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22.1: Overview of Nitrogen Metabolism - Biology


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Organic chemistry is usually described as the chemistry of carbon containing molecules. But isn't that definition a bit carboncentric, especially since the prevalence of oxygen-containing molecules is staggering? What about nitrogen? We live in a dinitrogen rich atmosphere (80%), and all classes of biomolecules (lipids, carbohydrates, nucleic acids and proteins) contain nitrogen. Dinitrogen is very stable, given its triple bond and nonpolarity. We rely on a few organisms to fix N2 from the atmosphere to form ammonium (NH4+), which through nitrification and denitrification can form nitrite (NO2-), nitrate (NO2-), nitric oxide (NO) and nitrous oxide (N2O),the latter being a potent greenhouse gas. We'll concentrate on the metabolic fate of amino groups in amino acids and proteins in the next section. Before exploring their fates, look at the figure below which shows an overall view of the biological nitrogen cycle. The study of biochemistry should encompass more than homo sapiens and expand to the ecosystem in which we are such a small but damaging part.

Let's break down the diagram from a biochemical perspective. There are aerobic and anaerobic processes (conducted by bacteria). Nitrogen-containing substances include both inorganic (ammonium, nitrate, nitrite) and organic (amino acids, nucleotides, etc) molecules. The reactions shown are oxidative and reductive (note: the oxidation number of the nitrogen atoms in the molecules are shown in red). Most of the reactions are carried out underground by bacterial and archaeal microorganisms.

Here are some of the major reactions:

  • N2 fixation (a reduction): N2 from the air is converted by bacteria to ammonium (NH4+) by the enzyme nitrogenase of soil prokaryotes. The energetically disfavored reaction requires lots of ATPs. Ammonium once made can then be taken up by primary producers like plants and incorporated into biomolecules such as amino acids, which animals consume. For those who may still believe that people have marginal effects on our biosphere, consider this. We may soon fix more N2 to NH3 through the industrial Born-Haber reaction (used for fertilizer and explosive productions) that all made by the biosphere. Much of the nitrogen in use comes from the Born-Haber reaction. The excess NH4+ (upwards of 50%) produced industrial and which enters the soil in fertilizers (mostly as NH4NO3) has overwhelmed nature's ability to balance the nitrogen cycle and is not taken up by plants. It is metabolized by microrganisms to nitrite and nitrate.
  • Nitrification: Ammonium is converted to nitrite by ammonia-oxidizing aerobic microorganisms and further to nitrate by a separate group of nitrite-oxidizing aerobic bacteria. Here are the reactions (Rx 1 and 2) to produce nitrate through a hydroxylamine intermediate, followed by the formation of nitrate (Rx 3).

NH3 + O2 + 2e- --> NH2OH + H2O Rx 1

NH2OH + H2O --> NO2- + 5H+ + 4e- Rx 2

NO2- + 1/2 O2 --> NO3- Rx 3

These added ions exceed soil capacity and end up runoff water, polluting our rivers and lakes.

  • Denitrification: This anaerobic reaction pathway reproduces N2 from nitrate Here is the net reaction:

2NO3- + 10e- + 12H+ --> 2N2 + 6H2O

  • Anammox reaction: This more recently discovered bacterial anaerobic reaction pathway converts ammonium and nitrate to N2. Here is the net reaction

NO2- + NH3+ --> N2 + 2H2O

  • Ammonification (not to be confused with mummification) occurs when plants and animals decompose, which returns ammonium to the soil for reuse by plants and microbes.

These reactions are shown in the abbreviated Nitrogen Cycle below.

Nitrogen metabolites are nutrients for plants and perhaps the most import nutrients in the regulation of plant growth (primary productivity) and in regulating life diversity in the biosphere. All living organisms require feedstocks to produce energy and as substrates for biosynthetic reactions. Which are used depend on the organism. Plants are primary producers so they use their own synthesized carbohydrates for both energy production and for biosynthesis. For carnivores, proteins and their derived amino acids are the source of energy (through oxidation) and serve as biosynthetic precursors. For organisms that are omnivorous, the source of energy depends on the "fed" state. With abundant food resources, carbohydrates and lipids are the source of energy. Unlike carbohydrates and lipids, which can be stored as glycogen and triacylglycerides for future use, excess protein and their associated amino acids can not be stored, so amino acid can be eliminated or used for oxidative energy.

In the fed state, carbohydrates are the main source, while in the unfed state, lipids take a predominant role. Under starving conditions, the organisms own proteins are broken down and used for oxidative energy production and for any biosynthesis that remains. In diseased states like diabetes, which can be likened to a starving state in the presence of abundant carbohydrates, both lipids and amino acids become the sources of energy.

How are amino acids in animals oxidatively metabolized? Many pathways could be used to do so but it would seem logical that NH4+ would be removed and the carbons in the remaining molecule would eventually enter glycolysis or the TCA cycle in the form of ketoacids. NH4+ is toxic in high concentration. Ammonium is not oxidized to nitrite or nitrates in humans as occurs in the soil by microorganisms. It can be recycled back into nucleotides or amino acids, and excess amounts are eliminated from the organism. Both processes must be highly controlled. We will turn out attention to the oxidation of amino acids in the next section.

Nitrogenase: An Introduction

Beauty is in the eye of the beholder.

As the domain biochemistry covers the entire biological world, the extent of coverage of a given topic in textbooks can depend, in part, on the interest and experiences of the author(s) presenting the material. Is relevance a metric that should determine coverage? If so, books focused on human or medical biochemistry would surely omit photosynthesis. If topics are selected based on their importance for life, then photosynthesis must surely be covered. If so, then nitrogenase must also be included. If the degree of chemical difficulty for a chemical reaction and the amazing eloquence of the evolved biochemistry solution is considered, then both photosynthesis and nitrogen fixation must be presented. Even though nitrogen fixation is a reductive reaction, it shares strong similarities with the oxygen evolving complex of photosynthesis. They catalyze enormously important redox reactions that involve an abundant atmospheric gas using a very complicated and unique inorganic metallic cofactor that evolution has selected as uniquely suited for the job.

Every first year student of chemistry can draw the Lewis structure of dinitrogen, N2, which contains a triple bond and a lone pair on each nitrogen. If Lewis structures speak to them, they should be able to state that the triple bond makes N2 extraordinarily stable, thus explaining why we can breathe an atmosphere containing 80% N2 and not die. If they have taken biology, they are also aware that very few biological organisms can utilize N2 as a substrate, as this require breaking bonds between the nitrogen atoms, a chemical process reserved for nitrogen “fixing” bacteria found in rhizomes of certain plants. Lastly, they probably memorized that high pressure and temperature, in a process called the Haber-Bosch process, is used react N2 and H2 to form ammonia, NH3. As with any scientific advance, the Haber-Bosch process has brought both harm (its used for explosive weapons) and good (fertilizers). This process now fixes enough N2 in the form of fertilizers to support half of the world’s population, with nitrogenase supporting the rest. Effort is being devoted to genetically modify plants to make their own nitrogenase, eliminating the need for fertilizers but perhaps creating unforeseen problems of its own.

You might be surprised to find out that at room temperature the equilibrium constant favors ammonia formation, hence DG0 < 0. The reaction is favored enthalpically as it is exothermic at room temperature. It is disfavored entropically as should be evident from the balanced equation below: N2(g) + 3H2(g) → 2 NH3(g)

If the reaction is favored thermodynamically at room temperature, why doesn’t it proceed? This story sounds familiar as this same descriptor applies to oxidation of organic molecules with dioxygen. There we showed using MO theory that the reaction is kinetically slow. Same with NH3 formation. A superficial way to see this is that we must break bonds in the stable N2 to start the reaction, leading to a high activation energy, making the reaction kinetics sluggish.

One could jump start the reaction by raising the temperature, but that would slow an exothermic reaction. The Keq (or KD) and DG0 are obviously functions of temperature and for this reaction, and the reaction becomes disfavored at higher temperatures. The solution Haber found was high pressure, forcing the reaction to the side that has fewer molecules of gas, and high temperature to overcome the activation energy barrier and make the reaction kinetically feasible. A complex metal catalysts (magnetite - Fe3O4 -with metal oxides like CaO and Al2O3 which prevent reduction of the Fe with H2) provides an absorptive surface to bring reagents together and facilitate bond breaking in H2 and N2.

