealing with storage structural polysaccharides in detail (Table 4-7).

A. Storage Polysaccharides:

Starch is the principal storage carbohydrate in higher plants and is usually stored in the form of granules in the cytoplasm of seeds where it acts as a source of nutrient for the seedling growth. It is also stored in fleshy tuberous roots and woody twigs. It occurs in two forms (i) Amylose and (ii) Amylopectin (Fig. 22).

Amylose is a straight chain polymer consisting of D-glucose units (200-1000 glucose molecule) joined by (1 → 4) linkage. Amylose has both reducing and non-reducing ends and gives blue black colour with iodine. Iodine atom occupies the helical coil of glucose which is formed when amylose is suspended in water.

Degradation of amylose can be completed by amylases (α -and β-) widely distributed in plants, α-amylase hydrolyses (1 → 4) bonds at random giving free glucose and maltose units whereas β-amylase cleaves amylose into maltose units (Fig. 4-23).

The amylopectin is a branched molecule consisting of 2000-22800 glucose units attached with (1 → 4) bonds and side chains are attached with (1 → 6) bonds. It form stable gel like solution (Fig. 4-22, 25).

Molecular weight of amylopectin is 50000 to 1 million in contrast to amylose. Amylopectin can be enzymatically hydrolysed by the combined action of β-amylase and (1 → 6) glucosidase enzymes into maltose and glucose units.

Glycogen, a reserve substance of animals (Fig. 4-22A) is also found in Cyanophyceae, fungi and bacteria and is similar to amylopectin in structure. The difference between amylopectin and glycogen is the profuse branching of glycogen.

Paramylon, a characteristic feature of Euglenophyceae, Ochromonas has been studied recently. It comprises glucose units linked by (1 → 3) β-glycosidic units in a linear chain.

Starch synthesis:

Chloroplasts in leaf and amyloplastids in storage organs have stored starch which has different functions. However, enzymic reactions in their synthesis are similar and a close relationship between starch metabolism and sucrose metabolism exists (Fig. 4-26).

Three enzymes are involved in starch biosynthesis and these are ADP- glucose pyrophosphorylase, starch synthase and branching enzyme. Figure 4-26 gives schematic representation of the process.

ADP-glucose pyrophosphorylase (ADPGPP) present in the plastids with starch and has four distinct subunits two 55 kDA and 2 60 kDA. Based on the distribution of ADPGPP subunits three groups are recognized: small subunit in all cells large subunit in non-photosynthetic cells and different large subunits in photosynthetic cells.

ADPG synthesis rate is correlated with the 3PGAP ratio in the plastids and hence starch and photosynthathates partitioning into sucrose in cytoplasm. It may be stated that amyloplastids lack F-1, 6-bisphosphatase and hence cannot produce hexose from triose phosphate. Further changes in Pi levels in amyloplastids influences ADPGPP activity and hence starch synthesis.

A (1 → 4) glucan chain oligosaccharides must be present in sufficient number to act as chain primer and used as acceptors for the growth of amylose through starch synthase reactions. In summary, specific primer molecule is required.

Glucose is transferred from ADP-glucose to the non-reducing end of a pre-existing a (1 → 4) glucan chain through starch synthase. The enzyme exists in two major genetically distinct forms: one bound to the surface of starch granule and second, is soluble fraction of amyloplast. The soluble fraction is concerned with the elongation of amylopectin while the gB enzyme synthesizes amylose using BDP-glucose as glucosyl donor.

Branching enzyme forms branches in amylopectin and closely associated with the soluble starch synthase.

α-(1-4)-glucan → α-(1-6) branched α-(1-4) glucan

As reported in potato, maize, rice and sweet potato the enzyme is usually found in two or three isoforms.

Starch degradation:

Different pathways exist in photosynthetic and non-photosynthetic tissues. In fact dynamic and regulation of enzymes vary.

In the storage organs like tubers starch degrading enzymes are dormant whereas in the germinating seeds the activity level of these enzymes increases enormously.

Hormones like gibberellins play a vital role in the induction of hydrolases. In the photosynthetic tissues
degradation process exists all the time though in a mild state. It is intriguing to mention that all the degradating enzymes are also present in the cytoplasm and their level is higher than plastids. The precise role of these enzymes is not well understood.

Following are the main degrading enzymes:

i. α-amylase — amylose symbol maltose (some glucose, maltotriose) amylopectin symbol short-chain oligosaccharides (Fig. 4-23, 25)

ii. β-amylase — removes maltose residues from non-reducing end of amylose chains

iii. α—glucosidase— removes α-glucosyl groups from terminal position of

iv. α-glucan chain debranching enzyme-capable of hydrolysing α (1-6) branch linkages.

v. α-(1, 4)-Glucanlyase-degrades maltose, malto-oligosaccharides 1, 5- anhydrofrutose

The first four enzymes are capable of degrading starch into glucose:

I, 4 glucan phosphorylase participate in the phosphorylytic route for cleavage of starch.

We know that amylopectin component of starch is branched chain molecule. The mechanism by which the side chains are established is not yet known.

An enzyme (Q) has been isolated from potatoes which along with phosphorylase produces α (1 → 6) glucosidic bond. The participation of new enzyme (Q) in the synthesis of amylopectin catalysed by ADP-glucose-starch transglucosidase has not been studied yet.

Oligosaccharide like sucrose can also be converted to starch with the help of nucleoside diphosphate glucose.

The two enzymes sucrose synthase and starch synthase catalyse the conversion of sugar to starch in the presence of ADP.

II. Fructosans or Fructans:

Certain plants do not store starch as reserve material but another type of homopolymer of fructose called fructans, especially in members of Compositae (Taraxicum and Parthenium) and Graminae.

Most commonly studied fructosan is inulin. It is stored in Helianthus tuberosus and Dahalia tubers. Fructosan consists of fructose units linked by (2 → 1) β- glucosidic bond.

All fructans are either inulin type (2 → 1) bond or phlein type (2 → 6) bond. Later are found in Graminae and some microorganism. (Fig. 4-27).

Biosynthesis of Fructans:

Fructans are thought to be synthesized from sucrose. New fructose moieties after separating from sucrose get attached to the fructose units by (2 → 6) β-glycosidic bonds.

Recent studies show that fructose units can also be transferred via uridine diphosphofructon (UDPF).

They are mannose homopolysaccharides found in yeast, mold, bacteria and higher plants, comprising chains of mannose units linked by (1 → 4) β(?) bond or (1 → 6) linkages may occur. The structure of mannans has not been studied in details.

B. Structural Polysaccharides:

Structural polysaccharides constitute up to 90 per cent of dry weight of the primary wall. The shape, elasticity and rigidity of the plant cell wall depend upon the polysaccharides they contain.

Although chemical composition of the polysaccharide has been studied in detail, yet data regarding biosynthesis are not sufficient.

Cellulose is the major constituent of cell wall polysaccharides in all the higher plants. It is a linear polymer of glucose units held together by β (1 → 4) linkages (Fig. 4-24).

Recent studies show that native cellulose is made up of approximately 14000 glucose moieties with molecular weight 2-3 millions.

Cellulose being an inert material requires prolonged heating with concentrated H2SO4 for complete degradation into glucose units.

“Saccharification of wood” and its partial hydrolysis into cellobiose can be done by the enzyme cellulase present in bacteria cellobiose in turn is attacked by the enzyme cellobiase which yields free hexose units.

Cellulose is insoluble in water, lacks chemical reactivity and is incapable of being degraded rapidly, so it lacks nutritive value.

Cellulose synthesis was thought to be accomplished by the enzyme preparation from bacterium Acetobacter xylinum.

Enzyme catalyses the transfer of D-glucose units from UDP-glucose to cellulose. In contrast enzyme preparation from Phaseolus aureus prefers the substrate GDP-glucose.

Transfer is carried out by an intermediate glycolipid (acts as transport) which carries UDP-glucose across lipophilic membranes.

II. Pectic Compounds:

Pectic substances consist of galactouronic acids linked by α (1 → 4) linkage. Three types of pectic substances have been observed in plants.

This has only galactouronic acid in a chain.

These have some methylated carboxyl groups in galactouronic acids.

This name is used for all the insoluble pectic substances. Not much is known regarding the structure and composition of protopectin.

These pectic compounds play dual role first they are major components of cell wall matrix and second they occur in large quantities in fruits (citrus, apple) from which they are mainly extracted. Being a gelling agent, they are used in food industry. Pectic substances are degraded by pectin esterases (cleave methyl group) and pectinases [break α (1→ 4) glycosidic bond] (See figure 4-27).

The probable starting substance for the synthesis of pectin compound is UDP-galactouronic acid. Latter is formed by the oxidation of the CH2 OH group of sugar. Oxidation of glucose gives glucuronic acid.

Organic Molecule # 5. Lipids:

We have already dealt with two major constituents of higher plants i.e. carbohydrates and proteins. They perform dual function they act both as structural components of cell wall and as reserve food materials. “Lipids are water insoluble organic biomolecules and can be extracted from the cells or tissues by one of the several non- polar solvents like ether, chloroform and acetone.”

Lipids can be classified on the basis of backbone structure into two main categories:

Organic Molecule # 6. Fatty Acids:

Chemically, fatty acids are the union of organic molecules attached to certain alcohol. They are straight chain hydrocarbons containing even number of carbon atoms.

Branched chain fatty acids seem to be absent in higher plants. Among the most predominating saturated fatty acids found in plants are palmitic acid (C16H32O2), stearic acid (C18H36O2) and other saturated fatty acids which are lauric acid (C12), myristic acid (C14).