We have seen that nature has seemed to involved mechanisms involving the very strange oxygen evolving complex of Mn, Fe, S and Ca to oxidize another very stable and ubiquitous molecule, H2O. Now we explore the amazing mechanisms behind the nitrogenase complex which fixes N2 to form NH3.

What might be needed to drive this reaction biologically? You might surmise the list to include:

  • a source of energy, most likely ATP, to drive this difficult reaction and you’d be right;

  • a source of electrons as the N atoms move from an oxidation state of 0 in elemental N to 3- in NH3; this source turns out to be a protein called flavodoxin or ferridoxin. Of course, these electrons also have interesting sources before they were in the electron carriers of these proteins;

  • some pretty amazing metal centers to accept and donate electrons in a controlled way; these centers are mostly FeS clusters with an additional cluster containing molybdenum (Mo). The clusters are named F, P, and M

  • a source of hydrogen; you might have guessed correctly that it’s not H2 gas (from where would that come?), but H+ ions which are pretty ubiquitously available.

  • a net reaction that is different that the Haber-Bausch process (N2 + 3H2 → 2 NH3).

Here is the actual reaction catalyzed by nitrogenase:

N2 + 8e- + 16ATP + 8H+ → 2NH3 + H2 + 16ADP + 16Pi.

Let’s think a bit about the reaction. As electrons are added the attractions between the nitrogen atoms must decrease. Eventually bonds between them must be broken. Protons could be easily added to maintain charge neutrality. A basic mechanism might involve intermediates as shown below:

Nitrogenase can also other small molecules with triple bonds, including :C=O: and H-C=C-H.

The Structure of Nitrogenase

Nitrogenase is a multiprotein complex containing the following:

  • Two identical subunits, E and F, which have binding sites for the mobile carrier of electrons (the protein ferrodoxin or flavodoxin), ATP, and an FeS cofactor (4Fe-4S, called the F cluster) which accept electrons. These subunits are hence called the nitrogenase reducatase subunits

  • a heterodimer of an alpha and beta subunits. The a (alpha chain) binds the 8Fe-7S F cluster and the Fe-S-Mo M cluster. These subunits comprise the (di)nitrogenase subunits

The overall structure of the protein complex with bound ATP and metal centers are shown below.

An interactive Jsmol structure of nitrogenase is found in the link below.

JSMol (HTML5)

Protopedia: Nitrogenase

An enhanced view of the bound cofactors and ATP are shown in the same spatial orientation in the figure below.

The metal centers are shown below in more detail in both line and space fill views.

Mo is bound to 3 sulfur ions and two a OH and carboxyl group of 3-hydroxy-3-carboxy, adipic acid in the crystal structure of shown above.

The M cluster has an interstitial carbide ion that derives from -CH3 attached to the sulfur of S-adenosyl-methionine (SAM) allowing the carbide to be labeled with either 13C or 14C for mechanistic studies. These labeled carbides are not exchanged or used as a substrate when the enzyme undergoes catalytic turnover. Hence it seems that the carbide probably just stabilizes the M cluster. it won't be shown in the figures below showing more detailed mechanisms.

Nitrogenase Reaction: Part 1 - Addition of Electrons and Protons

The sequential path of electrons from the reductase subunit containing the F cluster to the P and M clusters in the nitrogenase subunit should be apparent from the figures above. We will concentrate on the binding of N2 and how it receives electrons from the M cluster. The figure below shows the FeMo-cofactor and some adjacent amino acid residues. The Mo is not shown in space fill.

Question 1: If Val 70 is mutated to Ile, substrate appears not to access the cluster. Which part of the cluster most likely interacts with N2?

Question 2: His side chains are often found at enzyme active sites. What role might they play in catalysis?

The Lowe and Thorneley (LT) model has been proposed as a mechanism for dinitrogen reduction. In this model an electron and proton are added to the oxidized form of the enzyme (Eo) to produce E1. This is repeated 3 more times to form sequentially, E2, E3 and E4. Only then does N2 bind and the reduction of N2 occur. Two of the added electrons are accepted by H+ ions which form H2, which is liberated on N2 binding.

Question 3: Based on oxidation numbers of N and H in N2, NH3, H+ and H2 and the mechanism above, would you expect 8 electrons to be needed?

The crystal structure shows 2 ATPs bound to the reductase subunit. The stoichiometry of the reaction shows 16 ATP used. Simple math suggests that 2 ATP are cleaved to support the entry of one electron into the complex.

Part 1 - E1-E4: A potential structure for the E4 intermediate is shown below. Note the carbide is not shown. This is often called the Janus intermediate as it is half-way through the catalytic cycle. It is named for Janus, the Roman god of beginnings and transitions, and has been ascribed to gates, doors, doorways and passages. Janus is typically shown with two faces, one looking to the future and one to the past.

Question 4: How can you tell that this figure represents E4?

The hydrides bridge 2 Fe ions so these are examples of three-center, two-electron bonds.

How does this reaction occur? We must look to organometallic chemistry to help us understand the mechanism of this and subsequent steps. Clearly a hydride equivalent has been added to the metals, associated with the oxidation of metal ions in the center. This particular reaction is called an oxidative addition. Presumably the sulfur ions act as Lewis bases as they gain protons from a Lewis acid, probably HIs 195.

Oxidative addition reactions

A figure showing oxidative additions for three different types of reactants are shown below. Note the specific example for insertion of H2, an example somewhat similar to the hydride additions to the M cluster. Oxidative reduction occurs most readily when the two oxidation states of the metal ion are stable. It is likewise favored for metal centers that are not sterically hindered (makes sense if A-B is to be added) and if A-B has a low bond dissociation energy.

Question 5: What role might the H+ ions have in the E4 complex shown above? Why might they be relatively close to the hydrides?

One way to study reaction intermediates is to trap them. If N2 can't access the binding site and the temperature is reduced, the accumulated hydrides and H+ in E4 might interact as the reaction goes back to E1.

Question 6: What might be a likely product in such a case? What mutant might be used to attempt this experiment.

Nitrogenase Reaction: Part 2 - Reduction of N2

Step E4 to E5 seems a bit bizarre as H2 gas is released. This would seem to waste ATP but it must be obvious that we should trust evolution which has created this enzyme. Do you think that this reaction would be facilitated by having the hydrides on one Fe ion, Fe6? The mechanism is another classic organometallic reaction, reductive elimination, the reverse of oxidative addition.

Reductive elimination reactions

In this reaction, a molecule is eliminated or expelled from the complex as the metal ion is reduced and adds two electrons. The figure below shows reductive elimination. Reductive elimination occurs most readily in higher oxidation state metal centers which can be stabilized on reduction. It occurs most readily from electron rich ligands and if the other surrounding ligands are bulky. The dissociating species must also be cis to each other as they form a bond to each other when they leave.

Oxidative addition and reductive elimination at metal centers are often coupled together in organometallic catalytic cycles with some rearrangement or other modification occurring between them. Think about it. The FeS clusters must return to their original oxidation state after the complete LT cycle. We will encounter another organometallic reaction after the addition of N2, migratory insertion, in the second half of the reaction.

Is H2(g) really released? To study this, investigators have used an alternative substrate, acetylene, HC=CH, in the presence of D2 and N2 in an aqueous system.

The acetylene was reduced and formed C2H2D2 and C2H3D. Hence E4 must have had 2 Ds in it, and E2 probably 1. These results support the reversible reductive elimination mechanism for the E4 to E4:N2 reaction above. Previously it had been shown the H+ are reduced by D2 in the presence of D2 and N2 in an aqueous system, so these results are consistent. In additional support, deuterium from D2 is not incorporated into products (C2H2D2, C2H3D or HD) in the absence of N2.

Let's return for a moment to the bridging hydrides as shown again in the figure below.

To form H2, the H- hydrides must be protonated.

Question 5: The hydrides in the model above are shown as bridging (involved in 3 center, 2 electron bonds) and not as terminal (as shown for the H+). Which would be more resistant to protonation and hence reductive elimination, bridging or terminal hydrides?