Among the unsaturated ones, oleic acid (C18), linoleic acid (C18) and arachidonic acid (C20) are present. Linolic and linolenic acids are found in linseed oil, while others are found in cellular organelles (Table 4-8).

In summary the fatty acids are of two types: saturated and unsaturated.

Saturated fatty acids:

These may be considered as based on acetic acid as the first member of the series. Cells synthesize fatty acids from 2-carbon or even-numbered building units of acetic acid. Examples of saturated fatty acids are given in table 4-8. Acetic, propionic, butyric acid, palmitic acid are some important examples of this category.

The carbon atoms of fatty acids are numbered in sequence from the-COOH carbon (C no 1). The second carbon of the acid counted after the carboxyl group is referred to as α-carbon. Carbon no 3 is β-Carbon. Carbon 4 is Ƴ.

Unsaturated fatty acids:

In such fatty acids the melting point is greatly lowered and all the common unsaturated fatty acids of nature are liquids at room temperature. These are further subdivided according to the degree of unsaturation:

These FA have one double bond. Examples are oleic acid, linoleic, α- linolenic acid. Oleic acid is most abundant fatty acid in nature.

Polyunsaturated acids:

These FA have two, three or four double bonds.

Two double bonds-linoleic acid, found in peanuts, maize, cotton seeds and soybeans.

Three double bonds-linolenic acid found in linseed oil.

Four double bonds arachidonic acid found in peanut oil.

Arachidonic acid on metabolism gives rise to prostaglandins and plays a significant role in biochemical activity on smooth muscles, blood vessels and adipose tissue.

Fatty acids are characterized by several important properties and these include melting point saponification, etc.

I. Triacylglycerols:

Triacylglycerols are also called as neutral fats and are the commonest lipids in plants. “They are the esters of trihydric alcohol, glycerol and long chain fatty acids.”

Triglycerides which are solid at room temperature are called fats whereas those which are liquid at room temperature are called oils.

They are found as plant reserve material in the form of intracellular oil droplets in storage organs like seeds, fruits and sometimes in the chloroplasts also.

II. Phosphoglycerols:

They are also called phospholipids or glycerol phosphatides and are frequently associated with cell membranes. They may also occur in the cytoplasm.

“Phospholipids, as their name indicates, are derivatives of glycerol in which one of the three fatty acids is replaced by phosphoric acid.” The compound having two fatty acids and one phosphoric acid is called phosphatidic acid.

The other wide spread phosphoglycerides in plants are: phosphatidylethanolamine (PEA), phosphatidylserine (PS) and phosphatidylcholine (PC).

The latter is abundantly found in soybean. Phospholipids are also called as amphipathic or polar lipids since they possess polar head in addition to their non-polar tail.

III. Sphingolipids:

They are important membrane components of plants and animals. On hydrolysis they yield one molecule of phosphoric acid, one of fatty acid and one molecule of sphingosine.

In plants and microorganisms phytosphingosine is the major base of sphingolipids. Fatty acids are attached through the amide linkage to the amino group of sphingosine (Fig. 4-29).

Glucocerebrosides (sphingolipids) are found in plant cell as a part of chloroplast membrane. In cerebroside esterified alcohol group is replaced by sugar molecule.

Waxes resemble fats being water insoluble and are solid esters of higher fatty acids with long chain alcohol (24-36 carbon atoms).

Waxes contain higher aliphatic alcohol instead of glycerol. Recently it has been shown that waxes contain other constituents also.

Waxes are found in plants as components of cell wall and on cuticle of leaf and fruit. They can also occur in the cytoplasm e.g., in seeds of Simmondsia californica also.

Biosynthesis of Fatty Acids:

The biosynthesis of saturated fatty acids occurs in all the organisms from the ultimate precursor acetyl-CoA.

Complete biosynthesis of fatty acids takes place in cytosol whereas fatty acid oxidation proceeds in the mitochondria.

Overall scheme of reactions is catalyzed by the complex of seven proteins in the cytosol: the fatty acid synthetase complex.

Ultimate source of all carbon atoms of fatty acids is acetyl CoA which is produced from carbohydrates and amino acids.

Acetyl-CoA formed in mitochondria cannot move outside into the cytosol. However, its acetyl group passes through the membrane in some other form.

Citrate produced during tricarboxylic cycle moves to cytoplasm from mitochondria through tricarboxylate transport system.

Acetyl-CoA is regenerated with the help of citrate cleavage enzyme and one molecule of citrate + CoA + ATP is produced. Acetyl-CoA acts as a primer.

Molecules of malonyl-CoA (intermediate precursor of 14 and 16-carbon atoms of palmitic acid) are successively attached to the primer molecule of acetyl CoA accompanied by decarboxylation.

Before starting fatty acid biosynthesis, an important preparatory reaction, the formation of Malonyl CoA takes place in the cytosol in two ways:

There are different systems for synthesis of fatty acids:

A. Extra Mitochondrial system for de novo synthesis (formation of long chain fatty acids):

The cytoplasmic, multienzyme complex converts acetyl-CoA to long chain fatty acids when supplemented with ATP, CO2, Mn 2+ and NADPH. The donor of two-carbon units is in the elongation step is malonyl- ACP.

The main reactions in FA synthesis are:

I. Initiating reactions (malonyl CoA formation acetyl-ACP formation Malonyl-ACP formation).

II. Principal reactions (condensation reduction I dehydration reduction II).

B. Mitochondrial system for fatty acid synthesis:

Mitochondria have an enzyme system that catalyzes elongation of preformed fatty acids by successive addition of acetyl CoA units.

It may be added that mitochondrial system does not need CO2 fixation and its products are chiefly stearic acid, C20 and C24 fatty acids.

It may be noted that this pathway is essentially the reverse of the pathway for oxidation of fatty acids.

Thus, the system needs the addition of ATP, NADH and NADPH. ADP is required for the formation of acyl-CoA from endogenous fatty acids.

The whole pathway comprised four steps as given below:

In plants vast majority of fatty acid biosynthesis de novo takes place in the plastids (chloroplasts or amyloplasts or leucoplasts).

Compared with plastids the rate of FA synthesis in mitochondria is very low. Briefly it requires two enzymes: acetyl CoA carboxylase and fatty acid synthetase.

In higher plants acetyl CoA in the plastids is of two forms: single large polypeptide and four different polypeptides.

In view of the fact that activity of acetyl CoA is a rate limiting attempts are being made to introduce extra copies of acetyl CoA carboxylase gene to boost fatty acids biosynthesis in oil seeds.

Acetyl CoA carboxylase, an allosteric enzyme catalyzes the rate limiting step in biosynthesis of fatty acids. For its complete enzyme activity it requires citrate or isocitrate as its positive modulation.

Formation of Malonyl CoA and fatty acid synthesis is given below:

Fatty acid Synthesis:

Reaction Sequence for Fatty acid Synthesis:

It occurs in three steps:

I. Initiating Reaction:

Once malonyl-CoA is formed, further six steps are catalysed by the six enzymes of fatty acid synthetase complex.

The seventh protein has no enzymatic activity itself and is an acyl carrier protein (ACP) to which growing fatty chain is attached covalently.

Initiation reaction starts with the transfer of acetyl group of acetyl CoA to sulphydryl group of ACP by the action of enzyme ACP-acyl transferase.

Then acetyl group from ACP goes to specific cystein residue of the enzyme, β-ketoacyl ACP synthase. Now fatty acid synthetase complex becomes ready to carry the next process of chain elongation.

The first reaction (malonyl transfer) is catalysed by the enzyme, ACP- malonyl transferase. Malonyl-CoA formed from acetyl-CoA reacts with sulphydryl group of 4′ – phosphopantetheine arm of ACP to form malonyl-S-CAP with the release of free CoA (Fig. 4-30, 31).

In the next reaction (condensation) acetyl group becomes attached to the malonate residue by the action of enzyme, β-keto acyl ACP synthase releasing free carboxl group. The latter undergoes decarboxylation.

This reaction leads to the formation of 4C-compound 4-carbon compound undergoes following reductions: 1st reaction, dehydration and 2nd reduction and leads to the production of saturated fatty acids.

Further lengthening of chain step required is acyl transfer. In this fatty acid residue is transferred to—SH group again to which acetyl residues are attached during initiation reaction followed by malonyl transfer and condensation reaction.

After seven complete cycles palmitoyl-CoA is the end product. Palmitic acid can be converted to stearic acid.

In some of the organisms, the fatty acid synthesis stops with the formation of palmitic acid, production of stearic acid does not occur which has only 2-carbon atom more than palmitic acid.

The specificity of chain elongation depends on the binding site of the enzyme β-ketoacyl synthase which accepts the tetradeconyl group of 14-carbon from ACP but does not accept hexadeconyl group.

Moreover, palmitoyl-CoA may function as feed-back inhibitor of fatty acid synthetase system.

These chain elongation steps are restricted to mitochondria and microsomes since the enzymes involved are bound to this membrane system.

III. Termination Reaction:

When a fatty acid has attained a definite length, terminal reaction product is released from synthetase complex.

Now the latter is able to synthesize new fatty acid molecule. However, the factors which affect chain length and also the time at which the synthesis ceases are not known.

Plant fatty acid synthetase is different from those of animal comprising single protein having six different active sites.

The complete reaction scheme of falty acid synthetase complex is shown in (Fig. 4-32):

The normal products of the plant FA synthetase complex are either C16 FA (palmitic acid) or C18 FA (stearic acid), esterified to an ACP.

In most plant cells FA with chain lengths less than 16 carbons are rare though found in the oil of some seeds like nutmeg.

In such instances the FA synthetase possibly contains an extra medium-chain acyl-ACP thioesterase activity which terminates acyl chain prematurely resulting in the formation of FA with C8, C10, C12 and C14 FA.

Such FA have detergent-like activity. These FA are contained in oil bodies located in the endosperm and embryo of oil seeds.

It is possible to introduce double bonds through desaturates in 18-carbon FA. Thus, stearoyl-ACP desaturase introduces a double bond into the A9 position of stearic acid to form oleoyl-ACP.

The final reactions are the conversions of oleoyl-ACP to oleoyl-CoA and the extrusion of the latter from plastids.

In fact oleoyl-CoA is considered as a central metabolite in oil metabolism in plants. In summary, all the reactions from acetyl-CoA to oleoyl CoA occur in the plastids, the later reactions of oleoyl-CoA take place outside the plastids.

It may elongate further, experience desaturation or have hydroxyl moieties introduced in it to yield acyl- CoAs.

In higher plant cell free systems which synthesize long chain saturated fatty acid has been isolated.

It is still unknown whether fatty acid synthetase present in it is comparable to yeast, bacteria or not, though acyl carrier protein isolated from avogado fruits and spinach leaves are found to be different from yeast.

8 Acetyl CoA + 7 ATP + 14 NADPH → Palmitate

Biosynthesis of Unsaturated Fatty Acids:

Biosynthesis of unsaturated fatty acid has not been completely studied in higher plants. During their synthesis double bonds are introduced into previously formed saturated fatty acids by the enzyme desaturases.

Enzymes acyl-CoA desaturase and stearyl CoA desaturase have been isolated from yeast and Euglena, respectively.

Significant examples of formation of unsaturated fatty acids can be seen in seeds and greening cells.

They are also synthesized during chloroplast differentiation as revealed by the labelling experiments.

They may also undergo secondary conversion of various plant species e.g. conversion of double bonds to triple or additional chain elongation.

Biosynthesis of Natural Fats (Triacylglycerols):

Triacylglycerols which function as storage lipids in higher plants require two major precursors for their synthesis L- glycerol 3-phosphate and fatty acyl-CoA.

L-glycerol- β-P is derived from dihydroxy-acetone phosphate by the process of hydrogenation in the presence of NADH + H+ or from glycerol by the action of enzyme glycerol kinase as shown below:

Free hydroxyl groups of glycerol 3-P are acylated by two molecules of fatty acyl CoA yielding first lysophosphatidic acid and then phosphatidic acid.

In some microorganism fatty acyl- ACP acts as R donor of fatty acyl instead of fatty acyl CoA. Phosphatidic acid undergoes hydrolysis giving diacyl glycerol with the action enzyme phosphatidate phosphatase.

Diacylglycerol reacts with another molecule of fatty acyl-CoA to form a triglyceride. The reaction is catalysed by the enzyme diacylglycerol acyl transferase. In higher plants, triglyceride may be having 2- 3 different fatty acids.

Reaction sequence is as follows:

Lipid which acts as a storage material can also be used as an energy rich fuel by the cells to meet their demand for energy e.g. during seed germination, lipid content decreases and their products of degradation are subjected to biological oxidation and then for ATP production.

The degradation of fats starts with the breakdown of triglycerides into glycerol and fatty acids.

The enzymes specifically catalysing fat cleavage are lipases, the hydrolytic enzymes belonging to the group of esterases. The latter cleave ester bonds in the fat molecule along with the uptake of H2O.

Two products, glycerol and fatty acids, undergo various metabolic changes. Glycerol enters the carbohydrate metabolism.

In etiolated cotyledons label from glycerol-1-3-14C can also be degraded to dihydroxyacetone-P and pyruvic acid by glycolytic reactions.

Another cleavage products, fatty acid is subjected to β-oxidation yielding activated C2 fragments : latter serving as substrate for TCA cycle (Figs. 4-33, 34).

Several plant structures e.g. pollen and seeds abound in fats. On incubation, these fats are hydrolysed and consumed as source of energy.

This is accomplished through oxidative degradation to be described later on. Fats can also be converted into carbohydrates as follows (Figs. 4-33, 34).

Here isocitrate is broken down to succinate and glyoxylate by isocitrate lyase (ICL). Succinate is converted to malate through TCA cycle.

Fatty acids can also give rise to acetyl CoA. Glyoxylate also gives rise to malate and the key enzyme involved is malate synthetase.

The process increases the malate amount and this in turn increases supply of oxaloacetate. The latter is partially condensed with CoA to give rise to citrate.

The remainder of OAA through series of steps (phosphorylation and decarboxylation) yields PEP which in its turn is converted to hexoses.

The two enzymes malate synthetase (MS) and isocitrate lyase (ICL) are located in glyoxysomes.

The fatty acids produced from fats can be further degraded through β-or α-oxidation depending upon whether β-carbon atom or α-carbon atom are the points of introduction of oxygen function. The two degradative processes are described briefly.

Fatty acids are produced in the cytosol due to their biosynthesis. However, all the enzymes related to β-oxidation system are localized in the inner membrane of mitochondria.

Now the question is how these fatty acids enter mitochondria for their degradation. There are three steps by which the entry of fatty acid is facilitated (Fig. 4-35, 36).

I. Activation of Fatty Acids:

Free fatty acids are esterified with extra-mitochondrial-CoA giving rise to high energy fatty acyl- CoA with the consumption of ATP.

The reaction is catalysed by acyl thiokinase.

R – COOH + ATP + CoA – SH RCO – S – CoA + AMP + PPi

II. Transfer to Carnitine:

The acyl group from fatty acyl-CoA is transferred to carnitine, a specific carrier molecule which transports it through the internal barrier of inner mitochondrial membrane to the actual site of degradation.

III. Transfer to Intramitochondrial-CoA:

Fatty acid degradation occurs in matrix and requires acyl-CoA as a substrate and acyl group of fatty acyl carnitine is transferred to intramitochondrial CoA and is converted to acyl-CoA in the matrix.

Following the formation of Acyl-CoA all the reactions of fatty acid oxidation occur in mitochondria in the following steps (Fig. 4-36):

(i) Dehydrogenation Step:

The reaction is catalysed by the enzyme acyl-CoA dehydrogenase. Hydrogen is removed from fatty acid giving α -, β-unsaturated acyl derivatives of CoA.

Four different types of enzymes exist and their specificity depends upon the chain length of fatty acid. They have FAD as prosthetic group. The flavoprotein transfers the electron to the respiratory chain.

In this reaction, water is added to unsaturated fatty acyl derivatives of CoA resulting into β- hydroxy fatty acid by the action of enzyme Enoyl CoA hydratase.

The secondary alcohol group of β- hydroxy fatty acid undergoes oxidation in the reaction catalysed by β- hydroxy acyl CoA dehydratase.

(III) Dehydration (2nd) Cleavage Step:

L-3-hydroxy acyl CoA is dehydrogenated to form β- keto acid by the action of enzyme 3- hydroxyacyl-CoA dehydrogenase. β- keto acid being unstable releases C2 fragment as acetyl CoA.

The reaction is catalysed by the enzyme thiolase or is called thiolysis or a thiolytic cleavage. The molecule residue shortened by 2-carbon atom again enters the reaction cycle. The process is repeated until the carbon chain completely splits yielding acetyl CoA. After this another molecule of fatty acid is fed for degradation.

Recently it has been shown that p-oxidation can occur outside the mitochondria e.g. in seeds of Ricinus. The specific enzymes involved are located in the glyoxysomes.

Another mechanism comparatively less important than p-oxidation for the degradation of fatty acid has been observed in cotyledons and young leaves (Fig. 4-37).

In this type of oxidation only free fatty acids serve as substrates. It occurs in the following step:

(i) Carboxyl group of the free fatty acid is eliminated by oxidation with H2O2 by the action of enzyme fatty acid peroxidase.

(ii) The resulting aldehyde compound is further oxidised by a dehydrogenase enzyme.

Degradation of Unsaturated Fatty Acids:

Unsaturated fatty acids are oxidised by the same general pathway as saturated fatty acids except with certain modifications.

The problems of unsaturated fatty acids are that double bonds of unsaturated fatty acids are in cis-configuration whereas ∆ 2 -unsaturated acyl-CoA derivatives have trans­configuration.

Moreover, another problem with unsaturated fatty acid is that removal of 2-carbon atom fragments from carboxyl end yields ∆ 3 -unsaturated fatty acyl CoA rather than Δ 2 -fatty acyl CoA.

These problems have been overcome by the enzyme enoyl-CoA isomerase which catalyses shift of double bonds from ∆ 3 -cis to ∆ 3 -trans configuration reversibly. The ∆ 2 -trans unsaturated fatty acids act as normal substrate fatty acid oxidation (Fig. 4-38).

Simple lipids contain no fatty acids and occur in the smaller amounts in cells and tissues. Simple lipids are classified in two major classes, the terpenes and the steroids.

They are the derivatives of saturated tetracyclic hydrocarbons, perhydrocyclopentanol pentanophenanthrene containing three fused cyclohexane rings and a single cyclopentane ring.

The precursor for all steroids is triterpenoid, squalene which readily cyclize to form ring structures.

Steroids are widely distributed in the plant and animal kingdoms and those occurring in plants are known as phytosterols (Fig. 4-39). They are present in a very small quantity and their function is not completely understood.

Cholesterol and phytosterol occur rarely in higher plants. They contain other types of sterols like stigma sterols and sitosterol. Another group of sterols present in fungi and yeast are mycosterols. Sterols are not present in bacteria.

Organic Molecule # 7. Secondary Plant Products:

Compared with the primary metabolic products (carbohydrates, lipids, amino acids, proteins, nucleic acids, etc.) there are several secondary metabolic products also characterized in plants and these are phenols, terpenoids, alkaloids, etc. While the former products are essentially required for growth of plants, the latter are accessory plant metabolites and seemingly not involved in the biosynthesis of primary plant metabolites.