The bridging hydrides are now stable enough to react with the desired substrate, N2. The M cofactor is large enough to create a face of four Fe ions (see figure below) which are coordinated to a carbide on the bottom face. To create this face of Fe ions requires a cluster of sufficient size with the 6 Fes and 1 central C forming a trigonal prismatic geometry.

Oxidation States of Nitrogenase Fe centers

First Half: It would be difficult to assign specific oxidation states to each Fe ion in the M complex. Instead we can assign relative changes in the oxidation states as the reaction proceeds from E0 to E4. In each step of the LT model, 1 electron is added. We will first assign this to a average Fe ion, M0, with an arbitrarily assigned oxidation state of 0. On addition of 1 electron, the oxidation state would go from M0 to M-1 as the metal is reduced. The M-1 state is then oxidized as an electron is transferred to H+, and when 2 electrons are transferred, and a single H- is made. The diagram below shows the change in oxidation state in going from E0 to E4.

The red boxes highlight thermodynamic cycle-like steps which show how the changes in redox state of the Fe ions (M) could be visualized. Note that in going from E0 to E4, the actual oxidation state of M changes from 0 to +1 to 0 to +1 and back to 0. That is quite amazing given that 4 electrons have been added. Note also that in the red box going from step E0 to E1, M goes from -1 to +1 which corresponds to our description of an oxidative addition when the metal center looses two electrons. This mechanism shows that nitrogenase could be considered a "hydride storage device".

Second Half (facing forward to production NH3):

How does N2 initially interact with E4? It must depend on how the hydrides are released as H2, which evidence shows occurs by reductive elimination (re) and not hydride protonation (hp). On addition, the N2 very quickly is converted to diazene, HN=NH, with the departing H2 taking with it 2 H+s and 2 electrons (or reducing equivalents). These events could occur as shown in the figure below.

Now, with N2 bound as diazene (N2H2) and H2 released, the rest of the reaction could occur as shown below. One new step, a migratory insertion, is shown.

The two halves of the reaction are similar with bridging hydrides utilized. The E4 Janus intermediate links the two halves together.


Adolf Butenandt Institute, Molecular Biology, Ludwig-Maximilians-Universität M¨︁nchen, Schillerstr. 44, 80336 M¨︁nchen, Germany

Ludwig-Thoma-Strasse 22B, 85232 Bergkirchen, Germany

Summary

• The principal knowledge of the metabolic capabilities will help us understand the peculiarities that yeast reveals in the breakdown of organic compounds, production of new cell-specific components, and generation of energy necessary in anabolic pathways. First, we consider the major sources for energy production in S. cerevisiae – the hexose carbon compounds. Since this yeast (as well as many others) can adapt its metabolism to aerobic or aerobic conditions, we have to differentiate between respiration and oxidative phosphorylation, on the one hand, and alcoholic fermentation, on the other hand. In this context, we describe the effects of glucose repression and diauxie. The possibilities of how yeast utilizes other hexose sugars, non-hexose carbon sources, or complex carbon sources are outlined. Gluconeogenesis and carbohydrate biosynthesis are explained in view of yeast's potential to store different forms of carbohydrate for retrieval of energy. Following this, we deal with the utilization and manufacturing of “unusual” hexoses and amino sugars that play an important role in the biosynthesis of cell-specific macromolecules. A particular section is devoted to yeast compounds that contain inositol as a constituent, such as InsPs and the various phosphatidylinositol derivatives. The regular order of post-translational N- and O-linked glycosylation of proteins is presented in some detail. Similar attention is given to the structural carbohydrates that have an outstanding role in yeast cell wall organization.

• Next, we consider fatty acid and lipid metabolism, which in yeast reveals some specific features. In discussing the glycolipids, we focus on sphingolipids and GPI, which latter have a dominant role as lipid membrane anchors. This section includes a survey of the isoprenoid derivatives, which are particularly synthesized and utilized in yeast.

• Nitrogen metabolism considers the utilization of organic and inorganic sources in catabolic pathways, whereby the fact that yeasts can live on ammonium as a sole nitrogen source comes as a real surprise. Employing urea as a nitrogen source is restricted to yeast species other than S. cerevisiae. Yeast has the capacity to biosynthesize virtually all amino acids from simple carbon sources plus a nitrogen source, assimilation of sulfur from sulfate for the few sulfur-containing amino acids (cysteine, methionine, homocysteine), together with constituents from some cofactors. One of the most important activities in nitrogen metabolism concerns protein biosynthesis. We do not present a detailed picture in this overview, but point out further reading/references pertinent to this extremely important field.

• The following section then presents a concise overview of the manufacturing and breakdown of nucleotide compounds in yeast, most of whose features are common to all organisms. Except in degradation pathways, there are some unusual aspects in fungi. We add a description of nucleotide-modifying enzymes, which have been studied in S. cerevisiae in great detail.

• Sections dealing with the metabolism of phosphorus (phosphate) and sulfur in yeast as well as the capabilities of yeast to synthesize most of its “vitamins” and cofactors from endogenous sources complement our metabolic excursion.


Physiology of Lipid Metabolism

Pathway defects in lipoprotein synthesis, processing, and clearance can lead to accumulation of atherogenic lipids in plasma and endothelium.

Exogenous (dietary) lipid metabolism

Over 95% of dietary lipids are

The remaining about 5% of dietary lipids are

Cholesterol (present in foods as esterified cholesterol)

Dietary TGs are digested in the stomach and duodenum into monoglycerides (MGs) and FFAs by gastric lipase, emulsification from vigorous stomach peristalsis, and pancreatic lipase. Dietary cholesterol esters are de-esterified into free cholesterol by these same mechanisms.

Monoglycerides, FFAs, and free cholesterol are then solubilized in the intestine by bile acid micelles, which shuttle them to intestinal villi for absorption.

Once absorbed into enterocytes, they are reassembled into TGs and packaged with cholesterol into chylomicrons, the largest lipoproteins.

Chylomicrons transport dietary TGs and cholesterol from within enterocytes through lymphatics into the circulation. In the capillaries of adipose and muscle tissue, apoprotein C-II (apo C-II) on the chylomicron activates endothelial lipoprotein lipase (LPL) to convert 90% of chylomicron triglyceride to fatty acids and glycerol, which are taken up by adipocytes and muscle cells for energy use or storage.

Cholesterol-rich chylomicron remnants then circulate back to the liver, where they are cleared in a process mediated by apoprotein E (apo E).

Endogenous lipid metabolism

Lipoproteins synthesized by the liver transport endogenous triglycerides and cholesterol. Lipoproteins circulate through the blood continuously until the TGs they contain are taken up by peripheral tissues or the lipoproteins themselves are cleared by the liver. Factors that stimulate hepatic lipoprotein synthesis generally lead to elevated plasma cholesterol and TG levels.

Very-low-density lipoproteins (VLDL) contain apoprotein B-100 (apo B), are synthesized in the liver, and transport TGs and cholesterol to peripheral tissues. VLDL is the way the liver exports excess TGs derived from plasma free fatty acids and chylomicron remnants VLDL synthesis increases with increases in intrahepatic FFAs, such as occur with high-fat diets and when excess adipose tissue releases FFAs directly into the circulation (eg, in obesity, uncontrolled diabetes mellitus). Apo C-II on the VLDL surface activates endothelial LPL to break down TGs into FFAs and glycerol, which are taken up by cells.

Intermediate-density lipoproteins (IDL) are the product of LPL processing of VLDL and chylomicrons. IDL are cholesterol-rich VLDL and chylomicron remnants that are either cleared by the liver or metabolized by hepatic lipase into LDL, which retains apo B-100.

Low-density lipoproteins (LDL), the products of VLDL and IDL metabolism, are the most cholesterol-rich of all lipoproteins. About 40 to 60% of all LDL are cleared by the liver in a process mediated by apo B and hepatic LDL receptors. The rest are taken up by either hepatic LDL or nonhepatic non-LDL (scavenger) receptors. Hepatic LDL receptors are down-regulated by delivery of cholesterol to the liver by chylomicrons and by increased dietary saturated fat they can be up-regulated by decreased dietary fat and cholesterol. Nonhepatic scavenger receptors, most notably on macrophages, take up excess oxidized circulating LDL not processed by hepatic receptors. Monocytes rich in oxidized LDL migrate into the subendothelial space and become macrophages these macrophages then take up more oxidized LDL and form foam cells within atherosclerotic plaques.