They are biosynthesized in a selected few species of plants. Some of the phytohormones (gibberellins, abscisic acid) are terpenoids and belong to secondary metabolites.

Similarly, volatile oils are also a class of secondary plant metabolites. The importance of these secondary metabolites in regulating various physiological processes is well known.

There are many other secondary metabolites e.g., alkaloids, rubber, tannins which are species specific and presumably play some significant role in the biochemical processes within a plant. There still remains vast area regarding their function which deserves attention.

In the followings a brief account of the biosynthesis and functions of some of the important classes of secondary metabolites is given.

The phenols are second largest group of secondary plant products which are characterized by an aromatic ring that is attached to one or more hydroxyl groups.

They have variable constitution viz., simple phenols (hydroquinone), phenol carboxylic acid (gallic acid), phenylpropanes (coumarins, lignin) and flavan derivatives (flavonones, flavones, flavonals). Most of the flavan derivatives occur as sugar esters or glycosides and are found in vacuoles.

In general aromatic systems are synthesized by three different pathways (the shikimic acid, the acetate-malonate and acetate- mevalonate pathways).

Ligin is most widely distributed in plants and perhaps contributed to the transfer of aquatic plant life to land during evolution. Its biosynthesis begins from the cinnamic acids, p- coumaric acids, ferulic acid and sinapic acid through reduction and is converted into corresponding alcohols. The alcohols are transported as glucosides.

Carotenoids and flavan derivatives are some important flower pigments. While the former impart yellow to red colours, the latter compounds give white, yellow, red or even blue coloration. It may be stated that anthocyanins are red and blue flavan pigments.

The pigments may also be divided on cytological basis as follows:

(i) Chymochromic pigments (vacuole or cell sap soluble).

(ii) Plasmochromic pigments (plastidal e.g. chlorophylls, xanthophyll, carotenoids).

(iii) Membranochromic pigments (impregnated in cell walls).

The flower colour is determined by several factors like nature and amount of pigments, pH value, co-pigments, etc.

Phenolics are widely distributed in plants and their precise function is not specifically known. Majority of the phenolics are glycosylated and are, therefore, water soluble. Some of the phenolics are sort of waste products during the amino acids metabolism.

Some of the coumarins inhibit seed germination and even cell elongation by affecting IAA- oxidase. Some of the phenolics behave as phytoalexins and provide defensive mechanisms to plants against pathogens.

Lignin provides tensile strength and cementing of cell walls. The role of flavonoids in insect- attraction to aid pollination is widely acclaimed. Some of the flavonoids stimulate the oxidation of IAA whereas others inhibit such an oxidation.

These organic substances are produced from acetyl-CoA via the acetate-mevalonate pathway.

Basically they are constructed from 5C building units.

Depending upon the number of 5C units, the terpenoids are variously sub-grouped e.g.:

In addition tri- and tetraterpenes are also found in plants. Rubber, however, is a polyterpene with n × 5C units.

Hemiterpenes are components of quinones and help in oxidation- reduction reactions. A few monoterpenes are components of essential volatile oils in plants and impart specific odour to plants. These include camphor, carvone and some are antibiotics.

Some of the monoterpenes contribute towards defence or sex attraction signals. Sesquiterpenes include several essential oils as well as ABA.

The role of ABA in dormancy, abscission of leaves and fruits is well known. Gibberellins are diterpenes and contribute significantly towards several physiological processes.

Tetraterpenes include carotenoids and xanthophyll.

These are secondary plant products possessing N-containing heterocyclic ring. They are derived from several amino acids like lysine, tryptophan, tyrosine and phenylalanine. To date about 3000 alkaloids have been reported.

Nictotine and nornicotine are found in Nicotiana tabacum.

Phenylalanine and tyrosine are utilized in the synthesis of some alkaloids in several species of Amaryllidaceae and also in Papaver species. Papaver somniferum is a source of papaverine and morphine. Caffeine is found in tea, cocoa and coffee plants.

Most of the alkaloids are sources of drugs. They provide protection to the plant. Conine is obtained from hemlock (Conium maculatum) and was used in poisoning Socrates.

Reserpine is used for curing hypertension and is distributed in Rauwolfia. The latex from opium poppy capsules yields morphine, codeine, and heroin. Quinine (antimalarial drug) and colchicine are some of the other important alkaloids.

The biosynthesis of porphyrins is associated with the citric acid cycle and metabolism of amino acids.

Succinyl CoA and glycine are their precursors which combine to produce an intermediate acid called ∆-aminolaevulinic acid as follows:

Succinyl -CoA + glycine δ – aminolevulinic → porphobilinogen → uroporphyrinogen III → Protoporphyrin IX

The above scheme also shows formation of chlorophyll a and chlorophyll b from protoporphyrin IX and also biosynthesis of cell hemins when the latter combines with Fe ++ .

In blue-green algae open chain tetrapyrroles combine with proteins to form phycobili-proteins. Phytochrome system is a phycobiliprotein. Like the prophyrins, phycobiliproteins are derived from succinyl CoA and glycine.

Some of the amino group carrier present in the cell is pyridoxal phosphate. It is a prosthetic group in starch phosphorylase and transaminase enzymes. In the latter, pyridoxal undergoes reversible transformation into pyridoximine phosphate.

Derivatives of folic acid called tetrahydrofolic acid are also reported. It is also an important coenzyme which comprises peridine, p-amino benzoic and L-glutamic acid. The co-enzyme participates during amino acid and purine metabolism.


Metabolism Directory

I collaborate with researchers across campus to facilitate mass spectrometry based omics analysis through the Laboratory for Biomolecular Mass Spectrometry (LBMS). My scientific interests are on improving methodologies related to multi-omics data acquisition and integration.

Zhao, Xinyu

My lab is investigating the impact of metabolic and mitochondrial deficits on stem cell differentiation, brain development, and neurodevelopmental disorders, including autism, fragile X syndrome, and schizophrenia.

Merrins, Matthew

My lab is focused on understanding oscillatory metabolic signaling in the pancreatic islet and its deficiency in type 2 diabetes. My lab specializes in single-cell approaches – principally live-cell imaging and electrophysiology – and since the lab’s inception we have developed a number of exciting tools which have given us new insights into diabetes pathophysiology. One example is fluorescence lifetime imaging of NADH (FLIM-N), which allows us to distinguish metabolic activity in each subcellular compartment of the pancreatic beta cell, including the mitochondria. Using FLIM-N and FRET imaging, we hope to understand the bidirectional communication between mitochondrial metabolism and the cell cycle machinery, mediated by cyclin dependent kinases. We are also focused on characterizing a number of mitochondrial proteins initially identified by RNA-seq as type 2 diabetes-associated loci in this case, we are using Phy-PIF optogenetics to elucidate the mechanisms by which these proteins alter mitochondrial dynamics, metabolic oscillations, and insulin secretion.

Eisenstein, Rick

We study mechanisms of iron sensing and control of iron homeostasis in vertebrates by iron regulated RNA binding proteins, the iron regulatory proteins (IRP). IRP maintain iron homeostasis by controlling the fate of mRNA encoding proteins needed for iron metabolism or the responses to iron deficiency. We investigate how iron metabolism and erythropoiesis is coordinated particularly how IRP1 senses iron or oxygen status and controls the translation of hypoxia inducible factor 2-α (HIF-2α) mRNA. HIF-2α, a transcription factor, promotes adaptive responses to hypoxia by enhancing both red blood cell production and dietary iron acquisition for hemoglobin production. We use animal, cell culture and model systems (yeast) to define the physiological roles of IRP1, its selective control of mRNA fate and the iron trafficking pathways it responds to. Genome editing, flow cytometric analysis of hematopoiesis, RNA binding, gene by diet interaction studies, tissue specific knockouts and in vivo imaging.

Keller, Mark

I have worked closely with Professor Alan Attie for more than 16 years to integrate genetics, physiology and metabolism to better understand the molecular basis of disease susceptibility. The lab exploits natural genetic variation contained within in-bred mouse strains as a platform to link metabolic disease with genetics. One major project is focused on surveying diet-induced metabolic syndrome in a newly developed mouse resource called the Diversity Outcross (DO). Each DO mouse derives from 8 distinct parental mouse strains that together represent the genetic diversity contained within the human population. The degree of metabolic syndrome we observe in the DO population is remarkable, illustrating that phenotypic variation is driven by genetic variation. We employ various omics-based measurements (e.g., transcriptomics, proteomics, microbiomics, metabolomics) to generate robust causal models that will help to unravel the enormously complex relationship between the genetics of each DO mouse and their relative susceptibility to metabolic syndrome.

Yen, Eric

We are interested in understanding how cellular metabolism modulates systemic energy balance in response to diet. Our research centers on the synthesis of triacylglycerol, which serves as a storage and transport molecule of bioactive fatty acids and excess calories. Using genetically engineered mice, we examine the physiological functions of enzymes involved in the process. One current focus is on monoacylglycerol acyltransferase 2, which mediates the absorption of dietary fat in the small intestine. Mice lacking the enzyme are protected against obesity and other metabolic disorders normally induced by high-fat feeding. Interestingly, these mice absorb a normal quantity of fat but exhibit increases in energy expenditure. We are now combining biochemical and systems biology approaches to understand the underlying molecular mechanisms. The ultimate goals are to better understand the fundamental process of fat assimilation and to explore new approaches to prevent obesity and other metabolic diseases associated with excessive energy storage.

Engin, Feyza

Our lab is interested in understanding the role of organelle stress and stress responses in disease pathogenesis. The endoplasmic reticulum (ER) orchestrates protein synthesis, folding and trafficking in the cell and disruption of the ER's adaptive capacity results in activation of the unfolded protein response (UPR). Under chronic stress conditions, UPR engage many different inflammatory and stress signaling pathways that are critical for disease pathologies including insulin resistance, obesity and type2 diabetes. Interestingly, we recently discovered that ER stress plays a significant role in the pathogenesis of not only type 2 diabetes, but also autoimmune diabetes. Currently, we are exploring the molecular mechanisms leading to ER stress in type 1 diabetes and investigating the role of different UPR branches in metabolic homeostasis. We are also investigating how disruption of ER calcium homeostasis may affect interactions between ER and mitochondria in the context of metabolic and autoimmune diseases.

Klein, Mark

Sirtuin 6 is an NAD-dependent protein deacylase that regulates lipid and glucose homeostasis. Recently, we have discovered that Sirt6 can be activated by endogenous compounds in vitro. I am continuing to discover endogenous activators and aim to understand if this activation plays a role on Sirt6 metabolic regulation in vivo.

Hernandez, Laura

Our research is focused on how serotonin controls maternal metabolism to support lactation. We focus on two areas of how the mammary gland controls maternal metabolism during lactation: calcium homeostasis and energy homeostasis. We utilize rodent and cow in vivo models and as well in vitro techniques.

Knoll, Laura

Our research centers on studying the host/pathogen interactions for the intracellular parasite Toxoplasma gondii. Toxoplasma is a highly successful parasite that exists as a life-long infection in a large percentage of the world’s warm-blooded animals, including almost half the human population. In a healthy individual, Toxoplasma has evolved to stimulate, but not over-stimulate the host’s immune response. Toxoplasma causes encephalitis in immunocompromised patients and is a member of the coccidian family that includes Plasmodium. We are combining next generation sequencing, proteomics and metabolomics to uncover the host and parasite metabolic pathways that are necessary for Toxoplasma to establish and maintain chronic infection. Because Toxoplasma is auxotrophic for many essential nutrients, we are finding that infection dramatically manipulates host cell metabolism. Toxoplasma also has both a mitochondria and a remnant chloroplast, so it has several unusual metabolic products. These studies will generate new anti-parasitic targets that will help develop novel therapies.

Coe, Christopher

My research spans a number of topics in behavioral medicine and health psychology, which involve metabolism. Using a nonhuman primate model, we are investigating the relationship between the gut microbiome and systemic physiology during infancy. We also are studying novel iron supplements to treat iron deficiency anemia, and include metabolomic approaches to assess the benefits of treatment. Our research with human participants is focused more on the other end of the life span, and the biology of aging. My lab oversees the biomarker assessments for 2 surveys of health and aging in the US and Japan (MIDUS and MIJDA). Both include a number of outcomes related to metabolism, especially with respect to obesity and glucoregulation.

Sutula, Thomas

My interest and ongoing work addresses the influences of glycolysis and metabolism on neuronal and circuit function in the brain.

Converse, Alexander

Positron emission tomography (PET) using [18F]fluorodeoxyglucose (FDG) to noninvasively image glucose metabolism

Baker, Mei

My research interest is public health genetics and genomics, with a focus on applying and translating advanced biochemical and molecular technologies into routine newborn screening practice to enable public health laboratories to screen for new conditions and improve screening performance for the exiting screened conditions.

Donohue, Timothy

Bacterial metabolism and energy generation, this include central carbon and nitrogen metabolism, aromatic utilization and control of these pathways. We use genetic, biochemical genomic and other approaches in a wide set of microbes, but pure cultures and consortia or simple microbiomes

Fan, Jing

Our lab is interested in understanding how mammalian cellular metabolism is reprogrammed in response to changes in the environment and cellular state, and how activities in key metabolic pathways can in turn affect cell function. To study this, we combine systems biology approaches, especially fluxomics and metabolomics, with computational modeling and biochemical and genetic techniques. Particularly, our current works focus on (1) understanding the metabolic adaptations in cancer cells in acidic microenvironment, and (2) investigating the metabolic regulation during macrophage polarization

Eide, David

My lab studies the mechanisms cells use to respond and adapt to metal nutrient deficiencies. We study these processes in the yeast Saccharomyces cerevisiae and focus primarily on zinc deficiency. In this yeast, the Zap1 transcription factor responds to zinc deficiency to activate the expression of

80 genes. The products of these genes include several zinc transporters responsible for zinc homeostasis. Many Zap1-regulated genes also encode proteins involved in the adaptation to zinc deficiency including proteins involved in oxidative stress resistance, protein homeostasis, sulfur metabolism, and phospholipid synthesis.

Newton, Michael

I do statistics and biostatistics, especially in genomic apps, and have collaborated on various projects that integrate different data sources

Carey, Hannah

Metabolic adaptations in hibernating mammals, including basic mechanisms and biomedical implications. Current focus on gastrointestinal/liver physiology and the microbiome.

Nelson, David

I am interested in elucidating the interaction between cellular processes and whole body energy metabolism. In our research group, we use molecular genetics to modulate the function of genes involved in lipid metabolism. My primary focus is on the role of intestinal monoacylglycerol acyltransferase 2 (MGAT2), an enzyme that catalyzes the conversion of MAG to DAG (an essential step in the efficient absorption and assimilation of dietary fat), in mediating systemic responses to diet. Using indirect calorimetry, I am linking alterations in food intake, oxygen consumption (VO2), and substrate utilization (RER) with diet and environmental challenges in genetically engineered mice. In particular, I am investigating MGAT2’s role in intracellular lipid trafficking and effects on enteroendocrine signaling and the kinetics of nutrient absorption and delivery. Through this research, we hope to better understand how alterations in the kinetics of intestinal lipid metabolism affect whole body energy metabolism, obesity, and related morbidities.

Jefcoate, Colin

The Laboratory addresses signaling processes involved in adaptation to Diet, Chemicals and Stress. Mesenchymal cells are key mediators, which include multi-potential progenitor cells that deliver physical structure, energy control (adipose), steroid production and local support factors. The Laboratory played key roles in the discovery of two regulators of these processes Cytochrome P450 1B1 (CYP1B1) and the Steroidogenesis Acute Regulator (StAR). CYP1B1 is expressed in mesenchymal progenitors and vascular cells, but also controls oxidative stress, vascular adhesion and monocyte differentiation. We probe CYP1B1 functions through general and selective deletions of CYP1B1 from mice, facilitated by the development of a flox/flox Cyp1b1 mouse (gene expression/associated physiological changes). Hormonal activation of StAR, a labile protein, directs delivery of cholesterol to Cyp11a1 in mitochondria, thereby initiating adrenal glucocorticoid synthesis, a mediator of liver energy homeostasis or testosterone synthesis in fetal and adult testis, a notable source of endocrine disruption.

Fox, Brian

Protein structure and function. Oxidation and reduction reactions. Fatty acid metabolism. Production of biofuels and other products from cellulosic biomass.

Colopy, Sara

My laboratory is focused on understanding the pathogenesis of urinary tract infection and bladder epithelial healing. One current area of research is looking at the pathogenesis of urinary tract infections in type II diabetic mice.

Landick, Robert

We study the regulation of RNA synthesis by RNA polymerase and the use of gene expression engineering to create novel microbial systems and communities to produce beneficial compounds and materials. These studies link to metabolic regulation through a diverse set of protein, RNA, and small molecule regulators that interact with RNA polymerase to control gene expression, in the search for new inhibitors of RNA polymerase as lead compounds for drug discovery, and via ways that changes in gene expression can redesign metabolism for useful purposes.

Denu, John

Linking metabolism with the epigenome: Chromatin remodeling enzymes rely on co-enzymes derived from metabolic pathways, suggesting coordination between nuclear events and metabolic networks. Investigations are underway to understand the link between metabolism and the regulation of epigenetic mechanisms. We are testing the hypothesis that certain chromatin modifying complexes have evolved to exquisitely ‘sense’ metabolite levels and respond accordingly, modifying specific chromatin loci for altered gene expression. Sirtuins and reversible protein acetylation: Accumulating evidence suggests that reversible protein-lysine is a major regulatory mechanism that controls non-histone protein function. Sirtuins are a conserved family of NAD+-dependent protein deacetylases that have emerged as important players in modulating protein acetylation. Compelling genetic evidence implicates sirtuins in genome maintenance, metabolism, cell survival, and lifespan. The NAD+-dependence suggests that specific protein deacetylation is inextricably linked to metabolism. We are examining the central hypothesis that reversible protein acetylation is a major regulatory mechanism for controlling diverse metabolic processes, and that at the molecular level, site-specific acetylation alters the intrinsic activity of targeted proteins.

Markley, John

Our major interest is in NMR-based investigations of metabolites and their interactions. NMR allows unbiased detection and quantification of the 30-80 most abundant metabolites in biological fluids and tissue extracts. We created a database of one- and two-dimensional spectra of metabolites and other small molecules of biological importance, which has grown to include

1400 compounds. We developed efficient technology for high-throughput data collection, automated assignment and quantification of individual species and visual inspection of the results. We recently developed a platform for rapid screening of proteins against a panel of 500 metabolites to determine whether the protein binds or chemically modifies these small molecules. In favorable cases, we can use NMR to validate hits and to determine ligand binding sites. In the realm of natural products, we provide technology for LC-MS with solid phase extraction so that molecules with a mass of interest can be isolated for subsequent NMR analysis.