The size of LDL particles varies from large and buoyant to small and dense. Small, dense LDL is especially rich in cholesterol esters and is associated with metabolic disturbances such as hypertriglyceridemia and insulin resistance.

High-density lipoproteins (HDL) are initially cholesterol-free lipoproteins that are synthesized in both enterocytes and the liver. HDL metabolism is complex, but one role of HDL is to obtain cholesterol from peripheral tissues and other lipoproteins and transport it to where it is needed most—other cells, other lipoproteins (using cholesteryl ester transfer protein [CETP]), and the liver (for clearance). Its overall effect is anti-atherogenic.

Efflux of free cholesterol from cells is mediated by ATP-binding cassette transporter A1 (ABCA1), which combines with apoprotein A-I (apo A-I) to produce nascent HDL. Free cholesterol in nascent HDL is then esterified by the enzyme lecithin-cholesterol acyl transferase (LCAT), producing mature HDL. Plasma HDL levels may not completely represent reverse cholesterol transport, and the protective effects of higher HDL levels may also be due to anti-oxidant and anti-inflammatory properties.

Lipoprotein (a) [Lp(a)] is LDL that contains apoprotein (a), characterized by 5 cysteine-rich regions called kringles. One of these regions is homologous with plasminogen and is thought to competitively inhibit fibrinolysis and thus predispose to thrombus formation. The Lp(a) may also directly promote atherosclerosis. The metabolic pathways of Lp(a) production and clearance are not well characterized, but levels increase in patients with chronic kidney disease, especially in patients on dialysis.


Journal of Environmental Biology

Background: Journal of Environmental Biology (JEB) is one of the oldest peer reviewed research journal published from India since 1980.
In the year 1978, an International Conference was organized at Muzaffarnagar, India. Participating delegates of the conference realized the need of an international research Journal to cater to the needs of Environmental Biologists and Toxicologists, and the organizer of the conference Dr. R. C. Dalela, was entrusted the responsibility to take the necessary steps. After several meetings, Journal of Environmental Biology was launched on 7 th October 1980 with Dr. Dalela as Editor-in-chief, and was registered with Government of India (GOI).
In 1996, the editorial office of the Journal shifted to Lucknow and the registration was revised by GOI, accordingly. Website (www.jeb.co.in) of journal was launched in 2006 with Open Access facility free-of-charge.

Thus, the Journal has actively been involved for the last four decades in publishing quality research papers related to all areas of Environmental Sciences and Toxicology from all over the world.

Journal of Environmental Biology has gained recognition due to its well-established selection and review mechanism that comprises of well-organized editorial board members collaborating with critical reviewers, and the Journal's own R&D division scientists. The journal is peer-reviewed and follows a systematic path of double-blind review system. Please refer to the Publication Policies to have a complete view of the review process.

Rankings: Current rankings of the Journal of Environmental Biology across various standard journal metrics are as follows:

SCImago Journal Rank indicator (SJR)

Aims and Scope: Journal of Environmental Biology is a broad-based, peer-reviewed International Journal that publishes in English Language original research papers and research reviews (with prior permission) from all areas of Environmental Biology such as :

- Environmental Science - Biological Science - Environment Engineering

- Environmental Health -Biotechnology - Microbiology

- Biochemistry - Toxicology - Ecology

- Agricultural Sciences including Forestry and Fish & Fisheries

Periodicity: The journal is published bimonthly (six issue in a year): January, March May, July, September and November.

Website: The website of the Journal is www.jeb.co.in .

Open access: Immediately after the release of the print version, the papers are also released online for open access. Online copy of both the abstract as well as the full length paper can be read and downloaded FREE of cost.

DOI (Digital Object Identifier): We are the member of the Crossref organization, in order to get a DOI prefix, so we can create Crossref DOIs and register content. All the papers published in Journal of Environmental Biology are issued DOI.

Sponsor: Journal is sponsored by an educational trust Dalela Educational Foundation (DEF) and its secretary Dr. Divakar Dalela ( [email protected] ) is represented in the Editorial Board of the Journal as Executive Editor.

The Journal does not receive any financial grants from any Government Organizations, or any Non-Government Organizations , except academic and research oriented expertise inputs on request from DEF without any financial support. The Journal is independent and financially depends on the subscriptions and the Publication charges from authors.

Management: Journal of Environmental Biology is published by Kiran Dalela and its marketing and distribution is done through Triveni Enterprises. Managing Editor and Publisher manages the affairs through Directors and Executives at its management level. A high-level overview of the organization is as follows:

Secretariat: The secretariat of Journal is currently located with a good infrastructure, in rented premises, and is managed with three Scientists and two scientific assistants, two computer assistants and one dispatch-in-charge, who are permanently employed. The secretariat is well-advised by three eminent advisors/consultants.


Purine and Pyrimidine Metabolism

One of the important specialized pathways of a number of amino acids is the synthesis of purine and pyrimidine nucleotides. These nucleotides are important for a number of reasons. Most of them, not just ATP, are the sources of energy that drive most of our reactions. ATP is the most commonly used source but GTP is used in protein synthesis as well as a few other reactions. UTP is the source of energy for activating glucose and galactose. CTP is an energy source in lipid metabolism. AMP is part of the structure of some of the coenzymes like NAD and Coenzyme A. And, of course, the nucleotides are part of nucleic acids. Neither the bases nor the nucleotides are required dietary components. (Another perspective on this.) We can both synthesize them de novo and salvage and reuse those we already have.

Nomenclature

Nitrogen Bases

There are two kinds of nitrogen-containing bases - purines and pyrimidines. Purines consist of a six-membered and a five-membered nitrogen-containing ring, fused together. Pyridmidines have only a six-membered nitrogen-containing ring. There are 4 purines and 4 pyrimidines that are of concern to us.

Purines

  • Adenine = 6-amino purine
  • Guanine = 2-amino-6-oxy purine
  • Hypoxanthine = 6-oxy purine
  • Xanthine = 2,6-dioxy purine

Adenine and guanine are found in both DNA and RNA. Hypoxanthine and xanthine are not incorporated into the nucleic acids as they are being synthesized but are important intermediates in the synthesis and degradation of the purine nucleotides.

Pyrimidines

  • Uracil = 2,4-dioxy pyrimidine
  • Thymine = 2,4-dioxy-5-methyl pyrimidine
  • Cytosine = 2-oxy-4-amino pyrimidine
  • Orotic acid = 2,4-dioxy-6-carboxy pyrimidine

Cytosine is found in both DNA and RNA. Uracil is found only in RNA. Thymine is normally found in DNA. Sometimes tRNA will contain some thymine as well as uracil.

Nucleosides

If a sugar, either ribose or 2-deoxyribose , is added to a nitrogen base, the resulting compound is called a nucleoside . Carbon 1 of the sugar is attached to nitrogen 9 of a purine base or to nitrogen 1 of a pyrimidine base. The names of purine nucleosides end in -osine and the names of pyrimidine nucleosides end in -idine. The convention is to number the ring atoms of the base normally and to use l', etc. to distinguish the ring atoms of the sugar. Unless otherwise specificed, the sugar is assumed to be ribose. To indicate that the sugar is 2'-deoxyribose, a d- is placed before the name.

  • Adenosine
  • Guanosine
  • Inosine - the base in inosine is hypoxanthine
  • Uridine
  • Thymidine
  • Cytidine

Nucleotides

Adding one or more phosphates to the sugar portion of a nucleoside results in a nucleotide . Generally, the phosphate is in ester linkage to carbon 5' of the sugar. If more than one phosphate is present, they are generally in acid anhydride linkages to each other. If such is the case, no position designation in the name is required. If the phosphate is in any other position, however, the position must be designated. For example, 3'-5' cAMP indicates that a phosphate is in ester linkage to both the 3' and 5' hydroxyl groups of an adenosine molecule and forms a cyclic structure. 2'-GMP would indicate that a phosphate is in ester linkage to the 2' hydroxyl group of a guanosine. Some representative names are:

  • AMP = adenosine monophosphate = adenylic acid
  • CDP = cytidine diphosphate
  • dGTP = deoxy guanosine triphosphate
  • dTTP = deoxy thymidine triphosphate (more commonly designated TTP)
  • cAMP = 3'-5' cyclic adenosine monophosphate

Polynucleotides

Nucleotides are joined together by 3'-5' phosphodiester bonds to form polynucleotides. Polymerization of ribonucleotides will produce an RNA while polymerization of deoxyribonucleotides leads to DNA.