Lamming, Dudley

The Lamming laboratory's goal is to understand how nutrient-responsive signaling pathways can be harnessed to promote health and longevity. We are primarily focused on the physiological role played by the mechanistic target of rapamycin (mTOR), a protein kinase that through a diverse set of substrates regulates complex cellular processes, including growth, metabolism, and aging. Recent work has shown that rapamycin, an inhibitor of mTOR signaling, can improve both health and longevity in model organisms including mammals. Understanding and manipulating the mTOR signaling pathway through dietary, pharmaceutical or genetic interventions in mouse models may provide insight into the treatment of age-related diseases, including diabetes, Alzheimer's disease, cancer, and Hutchinson-Gilford Progeria Syndrome. Learn more at http://www.lamminglab.org/

Alexander, Caroline

We aim to determine the role of a skin-associated lipid layer, we call dermal white adipose tissue, on mammalian insulation, and therefore on glucose disposition and the frequency of thermogenic activation of brown adipose tissues. We propose that manipulation of this dWAT layer could confer health benefits to human subjects. Our research is enabled by the development of a high resolution, body wide quantitative MRI technique

Dowell, James

My research is focused on the metabolism of the brain. Specifically, I am interested in metabolic processing of glutamate in astrocytes, its regulation by protein kinases, and its involvement in neurodegeneration. I use both mass spectrometry-based metabolomics and proteomics platforms to study these processes.

Westmark, Cara

My research interests lie in the area of synaptic function as related to the over-expression of amyloid-beta protein precursor and amyloid-beta in Alzheimer’s disease, Down syndrome and fragile X syndrome. I study therapeutic approaches that reduce amyloid-beta and rescue seizure, behavioral, cognitive and biomarker phenotypes associated with the aforementioned disorders. During the course of these studies, I serendipitously discovered that diet, specifically soy-based diets, exacerbate seizure incidence and weight gain in juvenile mice as well as in infants. Soy is rich in phytoestrogens and contaminated with agrochemicals, which can act as endocrine disrupting chemicals. Surprisingly, there has been a paucity of studies regarding the long-term effects of consuming singe-source soy-based diets. Our research focus in metabolism is to study the effect of soy on neurological and metabolic phenotypes, particularly in developmental disability models, to validate dietary restriction of soy-based infant formula as a therapeutic intervention for autism and fragile X.

Li, Lingjun

Our research focuses on the development of mass spectrometry-based tools and systems biology strategy to understand metabolic profile changes during various disease conditions such as aging, lower urinary tract symptoms, and cardiovascular injury. We also employ imaging MS technology to understand symbiosis between bacteria/microbiome and host organisms.

Romero, Pedro

BMRB hosts a metabolite and small molecule NMR database useful for metabolomics studies, and it is involved in research and development of software tools to facilitate automatic metabolite identification from metabolomics profiles.

Dahlberg, James

Impact of physiology on metabolic states.

Suen, Garret

Our work focuses on understanding the rumen ecosystem with an toward improving animal production and utilizing this microbiome as a model for biofuel production. We utilize genome-enabled approaches to characterize and understand this community to determine if alterations to the rumen microbiome can result in increased animal production. From a biofuels perspective, our work seeks to determine how this highly optimized microbiome is capable of rapid biomass degradation and fermentation.

Keller, Nancy

My lab focuses on the genetics and chemistry of fungal natural products (NPs). We explore the role of NPs as virulence factors (particularly in Aspergillus and Penicillium species), as signaling molecules in intra- and inter-Kingdom milieus and for development as pharmaceuticals. We are interested in the crosstalk of primary and secondary metabolism in fungi and how natural product clusters are regulated by endogenous mechanisms (e.g. epigenetic regulation and global transcriptional regulators) or exogenous input from other organisms (e.g. other microbes or hosts) and abiotic factors.

Sinha, Divya

Human pluripotent stem cell derived retinal pigment epithelium (hPSC-RPE) and photoreceptor (hPSC-PR) cells are being utilized for in vitro modeling of retinal diseases as well as to develop cell replacement therapy for retinal degenerative diseases that lead to blindness. Since these retinal cell types are highly functional and mitochondria-rich in vivo, our efforts are focused on assessing and enhancing cell health of hPSC-derived RPE and photoreceptors in terms of mitochondrial metabolism under physiologic conditions.

Eells, Janis

Mitochondria play a key role in cellular metabolism and intracellular signaling. Mitochondrial dysfunction and the resulting oxidative stress are central in the pathogenesis of aging and degenerative diseases including diabetes, cardiovascular disease, macular degeneration and Alzheimer’s disease. Research in my laboratory is directed at understanding the mitochondrial signaling pathways that regulate the processes of cellular aging and degeneration with the long-term goal of learning how to protect cells and tissues against these degenerative processes. One mitoprotective strategy is photobiomodulation. Exposure to low energy photon irradiation in the far-red (FR) to near-infrared (NIR) range of the spectrum (630 – 900 nm), collectively termed “photobiomodulation” (PBM) can restore the function of damaged mitochondria, upregulate the production of cytoprotective factors and prevent apoptotic cell death. FR/NIR photons penetrate diseased tissues including the retina and optic nerve. Investigations in rodent models of retinal injury and disease have demonstrated the PBM attenuates photoreceptor cell death, protects retinal function and exerts anti-inflammatory actions. Recent clinical studies have documented amelioration of atrophic AMD.

Maeda, Hiroshi

The Maeda lab investigates how plants synthesize aromatic amino acids, which are essential nutrients in the human diet and key precursors of numerous plant natural products (e.g. alkaloids, quinones, phenolic compounds). We combine phylogenetic, biochemical, genetics, and protein structure analyses to understand how key enzymes in the aromatic amino acid pathways evolved in different plant lineages that produce distinct downstream specialized metabolites. Such evolutionary variations are then utilized to identify amino acid residues responsible for their catalytic and regulatory properties and also to enhance the production of natural products through metabolic engineering.

Gamm, David

Human pluripotent stem cell derived retinal pigment epithelium (hPSC-RPE) and photoreceptor (hPSC-PR) cells are being utilized for in vitro modeling of retinal diseases as well as to develop cell replacement therapy for retinal degenerative diseases that lead to blindness. Since these retinal cell types are highly functional and mitochondria-rich in vivo, our efforts are focused on assessing and enhancing cell health of hPSC-derived RPE and photoreceptors in terms of mitochondrial metabolism under physiologic conditions.

Cryns, Vincent

Tumor cells must adapt to metabolic stress intrinsic to their rapid growth in order to survive. Our group focuses on the molecular mechanisms by which tumor cells adapt to metabolic stress. We are also interested in targeting metabolic stress in tumor cells by depriving them of essential nutrients to metabolically prime them to respond to pro-apoptotic therapies. As a defining feature of cancer cells, metabolic stress offers unprecedented translational opportunities to selectively target cancer.

Parks, Brian

The Parks Lab is focused on addressing the question of how genetics and diet interact together to contribute to common metabolic diseases, such as obesity and diabetes. Through the use of large-scale integrative genetic studies in the mouse we have identified several candidate genes that mediate gene-diet interactions. Current work is focused on a novel candidate drug target, Agpat5, which improves common symptoms of obesity and diabetes. In addition to this work we have a strong interest in the development of systems genetics approaches for dissecting biological pathways and networks.

Brow, Dave

Metabolite-regulated gene expression in yeast. We identified a pathway for regulation of IMPDH synthesis by its end-product GTP via transcription attenuation. We are investigating additional levels of regulation using IMPDH-GFP and quantitative live-cell microscopy. Mutations in human IMPDH1 can result in an inherited form of blindness, retinitis pigmentosa, possibly by disrupting binding of IMPDH to its gene or mRNA. We are using yeast as a model system to study the effects of these disease mutations.

Amador-Noguez, Daniel

The production of biofuels from cellulosic biomass holds promise as a source of clean renewable energy that can reduce our dependence on fossil fuels. Attaining this goal will require engineered microorganisms capable of economical conversion of cellulosic biomass into biofuels. Effective microbe design relies on understanding the relevant metabolic pathways and their regulation, including how the integrated networks function as a whole. My research program integrates systems-level analyses, especially metabolomics, with computational modeling and genetic engineering to advance understanding of metabolism in biofuel producing microorganisms, particularly clostridium species such as C. acetobutylicum, C. cellulolyticum and C. thermocellum. The main research topics in my laboratory are: 1) Systems-level analysis of metabolic regulation in biofuel producing microorganisms and 2) Engineering symbiotic consortia for biofuel production.

Burkard, Mark

My research is focused on identifying unique abnormalities of cancer cells, including metabolic alterations, that confer sensitivity to anticancer therapy, thereby enabling precision medicine.

Hittinger, Chris

We study the evolutionary genomics of yeasts with particular emphases on biodiversity and carbon metabolism. Over the last half a billion years, different yeast species have evolved radically different metabolic strategies, from the rare highly fermentative lifestyle of Saccharomyces (i.e. Crabtree-Warburg Effect or aerobic fermentation) to yeasts that accumulate over half of their dry weight as fatty acids. We use genetic, genomic, phylogenetic, and metabolic approaches to understand how these differences are encoded in their genomes. We also have applied projects in brewing, cellulosic biofuels, and synthetic biology.

Cobra, Paulo Falco

The main project I'm currently working on focus on the investigation of two different species of the protozoa responsible for the leishmaniasis disease and how their metabolism is affect by adding two of the current drugs used to treat the disease to the growth medium used to cultivate them. There are other collaboration projects in different segments being investigated simultaneously.

Kemnitz, Joseph

calorie restriction and aging T2DM

Audhya, Anjon

The Audhya lab is interested in the contribution of membrane transport pathways (both secretory and endocytic trafficking) to cellular metabolism as it relates to growth, development, endocrine function, and the regulation of lipid homeostasis.

Kimple, Michelle

G protein-coupled receptor signaling pathways affecting pancreatic beta-cell function, growth, and survival in normal and pathophysiological states.

Loewe, Laurence

Understanding metabolic networks is complicated and mechanistic simulations have been used for integrating much of the known observations. Much would be gained by providing experimental biologists with the possibility to describe the metabolic networks they study in a way that facilitates automated computational analyses. My lab has built a prototype of the Evolvix model description language that makes it easy to construct such models and enables the efficient collection of time series data from arbitrarily complicated pure mass-action kinetics simulations. We are currently working to extend this language into the first general purpose programming language designed by biologists for biologists. The purpose of this effort is to enable automated analyses of metabolic and other biochemical reaction networks in order to investigate their robustness in a broader evolutionary systems biology context.

Bates, Donna

I work in the Conversion Area of GLBRC where I help coordinate research on making biofuels from lignocellulosic biomass. This research relies heavily on an understanding of microbial metabolic pathways and ways in which they can be manipulated.

Puglielli, Luigi

Role of the endoplasmic reticulum (ER) acetylation machinery and intracellular acetyl-CoA flux in developmental and degenerative diseases.

Craven, Mark

Developing and applying methods from machine learning, natural language processing, and optimization to infer models characterizing networks of interactions among genes, proteins, metabolites, clinical variables, environmental factors, and phenotypes of interest.

Livny, Miron

Distributed High Throughput Computing. Frameworks and software tools that enable researchers to run with the help of automation tools (workflows) large ensembles of interdependent jobs on large collection of distributed computing and data resources.

Sato, Trey

My group is interested in understanding the regulation of lignocellulosic sugar conversion into biofuels and bioproducts by Saccharomyces cerevisiae. We employ metabolomic, proteomic and transcriptomic tools to determine the molecular mechanisms by which genetically engineered and evolved yeast strains increase their rate of sugar metabolism.

Fathi, Fariba

NMR-based metabolomics studies including: NMR sample preparation identification metabolites in biofluid by 1HNMR, 2D methods(TOCSY and HSQC) measure the concentration of metabolites or binning methods investigation of linear and non-linear models for classification®ression identification of significant metabolites associated with disease metabolic pathway

Reeder, Scott

My research interests focus on the development and validation of advanced magnetic resonance imaging (MRI) methods to quantify tissue characteristics such as tissue triglyceride and iron concentration. Our group also works to develop methods to quantify body composition, including visceral and subcutaneous adipose tissue volumes, total body fat and muscle volumes. Many of these methods can be performed in children and adults, as well as animal models such as mice, rats, and large animals.

Aceti, David

Ligand screening to assign functions to orphan proteins

Ponik, Suzanne

Increased collagen density and organization are driving factors for breast cancer progression. However, the specific cellular mechanisms resulting in altered cell metabolism, proliferation and invasion are not yet clearly defined. I am interested in identifying the mechanisms involved in altered cellular metabolism in a dense collagen microenvironment.

Stewart, Ron

We are interested in the relationship between metabolism and stem cell differentiation.

Karasov, William

We study metabolism of vertebrate wildlife. The majority of the studies involve measurement of whole-animal metabolism (indirect calorimetry), often using the doubly labeled water method. These measurements provide insights into nutrition, feeding and population ecology of wildlife and also their exposure to toxicants in foods. We also study digestive physiology, including work related to gut microbiome.

Roy, Sushmita

My research generally spans the development and application of computational methods based on machine learning for inference and analysis of different types of molecular regulatory networks. Specifically we are interested in developing methods that enable us to ask three main questions: (A) what networks exist in a specific biological context (e.g. a cell type, tissue, species), (B) how do they change between cell types and how do they evolve across species, and (C) how do changes in the network affect overall cellular and organismal state.

Bugni, Tim

Bacteria produce a large repertoire of small molecules (

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Types of Enzymes

The human body includes six major groups, or classes, of enzymes.

Oxidoreductases enhance the rate of oxidation and reduction reactions. In these reactions, also called redox reactions, one of the reactants gives up a pair of electrons that another reactant gains. The electron-pair donor is said to be oxidized and acts as a reducing agent, while the electron-pair recipient is reduced is called the oxidizing agent. A more straightforward way of putting this is that in these kinds of reactions, oxygen atoms, hydrogen atoms or both are moved. Examples include cytochrome oxidase and lactate dehydrogenase.

Transferases speed along the transfer of groups of atoms, such as methyl (CH3), acetyl (CH3CO) or amino (NH2) groups, from one molecule to another molecule. Acetate kinase and alanine deaminase are examples of transferases.

Hydrolases accelerate hydrolysis reactions. Hydrolysis reactions use water (H2O) to split a bond in a molecule to create two daughter products, usually by affixing the -OH (hydroxyl group) from the water to one of the products and a single -H (hydrogen atom) to the other. In the meantime, a new molecule is formed from the atoms displaced by the -H and -OH components. The digestive enzymes lipase and sucrase are hydrolases.

Lyases enhance the rate of the addition of one molecular group to a double bond or the removal of two groups from nearby atoms to create a double bond. These act like hydrolases, except that the removed component is not displaced by water or portions of water. This class of enzymes includes oxalate decarboxylase and isocitrate lyase.

Isomerases speed up isomerization reactions. These are reactions in which all of the original atoms in the reactant are retained, but are rearranged to form an isomer of the reactant. (Isomers are molecules with the same chemical formula, but different arrangements.) Examples include glucose-phosphate isomerase and alanine racemase.

Ligases (also called synthetases) enhance the rate of the joining of two molecules. They usually accomplish this by making use of energy derived from the breakdown of adenosine triphosphate (ATP). Examples of ligases include acetyl-CoA synthetase and DNA ligase.


Materials and methods

Identifying the set of producible metabolites

As biosynthetic pathways leading to certain metabolites are still not completely characterized in the available reconstructions, we cannot expect these molecules to be accessible from the food set, even if otherwise no autocatalytic metabolite is present in the network. Thus, before performing the scope analysis, we first need to identify the sets of molecules whose net synthesis is possible in steady state (that is, producible metabolites). Note that those compounds, which cannot be synthesized from the food molecules in steady state would always be identified as inaccessible by the scope analysis (a non-steady-state approach), but the reverse is not necessarily true. Because flux balance analysis is widely used to assess the production capabilities of metabolic networks, we performed a series of flux balance analyses on each network to identify the set of producible metabolites in each organism. As the principles of flux balance analysis have been described elsewhere [44], here we only briefly note that it involves two fundamental steps: first, specification of mass balance constraints around intracellular metabolites (that is, assumption of steady-state) and second, maximization of the production of one or more compounds using linear programming. The assumption of a steady state of metabolite concentrations specifies a series of linear equations of individual reaction fluxes. Availability of nutrients and directions of individual reactions were included as boundary conditions (all possible external metabolites were available for uptake). For each intracellular metabolite, we identified the flux distribution that maximizes its production rate using the linear programming package CPLEX 9.0.0 (ILOG, Paris, France). If the maximal production rate of a given metabolite was zero, we considered it as a dead-end metabolite and not included in the set of producible metabolites.

Second, some biosynthetic pathways leading to producible metabolites involve reaction steps in which a non-producible cofactor participates (such a situation can occur if synthesis of the cofactor is incomplete in the reconstruction, but there is no net consumption of the cofactor by the pathway). As certain intermediates of these pathways would appear inaccessible in the scope analysis, we excluded them from the set of producible metabolites (even though they could be synthesized in steady state).

Scope analysis

In the first step of scope analysis [17], metabolites produced in reactions whose substrates are all present in the initial seed are added to the initial seed to form the seed set for the next step. In successive steps, metabolites that can be produced from metabolites already present in the set are added to the seed set. The expansion of the seed set is finished when no new compounds can be added, that is, there are no reactions in the metabolic network whose substrate molecules are all in the seed set, but at least one of the products is not. The final set of molecules is referred to as the scope of the input set.

Identifying autocatalytic compounds

If the scope of the input set did not include all metabolites that can be otherwise produced by the network, then we identified the smallest set of internal molecules that had to be added to the input set, so that the scope of this combined input included all required metabolites. To find the smallest set of such internal molecules, we searched for the metabolite that increased the scope to the highest extent (that is, a greedy algorithm). Next, we added this metabolite to the set of input molecules and performed the scope analysis again. The above steps were iterated until we arrived at an input set whose scope included all required metabolites.

Those molecules increasing the scope the most at various steps of the above procedure are either autocatalytic molecules (Figures S2, S3, S5 and S6 in Additional data file 1) or intermediates in the biosynthetic pathways leading to such molecules (Figure S5 in Additional data file 1). In other cases, the identity of the autocatalytic molecule is not self-evident from those compounds found to give the highest increase in the scope (Figures S1 and S7-S9 in Additional data file 1). In such cases, we analyzed the set of molecules, which became accessible after the addition of the identified molecule to the seed. These cases are further discussed in the description of the analysis of Synechocystis (Additional data file 1).


Metabolic Reconstruction

Combining the Predicted Reactions into Biochemical Networks

The entries in Enzyme Nomenclature, which are the preferred way to describe the catalytic activity of a protein, contain information about reactants and their stoichiometry. Theoretically, the information about reactants could be extracted and used to construct a biochemical network by joining the individual reactions through the common reactants. Although the use of the reactions recorded in the Enzyme Nomenclature is possible, this approach is highly likely to produce many erroneous connections, as a result of inconsistency in the compound naming and reaction descriptions, and because of the possibility of joining physiologically unrelated reactions, which happen to include common metabolites. Therefore, in practice, the EC numbers assigned to genes and proteins are used to map them to the reactions in one of the several metabolic pathway resources, such as KEGG, MetaCyc, or PUMA2 (use EMP pathway collection). The advantage of using these resources is that, in addition to recording the enzymatic reactions per se, they record the information about experimentally verified connections between reactions (i.e., biochemical pathways, as they have been described in the biochemical literature). Another reason as to why these metabolic pathway resources are so popular is that they provide an integration of genomic data with metabolic information and a baseline annotation including automated assignments of proteins to biochemical reactions, thus facilitating the analysis of genomes in a metabolic context.