Hydrolysis of Polynucleotides

Most, but not all, nucleic acids in the cell are associated with protein. Dietary nucleoprotein is degraded by pancreatic enzymes and tissue nucleoprotein by lysosomal enzymes. After dissociation of the protein and nucleic acid, the protein is metabolized like any other protein.

The nucleic acids are hydrolyzed randomly by nucleases to yield a mixture of polynucleotides. These are further cleaved by phosphodiesterases (exonucleases) to a mixture of the mononucleotides. The specificity of the pancreatic nucleotidases gives the 3'-nucleotides and that of the lysosomal nucleotidases gives the biologically important 5'-nucleotides.

The nucleotides are hydrolyzed by nucleotidases to give the nucleosides and P i . This is probably the end product in the intestine with the nucleosides being the primary form absorbed. In at least some tissues, the nucleosides undergo phosphorolysis with nucleoside phosphorylases to yield the base and ribose 1-P (or deoxyribose 1-P). Since R 1-P and R 5-P are in equilibrium, the sugar phosphate can either be reincorporated into nucleotides or metabolized via the Hexose Monophosphate Pathway. The purine and pyrimidine bases released are either degraded or salvaged for reincorporation into nucleotides. There is significant turnover of all kinds of RNA as well as the nucleotide pool. DNA doesn't turnover but portions of the molecule are excised as part of a repair process.

Purine and pyrimidines from tissue turnover which are not salvaged are catabolized and excreted. Little dietary purine is used and that which is absorbed is largely catabolized as well. Catabolism of purines and pyrimidines occurs in a less useful fashion than did the catabolism of amino acids in that we do not derive any significant amount of energy from the catabolism of purines and pyrimidines. Pyrimidine catabolism, however, does produce beta-alanine, and the endproduct of purine catabolism, which is uric acid in man, may serve as a scavenger of reactive oxygen species.

Purine Catabolism

Nucleotides to Bases

Guanine nucleotides are hydrolyzed to the nucleoside guanosine which undergoes phosphorolysis to guanine and ribose 1-P . Man's intracellular nucleotidases are not very active toward AMP, however. Rather, AMP is deaminated by the enzyme adenylate (AMP) deaminase to IMP . In the catobilsm of purine nucleotides, IMP is further degraded by hydrolysis with nucleotidase to inosine and then phosphorolysis to hypoxanthine .

Adenosine does occur but usually arises from S-Adenosylmethionine during the course of transmethylation reactions. Adenosine is deaminated to inosine by an adenosine deaminase. Deficiencies in either adenosine deaminase or in the purine nucleoside phosphorylase lead to two different immunodeficiency diseases by mechanisms that are not clearly understood. With adenosine deaminase deficiency , both T and B-cell immunity is affected. The phosphorylase deficiency affects the T cells but B cells are normal. In September, 1990, a 4 year old girl was treated for adenosine deaminase deficiency by genetically engineering her cells to incorporate the gene. The treatment,so far, seems to be successful.

Whether or not methylated purines are catabolized depends upon the location of the methyl group. If the methyl is on an -NH 2 , it is removed along with the -NH 2 and the core is metabolized in the usual fashion. If the methyl is on a ring nitrogen, the compound is excreted unchanged in the urine.

Bases to Uric Acid

Both adenine and guanine nucleotides converge at the common intermediate xanthine . Hypoxanthine, representing the original adenine, is oxidized to xanthine by the enzyme xanthine oxidase . Guanine is deaminated, with the amino group released as ammonia, to xanthine. If this process is occurring in tissues other than liver, most of the ammonia will be transported to the liver as glutamine for ultimate excretion as urea.

Xanthine, like hypoxanthine, is oxidized by oxygen and xanthine oxidase with the production of hydrogen peroxide. In man, the urate is excreted and the hydrogen peroxide is degraded by catalase. Xanthine oxidase is present in significant concentration only in liver and intestine. The pathway to the nucleosides, possibly to the free bases, is present in many tissues.

Gouts and Hyperuricemia

Both undissociated uric acid and the monosodium salt (primary form in blood) are only sparingly soluble. The limited solubility is not ordinarily a problem in urine unless the urine is very acid or has high [Ca 2+ ]. [Urate salts coprecipitate with calcium salts and can form stones in kidney or bladder.] A very high concentration of urate in the blood leads to a fairly common group of diseases referred to as gout. The incidence of gout in this country is about 3/1000.

Gout is a group of pathological conditions associated with markedly elevated levels of urate in the blood (3-7 mg/dl normal). Hyperuricemia is not always symptomatic, but, in certain individuals, something triggers the deposition of sodium urate crystals in joints and tissues. In addition to the extreme pain accompanying acute attacks, repeated attacks lead to destruction of tissues and severe arthritic-like malformations. The term gout should be restricted to hyperuricemia with the presence of these tophaceous deposits.

Urate in the blood could accumulate either through an overproduction and/or an underexcretion of uric acid. In gouts caused by an overproduction of uric acid, the defects are in the control mechanisms governing the production of - not uric acid itself - but of the nucleotide precursors. The only major control of urate production that we know so far is the availability of substrates (nucleotides, nucleosides or free bases) .

One approach to the treatment of gout is the drug allopurinol , an isomer of hypoxanthine.

Allopurinol is a substrate for xanthine oxidase, but the product binds so tightly that the enzyme is now unable to oxidized its normal substrate. Uric acid production is diminished and xanthine and hypoxanthine levels in the blood rise. These are more soluble than urate and are less likely to deposit as crystals in the joints. Another approach is to stimulate the secretion of urate in the urine.

Summary

In summary, all, except ring-methylated, purines are deaminated (with the amino group contributing to the general ammonia pool) and the rings oxidized to uric acid for excretion. Since the purine ring is excreted intact, no energy benefit accrues to man from these carbons.

Pyrimidine Catabolism

Ring Cleavage

In order for the rings to be cleaved, they must first be reduced by NADPH . Atoms 2 and 3 of both rings are released as ammonia and carbon dioxide. The rest of the ring is left as a beta-amino acid . Beta-amino isobutyrate from thymine or 5-methyl cytosine is largely excreted. Beta-alanine from cytosine or uracil may either be excreted or incorporated into the brain and muscle dipeptides, carnosine (his-beta-ala) or anserine (methyl his-beta-ala).

General Comments

Purine and pyrimidine bases which are not degraded are recycled - i.e. reincorporated into nucleotides. This recycling, however, is not sufficient to meet total body requirements and so some de novo synthesis is essential. There are definite tissue differences in the ability to carry out de novo synthesis. De novo synthesis of purines is most active in liver. Non-hepatic tissues generally have limited or even no de novo synthesis. Pyrimidine synthesis occurs in a variety of tissues. For purines, especially, non-hepatic tissues rely heavily on preformed bases - those salvaged from their own intracellular turnover supplemented by bases synthesized in the liver and delivered to tissues via the blood.

"Salvage" of purines is reasonable in most cells because xanthine oxidase, the key enzyme in taking the purines all of the way to uric acid, is significantly active only in liver and intestine. The bases generated by turnover in non-hepatic tissues are not readily degraded to uric acid in those tissues and, therefore, are available for salvage. The liver probably does less salvage but is very active in de novo synthesis - not so much for itself but to help supply the peripheral tissues.

De novo synthesis of both purine and pyrimidine nucleotides occurs from readily available components.

We use for purine nucleotides the entire glycine molecule (atoms 4, 5,7), the amino nitrogen of aspartate (atom 1), amide nitrogen of glutamine (atoms 3, 9), components of the folate-one-carbon pool(atoms 2, 8), carbon dioxide, ribose 5-P from glucose and a great deal of energy in the form of ATP. In de novo synthesis, IMP is the first nucleotide formed. It is then converted to either AMP or GMP.

Since the purines are synthesized as the ribonucleotides, (not as the free bases) a necessary prerequisite is the synthesis of the activated form of ribose 5-phosphate. Ribose 5-phosphate reacts with ATP to form 5-Phosphoribosyl-1-pyrophosphate (PRPP) .

This reaction occurs in many tissues because PRPP has a number of roles - purine and pyrimidine nucleotide synthesis, salvage pathways, NAD and NADP formation. The enzyme is heavily controlled by a variety of compounds (di- and tri-phosphates, 2,3-DPG), presumably to try to match the synthesis of PRPP to a need for the products in which it ultimately appears.

Commitment Step

De novo purine nucleotide synthesis occurs actively in the cytosol of the liver where all of the necessary enzymes are present as a macro-molecular aggregate. The first step is a replacement of the pyrophosphate of PRPP by the amide group of glutamine. The product of this reaction is 5-Phosphoribosylamine . The amine group that has been placed on carbon 1 of the sugar becomes nitrogen 9 of the ultimate purine ring. This is the commitment and rate-limiting step of the pathway.

The enzyme is under tight allosteric control by feedback inhibition. Either AMP, GMP, or IMP alone will inhibit the amidotransferase while AMP + GMP or AMP + IMP together act synergistically . This is a fine control and probably the major factor in minute by minute regulation of the enzyme. The nucleotides inhibit the enzyme by causing the small active molecules to aggregate to larger inactive molecules.

[PRPP] also can play a role in regulating the rate. Normal intracellular concentrations of PRPP (which can and do fluctuate) are below the KM of the enzyme for PRPP so there is great potential for increasing the rate of the reaction by increasing the substrate concentration. The kinetics are sigmoidal. The enzyme is not particularly sensitive to changes in [Gln] (Kinetics are hyperbolic and [gln] approximates KM). Very high [PRPP] also overcomes the normal nucleotide feedback inhibition by causing the large, inactive aggregates to dissociate back to the small active molecules.

Purine de novo synthesis is a complex, energy-expensive pathway. It should be, and is, carefully controlled.

Formation of IMP

Once the commitment step has produced the 5-phosphoribosyl amine, the rest of the molecule is formed by a series of additions to make first the 5- and then the 6-membered ring. (Note: the numbers given to the atoms are those of the completed purine ring and names, etc. of the intermediate compounds are not given.) The whole glycine molecule, at the expense of ATP adds to the amino group to provide what will eventually be atoms 4, 5, and 7 of the purine ring (The amino group of 5-phosphoribosyl amine becomes nitrogen N of the purine ring.) One more atom is needed to complete the five-membered ring portion and that is supplied as 5, 10-Methenyl tetrahydrofolate.

Before ring closure occurs, however, the amide of glutamine adds to carbon 4 to start the six-membered ring portion (becomes nitrogen 3). This addition requires ATP. Another ATP is required to join carbon 8 and nitrogen 9 to form the five-membered ring.

The next step is the addition of carbon dioxide (as a carboxyl group) to form carbon 6 of the ring. The amine group of aspartate adds to the carboxyl group with a subsequent removal of fumarate. The amino group is now nitrogen 1 of the final ring. This process, which is typical for the use of the amino group of aspartate, requires ATP. The final atom of the purine ring, carbon 2, is supplied by 10-Formyl tetrahydrofolate. Ring closure produces the purine nucleotide, IMP.

Note that at least 4 ATPs are required in this part of the process. At no time do we have either a free base or a nucleotide.

Formation of AMP and GMP

IMP can then become either AMP or GMP. GMP formation requires that IMP be first oxidized to XMP using NAD. The oxygen at position 2 is substituted by the amide N of glutamine at the expense of ATP. Similarly, GTP provides the energy to convert IMP to AMP . The amino group is provided by aspartate in a mechanism similar to that used in forming nitrogen 1 of the ring. Removal of the carbons of aspartate as fumarate leaves the nitrigen behind as the 6-amino group of the adenine ring. The monophosphates are readily converted to the di- and tri-phosphates.

Control of De Novo Synthesis

Control of purine nucleotide synthesis has two phases. Control of the synthesis as a whole occurs at the amidotransferase step by nucleotide inhibition and/or [PRPP]. The second phase of control is involved with maintaining an appropriate balance (not equality) between ATP and GTP . Each one stimulates the synthesis of the other by providing the energy. Feedback inhibition also controls the branched portion as GMP inhibits the conversion of IMP to XMP and AMP inhibits the conversion of IMP to adenylosuccinate.

One could imagine the controls operating in such a way that if only one of the two nucleotides were required, there would be a partial inhibition of de novo synthesis because of high levels of the other and the IMP synthesized would be directed toward the synthesis of the required nucleotide. If both nucleotides were present in adequate amounts, their synergistic effect on the amidotransferase would result in almost complete inhibition of de novo synthesis.

De Novo Synthesis of Pyrimidine Nucleotides

Since pyrimidine molecules are simpler than purines, so is their synthesis simpler but is still from readily available components. Glutamine's amide nitrogen and carbon dioxide provide atoms 2 and 3 or the pyrimidine ring. They do so, however, after first being converted to carbamoyl phosphate. The other four atoms of the ring are supplied by aspartate. As is true with purine nucleotides, the sugar phosphate portion of the molecule is supplied by PRPP.

Carbamoyl Phosphate

Pyrimidine synthesis begins with carbamoyl phosphate synthesized in the cytosol of those tissues capable of making pyrimidines (highest in spleen, thymus, GItract and testes). This uses a different enzyme than the one involved in urea synthesis. Carbamoyl phosphate synthetase II (CPS II) prefers glutamine to free ammonia and has no requirement for N-Acetylglutamate.

Formation of Orotic Acid

Carbamoyl phosphate condenses with aspartate in the presence of aspartate transcarbamylase to yield N-carbamylaspartate which is then converted to dihydroorotate.

In man, CPSII, asp-transcarbamylase, and dihydroorotase activities are part of a multifunctional protein .

Oxidation of the ring by a complex, poorly understood enzyme produces the free pyrimidine, orotic acid. This enzyme is located on the outer face of the inner mitochondrial membrane, in contrast to the other enzymes which are cytosolic. Note the contrast with purine synthesis in which a nucleotide is formed first while pyrimidines are first synthesized as the free base .

Formation of the Nucleotides

Orotic acid is converted to its nucleotide with PRPP. OMP is then converted sequentially - not in a branched pathway - to the other pyrimidine nucleotides. Decarboxylation of OMP gives UMP . O-PRT and OMP decarboxylase are also a multifunctional protein . After conversion of UMP to the triphosphate, the amide of glutamine is added, at the expense of ATP, to yield CTP .

Control

The control of pyrimidine nucleotide synthesis in man is exerted primarily at the level of cytoplasmic CPS II . UTP inhibits the enzyme, competitively with ATP. PRPP activates it. Other secondary sites of control also exist (e.g. OMP decarboxylase is inhibited by UMP and CMP). These are probably not very important under normal circumstances.

In bacteria, aspartate transcarbamylase is the control enzyme. There is only one carbamoyl phosphate synthetase in bacteria since they do not have mitochondria. Carbamoyl phosphate, thus, participates in a branched pathway in these organisms that leads to either pyrimidine nucleotides or arginine.

Interconversion of Nucleotides

The monophosphates are the forms synthesized de novo although the triphosphates are the most commonly used forms. But, of course, the three forms are in equilibrium. There are several enzymes classified as nucleoside monophosphate kinases which catalyze the general reaction:(= represents a reversible reaction)

Base-monophosphate + ATP = Base-diphosphate + ADP

e.g. Adenylate kinase: AMP + ATP = 2 ADP

There is a different enzyme for GMP, one for pyrimidines and also enzymes that recognize the deoxy forms.

Similarly, the diphosphates are converted to the triphosphates by nucleoside diphosphate kinase :

There may be only one nucleoside diphosphate kinase with broad specificity. One can legitimately speak of a pool of nucleotides in equilibrium with each other.

Salvage of Bases

Salvaging of purine and pyrimidine bases is an exceedingly important process for most tissues. There are two distinct pathways possible for salvaging the bases.

Salvaging Purines

The more important of the pathways for salvaging purines uses enzymes called phosphoribosyltransferases (PRT) :

PRTs catalyze the addition of ribose 5-phosphate to the base from PRPP to yield a nucleotide.:

Base + PRPP = Base-ribose-phosphate (BMP) + PPi

We gave already seen one example of this type of enzyme as a normal part of de novo synthesis of the pyrimidine nucleotides, - O-PRT.