Although all three resources listed above maintain relatively comprehensive collections of metabolic pathways comprising the main aspects of microbial metabolism, they differ in their approach to recording the metabolic pathway data. MetaCyc and PUMA2 pathway collections follow a similar approach and record pathways exactly as they were experimentally elucidated and described in the literature, that is, their pathways are concise, organism-specific, and may even be specific for certain growth conditions (see, e.g., six variants of lysine biosynthesis in MetaCyc or more than 30 versions of the Embden–Meyerhoff pathway in PUMA2). In contrast, KEGG maps represent generalized biochemical networks, in the sense that the same map includes reactions and pathway variants observed in different organisms and in different conditions. Moreover, KEGG maps are much larger than most pathways in either MetaCyc or PUMA2 and often contain both biosynthetic and catabolic reactions for the same compound (e.g., Histidine metabolism, Cysteine metabolism , and Methionine metabolism maps). The difference between the two approaches is illustrated best by Figure 2 , which compares the arginine biosynthesis pathway versions in MetaCyc, PUMA2, and KEGG.

Figure 2 . The differences in representation of arginine biosynthesis in three popular metabolic pathway resources (MetaCyc, PUMA2, and KEGG): MetaCyc and PUMA2 provide concise organism-specific pathways, as they were described in the literature, whereas KEGG combines the information from multiple organisms and conditions into a generalized map.

Both approaches have certain advantages and disadvantages. For instance, if an organism of interest has exactly the same pathway variant as the one recorded in MetaCyc or PUMA2, its presence will be detectable easily because the genes and proteins catalyzing all reactions in the pathway will be found. Moreover, both MetaCyc and PUMA2 pathways are quite intuitive in terms of biological interpretation, as in most cases the presence of a pathway can be correlated with an experimentally verifiable phenotype (e.g., the ability to grow on certain substrates or to produce certain metabolites). However, even a slight deviation from the recorded pathway (such as a novel combination of the known pathway variants) may result in ambiguous interpretation. On the other hand, detection of a new combinatorial pathway variant using KEGG maps is relatively easy as long as all reactions can be found on the same map. However, in many cases, inferring the phenotype of an organism may require significant knowledge of biochemistry because of the presence of multiple pathway variants and biosynthetic and catabolic reactions on the same map. It should be noted that in the past few years, some convergence between the two approaches was observed: MetaCyc is introducing superpathways, which represent combinations of the individual pathway variants (e.g., superpathway of leucine, valine, and isoleucine biosynthesis), whereas KEGG now includes KEGG Pathway Modules as part of KEGG BRITE. The latter represents a hierarchical classification whereby the modules represent the subsets of reactions on each map corresponding to certain pathway variants (for instance, compare the modules Methionine biosynthesis, aspartate ⇒ homoserine ⇒ Methionine and Cysteine biosynthesis, serine ⇒ cysteine to the maps).

The differences between the three resources described above are not limited to their distinct approaches to metabolic pathway recording: although each of them predicts the catalytic activities for the proteins encoded in the genomes, they use very different algorithms. In addition, each of them provides a more or less extensive user interface for searching and browsing of the pathway collection. For example, MetaCyc keeps a detailed and well-annotated record of the literature used in extraction and recording of their pathways and provides a software suite called Pathway Tools, which includes a module (PathoLogic) for inferring the metabolic pathways in an organism using its genome sequence with predicted protein-coding genes as an input. The inferences are based on a number of criteria, such as primary annotation (EC number assignments and protein product descriptions) and sequence similarity-based pathway hole filler. Pathway Tools also includes Pathway/Genome Navigator and Pathway/Genome Editor supporting query, visualization, and editing of Pathway/Genome Databases (i.e., genome-specific databases integrating genomic data with metabolic information).

Similarly, PUMA2 also provides some editing capabilities: users can modify connections between the reactions/pathways and proteins by changing their functional annotations (e.g., by assigning or deleting an EC number assigned to a protein). In addition, PUMA2 supplies many sequence analysis tools and provides an option of selecting pathways for further display and analysis based on the cutoff score reflecting the ‘completeness’ of a pathway (i.e., the percent of enzymatic reactions connected to protein catalysts). In contrast, KEGG performs functional annotations based on its own protein family classification (KEGG Orthology), which is used instead of EC numbers to connect proteins in the genome to biochemical reactions in the pathway maps. The assignment of proteins to KEGG Orthology groups is performed manually using pairwise best BLAST hits. However, users cannot change these assignments, because no annotation-editing capabilities are provided. Also KEGG has no mechanism for suggesting the pathways or pathway modules that are likely to be present in an organism, which has to be done by manual inspection of the individual metabolic maps. KEGG provides some downloadable software (KegTools), which includes modules for searching and browsing the KEGG BRITE functional hierarchies and analysis of functional genomics data through KEGG maps. It should be pointed out that there is a growing movement toward unification of pathway data and facilitation of data exchange, which resulted in establishing a BioPAX group in 2002. The group has developed an open file format specification for the exchange of pathway data to enable the integration of diverse pathway resources (see BioPAX web site for more details).

Although the three metabolic pathway resources described above provide the most comprehensive compendium of biochemical reactions and pathways of microbial metabolism, which benefits from many years of manual curation and careful analysis of the literature, there are numerous other pathway initiatives and public and commercial databases containing metabolic information. Examples of publicly available pathway resources include SEED, IMG, Pathway Database System, aMAZE, and PATIKA (Pathway Analysis Tools for Integration and Knowledge Acquisition) ( Table 2 ). One more resource, TIGR’s Genome Properties ( Table 2 ), although not being a pathway database per se, may be used for metabolic inferences, because many properties in this database correspond to metabolic pathways. Examples of commercial pathway databases include BioCarta, GeneGo’s MetaCore, Ingenuity Pathways Knowledge Base, and ERGO ( Table 2 ). Many of these resources have features complementary to each other, so exploring at least some of them may be helpful in improving the quality of metabolic reconstruction.

Table 2 . Select public and commercial metabolic pathway resources

Public pathway repositories
MetaCyc, BioCyc, and EcoCyc http://metacyc.org/ Caspi R, Foerster H, Fulcher CA, et al. (2006) MetaCyc: A multiorganism database of metabolic pathways and enzymes. Nucleic Acids Research 34: D511–D516.
KEGG http://www.genome.jp/ Kanehisa M, Goto S, Hattori M, et al. (2006) From genomics to chemical genomics: New developments in KEGG. Nucleic Acids Research 34: D354–D357.
PUMA2 http://compbio.mcs.anl.gov/research/group_detail.php?d=6 Maltsev N, Glass E, Sulakhe D, et al. (2006) PUMA2 – Grid-based high-throughput analysis of genomes and metabolic pathways. Nucleic Acids Research 34: D369–D372.
SEED http://theseed.uchicago.edu/FIG/start.cgi Overbeek R, et al. (2005) The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Research 33: 5691–5702.
IMG http://img.jgi.doe.gov/ Markowitz VM, et al. (2006) The integrated microbial genomes (IMG) system. Nucleic Acids Research 34: D344–D348.
Pathway database system http://nashua.cwru.edu/ Krishnamurthy L, Nadeau JH, Ozsoyoglu G, et al. (2003) Pathways Database System: An integrated system for biological pathways. Bioinformatics 19: 930–937.
aMAZE http://www.scmbb.ulb.ac.be/ Lemer C, et al. (2004) The aMAZE LightBench: A web interface to a relational database of cellular processes. Nucleic Acids Research 32: D443–D448.
PATIKA http://www.patika.org/ Demir E, Babur O, Dogrusoz U, et al. (2002) PATIKA: An integrated visual environment for collaborative construction and analysis of cellular pathways. Bioinformatics 18: 996–1003.
TIGR genome properties http://cmr.tigr.org/tigr-scripts/CMR/CmrHomePage.cgi Selengut JD, Haft DH, Davidsen T, et al. (2007) TIGRFAMs and Genome Properties: Tools for the assignment of molecular function and biological process in prokaryotic genomes. Nucleic Acids Research 35: D260–D264.
Commercial pathway repositories
BioCarta http://www.biocarta.com/
GeneGo MetaCore http://www.genego.com/
ERGO http://www.integratedgenomics.com/ Overbeek R, et al. (2003) The ERGO™ genome analysis and discovery system. Nucleic Acids Research 31: 164–171.
Ingenuity pathway knowledge base http://www.ingenuity.com/

IMG, integrated microbial genomes KEGG, Kyoto Encyclopedia of Genes and Genomes PATIKA, Pathway Analysis Tools for Integration and Knowledge Acquisition.

Automated mapping of the genes and proteins onto biochemical reactions and further onto metabolic pathways produces the initial reconstruction of an organism-specific metabolic network. The pathways detected in this process are likely to have numerous holes, including reactions to which no catalyst has been assigned isolated (or dead end) metabolites, which may be produced by one reaction, but not utilized by any other enzymatic or transport reactions and vice versa. There is also a strong possibility of false-positive pathway inferences, whereby pathway presence is suggested based on the erroneous assignment of enzymatic activity to a protein or on the detection of a reaction, which is shared by several pathways, such as mutually exclusive variants of a superpathway. Thus, manual refinement of the initial metabolic reconstruction is always necessary.