As a salvage process though, we are dealing with purines. There are two enzymes, A-PRT and HG-PRT. A-PRT is not very important because we generate very little adenine. (Remember that the catabolism of adenine nucleotides and nucleosides is through inosine). HG-PRT , though, is exceptionally important and it is inhibited by both IMP and GMP. This enzyme salvages guanine directly and adenine indirectly. Remember that AMP is generated primarily from IMP, not from free adenine.

Lesch-Nyhan Syndrome

HG-PRT is deficient in the disease called Lesch-Nyhan Syndrome , a severe neurological disorder whose most blatant clinical manifestation is an uncontrollable self-mutilation. Lesch-Nyhan patients have very high blood uric acid levels because of an essentially uncontrolled de novo synthesis . (It can be as much as 20 times the normal rate). There is a significant increase in PRPP levels in various cells and an inability to maintain levels of IMP and GMP via salvage pathways. Both of these factors could lead to an increase in the activity of the amidotransferase.

Salvaging Pyrimidines

A second type of salvage pathway involves two steps and is the major pathway for the pyrimidines, uracil and thymine.

Base + Ribose 1-phosphate = Nucleoside + Pi (nucleoside phosphorylase)

Nucleoside + ATP - Nucleotide + ADP (nucleoside kinase - irreversible)

There is a uridine phosphorylase and kinase and a deoxythymidine phosphorylase and a thymidine kinase which can salvage some thymine in the presence of dR 1-P.

Formation of Deoxyribonucleotides

De novo synthesis and most of the salvage pathways involve the ribonucleotides. (Exception is the small amount of salvage of thymine indicated above.) Deoxyribonucleotides for DNA synthesis are formed from the ribonucleotide diphosphates (in mammals and E. coli ).

A base diphosphate (BDP) is reduced at the 2' position of the ribose portion using the protein, thioredoxin and the enzyme nucleoside diphosphate reductase . Thioredoxin has two sulfhydryl groups which are oxidized to a disulfide bond during the process. In order to restore the thioredoxin to its reduced for so that it can be reused, thioredoxin reductase and NADPH are required.

This system is very tightly controlled by a variety of allosteric effectors. dATP is a general inhibitor for all substrates and ATP an activator. Each substrate then has a specific positive effector (a BTP or dBTP). The result is a maintenance of an appropriate balance of the deoxynucleotides for DNA synthesis.

Synthesis of dTMP

DNA synthesis also requires dTMP (dTTP). This is not synthesized in the de novo pathway and salvage is not adequate to maintain the necessary amount. dTMP is generated from dUMP using the folate-dependent one-carbon pool.

Since the nucleoside diphosphate reductase is not very active toward UDP, CDP is reduced to dCDP which is converted to dCMP. This is then deaminated to form dUMP. In the presence of 5,10-Methylene tetrahydrofolate and the enzyme thymidylate synthetase , the carbon group is both transferred to the pyrimidine ring and further reduced to a methyl group. The other product is dihydrofolate which is subsequently reduced to the tetrahydrofolate by dihydrofolate reductase.

Chemotherapeutic Agents

Thymidylate synthetase is particularly sensitive to availability of the folate one-carbon pool. Some of the cancer chemotherapeutic agents interfere with this process as well as with the steps in purine nucleotide synthesis involving the pool.

Cancer chemotherapeutic agents like methotrexate (4-amino, 10-methyl folic acid) and aminopterin (4-amino, folic acid) are structural analogs of folic acid and inhibit dihydrofolate reductase. This interferes with maintenance of the folate pool and thus of de novo synthesis of purine nucleotides and of dTMP synthesis. Such agents are highly toxic and administered under careful control.

Quiz Questions

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112 Prokaryotic Metabolism

By the end of this section, you will be able to do the following:

  • Identify the macronutrients needed by prokaryotes, and explain their importance
  • Describe the ways in which prokaryotes get energy and carbon for life processes
  • Describe the roles of prokaryotes in the carbon and nitrogen cycles

Prokaryotes are metabolically diverse organisms. In many cases, a prokaryote may be placed into a species clade by its defining metabolic features: Can it metabolize lactose? Can it grow on citrate? Does it produce H2S? Does it ferment carbohydrates to produce acid and gas? Can it grow under anaerobic conditions? Since metabolism and metabolites are the product of enzyme pathways, and enzymes are encoded in genes, the metabolic capabilities of a prokaryote are a reflection of its genome. There are many different environments on Earth with various energy and carbon sources, and variable conditions to which prokaryotes may be able to adapt. Prokaryotes have been able to live in every environment from deep-water volcanic vents to Antarctic ice by using whatever energy and carbon sources are available. Prokaryotes fill many niches on Earth, including involvement in nitrogen and carbon cycles, photosynthetic production of oxygen, decomposition of dead organisms, and thriving as parasitic, commensal, or mutualistic organisms inside multicellular organisms, including humans. The very broad range of environments that prokaryotes occupy is possible because they have diverse metabolic processes.

Needs of Prokaryotes

The diverse environments and ecosystems on Earth have a wide range of conditions in terms of temperature, available nutrients, acidity, salinity, oxygen availability, and energy sources. Prokaryotes are very well equipped to make their living out of a vast array of nutrients and environmental conditions. To live, prokaryotes need a source of energy, a source of carbon, and some additional nutrients.

Macronutrients

Cells are essentially a well-organized assemblage of macromolecules and water. Recall that macromolecules are produced by the polymerization of smaller units called monomers. For cells to build all of the molecules required to sustain life, they need certain substances, collectively called nutrients . When prokaryotes grow in nature, they must obtain their nutrients from the environment. Nutrients that are required in large amounts are called macronutrients, whereas those required in smaller or trace amounts are called micronutrients. Just a handful of elements are considered macronutrients—carbon, hydrogen, oxygen, nitrogen, phosphorus, and sulfur. (A mnemonic for remembering these elements is the acronym CHONPS.)

Why are these macronutrients needed in large amounts? They are the components of organic compounds in cells, including water. Carbon is the major element in all macromolecules: carbohydrates, proteins, nucleic acids, lipids, and many other compounds. Carbon accounts for about 50 percent of the composition of the cell. In contrast, nitrogen represents only 12 percent of the total dry weight of a typical cell. Nitrogen is a component of proteins, nucleic acids, and other cell constituents. Most of the nitrogen available in nature is either atmospheric nitrogen (N2) or another inorganic form. Diatomic (N2) nitrogen, however, can be converted into an organic form only by certain microorganisms, called nitrogen-fixing organisms. Both hydrogen and oxygen are part of many organic compounds and of water. Phosphorus is required by all organisms for the synthesis of nucleotides and phospholipids. Sulfur is part of the structure of some amino acids such as cysteine and methionine, and is also present in several vitamins and coenzymes. Other important macronutrients are potassium (K), magnesium (Mg), calcium (Ca), and sodium (Na). Although these elements are required in smaller amounts, they are very important for the structure and function of the prokaryotic cell.

Micronutrients

In addition to these macronutrients, prokaryotes require various metallic elements in small amounts. These are referred to as micronutrients or trace elements. For example, iron is necessary for the function of the cytochromes involved in electron-transport reactions. Some prokaryotes require other elements—such as boron (B), chromium (Cr), and manganese (Mn)—primarily as enzyme cofactors.

The Ways in Which Prokaryotes Obtain Energy

Prokaryotes are classified both by the way they obtain energy, and by the carbon source they use for producing organic molecules. These categories are summarized in (Figure). Prokaryotes can use different sources of energy to generate the ATP needed for biosynthesis and other cellular activities. Phototrophs (or phototrophic organisms) obtain their energy from sunlight. Phototrophs trap the energy of light using chlorophylls, or in a few cases, bacterial rhodopsin. (Rhodopsin-using phototrophs, oddly, are phototrophic, but not photosynthetic, since they do not fix carbon.) Chemotrophs (or chemosynthetic organisms) obtain their energy from chemical compounds. Chemotrophs that can use organic compounds as energy sources are called chemoorganotrophs. Those that can use inorganic compounds, like sulfur or iron compounds, as energy sources are called chemolithotrophs.

Energy-producing pathways may be either aerobic , using oxygen as the terminal electron acceptor, or anaerobic, using either simple inorganic compounds or organic molecules as the terminal electron acceptor. Since prokaryotes lived on Earth for nearly a billion years before photosynthesis produced significant amounts of oxygen for aerobic respiration, many species of both Bacteria and Archaea are anaerobic and their metabolic activities are important in the carbon and nitrogen cycles discussed below.

The Ways in Which Prokaryotes Obtain Carbon

Prokaryotes not only can use different sources of energy, but also different sources of carbon compounds. Autotrophic prokaryotes synthesize organic molecules from carbon dioxide. In contrast, heterotrophic prokaryotes obtain carbon from organic compounds. To make the picture more complex, the terms that describe how prokaryotes obtain energy and carbon can be combined. Thus, photoautotrophs use energy from sunlight, and carbon from carbon dioxide and water, whereas chemoheterotrophs obtain both energy and carbon from an organic chemical source. Chemolithoautotrophs obtain their energy from inorganic compounds, and they build their complex molecules from carbon dioxide. Finally, prokaryotes that get their energy from light, but their carbon from organic compounds, are photoheterotrophs. The table below ((Figure)) summarizes carbon and energy sources in prokaryotes.

Carbon and Energy Sources in Prokaryotes
Energy Sources Carbon Sources
Light Chemicals Carbon dioxide Organic compounds
Phototrophs Chemotrophs Autotrophs Heterotrophs
Organic chemicals Inorganic chemicals
Chemo-organotrophs Chemolithotrophs

Role of Prokaryotes in Ecosystems

Prokaryotes are ubiquitous: There is no niche or ecosystem in which they are not present. Prokaryotes play many roles in the environments they occupy. The roles they play in the carbon and nitrogen cycles are vital to life on Earth. In addition, the current scientific consensus suggests that metabolically interactive prokaryotic communities may have been the basis for the emergence of eukaryotic cells.

Prokaryotes and the Carbon Cycle

Carbon is one of the most important macronutrients, and prokaryotes play an important role in the carbon cycle ((Figure)). The carbon cycle traces the movement of carbon from inorganic to organic compounds and back again. Carbon is cycled through Earth’s major reservoirs: land, the atmosphere, aquatic environments, sediments and rocks, and biomass. In a way, the carbon cycle echoes the role of the “four elements” first proposed by the ancient Greek philosopher, Empedocles: fire, water, earth, and air. Carbon dioxide is removed from the atmosphere by land plants and marine prokaryotes, and is returned to the atmosphere via the respiration of chemoorganotrophic organisms, including prokaryotes, fungi, and animals. Although the largest carbon reservoir in terrestrial ecosystems is in rocks and sediments, that carbon is not readily available.

Participants in the carbon cycle are roughly divided among producers, consumers, and decomposers of organic carbon compounds. The primary producers of organic carbon compounds from CO2 are land plants and photosynthetic bacteria. A large amount of available carbon is found in living land plants. A related source of carbon compounds is humus, which is a mixture of organic materials from dead plants and prokaryotes that have resisted decomposition. (The term “humus,” by the way, is the root of the word “human.”) Consumers such as animals and other heterotrophs use organic compounds generated by producers and release carbon dioxide to the atmosphere. Other bacteria and fungi, collectively called decomposers , carry out the breakdown (decomposition) of plants and animals and their organic compounds. Most carbon dioxide in the atmosphere is derived from the respiration of microorganisms that decompose dead animals, plants, and humus.

In aqueous environments and their anoxic sediments, there is another carbon cycle taking place. In this case, the cycle is based on one-carbon compounds. In anoxic sediments, prokaryotes, mostly archaea, produce methane (CH4). This methane moves into the zone above the sediment, which is richer in oxygen and supports bacteria called methane oxidizers that oxidize methane to carbon dioxide, which then returns to the atmosphere.


Prokaryotes and the Nitrogen Cycle

Nitrogen is a very important element for life because it is a major constituent of proteins and nucleic acids. It is a macronutrient, and in nature, it is recycled from organic compounds to ammonia, ammonium ions, nitrate, nitrite, and nitrogen gas by many processes, many of which are carried out only by prokaryotes. As illustrated in (Figure), prokaryotes are key to the nitrogen cycle. The largest pool of nitrogen available in the terrestrial ecosystem is gaseous nitrogen (N2) from the air, but this nitrogen is not usable by plants, which are primary producers. Gaseous nitrogen is transformed, or “fixed” into more readily available forms, such as ammonia (NH3), through the process of nitrogen fixation . Nitrogen-fixing bacteria include Azotobacter in soil and the ubiquitous photosynthetic cyanobacteria. Some nitrogen fixing bacteria, like Rhizobium, live in symbiotic relationships in the roots of legumes. Another source of ammonia is ammonification , the process by which ammonia is released during the decomposition of nitrogen-containing organic compounds. The ammonium ion is progressively oxidized by different species of bacteria in a process called nitrification. The nitrification process begins with the conversion of ammonium to nitrite (NO2 – ), and continues with the conversion of nitrite to nitrate. Nitrification in soils is carried out by bacteria belonging to the genera Nitrosomas, Nitrobacter, and
Nitrospira
. Most nitrogen in soil is in the form of ammonium (NH4 + ) or nitrate (NO3 – ). Ammonia and nitrate can be used by plants or converted to other forms.

Ammonia released into the atmosphere, however, represents only 15 percent of the total nitrogen released the rest is as N2 and N2O (nitrous oxide). Ammonia is catabolized anaerobically by some prokaryotes, yielding N2 as the final product. Denitrifying bacteria reverse the process of nitrification, reducing the nitrate from soils to gaseous compounds such as N2O, NO, and N2.


Which of the following statements about the nitrogen cycle is false?

  1. Nitrogen-fixing bacteria exist on the root nodules of legumes and in the soil.
  2. Denitrifying bacteria convert nitrates (NO3 – ) into nitrogen gas (N2).
  3. Ammonification is the process by which ammonium ion (NH4 + ) is released from decomposing organic compounds.
  4. Nitrification is the process by which nitrites (NO2 – ) are converted to ammonium ion (NH4 + ).

Section Summary

As the oldest living inhabitants of Earth, prokaryotes are also the most metabolically diverse they flourish in many different environments with various energy and carbon sources, variable temperature, pH, pressure, oxygen and water availability. Nutrients required in large amounts are called macronutrients, whereas those required in trace amounts are called micronutrients or trace elements. Macronutrients include C, H, O, N, P, S, K, Mg, Ca, and Na. In addition to these macronutrients, prokaryotes require various metallic elements for growth and enzyme function. Prokaryotes use different sources of energy to assemble macromolecules from smaller molecules. Phototrophs obtain their energy from sunlight, whereas chemotrophs obtain energy from chemical compounds. Energy-producing pathways may be either aerobic or anaerobic.

Prokaryotes play roles in the carbon and nitrogen cycles. Producers capture carbon dioxide from the atmosphere and convert it to organic compounds. Consumers (animals and other chemoorganotrophic organisms) use organic compounds generated by producers and release carbon dioxide into the atmosphere by respiration. Carbon dioxide is also returned to the atmosphere by the microbial decomposers of dead organisms. Nitrogen also cycles in and out of living organisms, from organic compounds to ammonia, ammonium ions, nitrite, nitrate, and nitrogen gas. Prokaryotes are essential for most of these conversions. Gaseous nitrogen is transformed into ammonia through nitrogen fixation. Ammonia is anaerobically catabolized by some prokaryotes, yielding N2 as the final product. Nitrification is the conversion of ammonium into nitrite. Nitrification in soils is carried out by bacteria. Denitrification is also performed by bacteria and transforms nitrate from soils into gaseous nitrogen compounds, such as N2O, NO, and N2.

Visual Connection Questions

(Figure) Which of the following statements about the nitrogen cycle is false?


22.1: Overview of Nitrogen Metabolism - Biology

Explained by: Brenton W. Thomas

Overview: The nitrogen cycle involves three major steps: nitrogen fixation, nitrification, and denitrification. It is a cycle within the biosphere which involves the atmosphere, hydrosphere, and lithosphere. Nitrogen is found in several locations, or reservoirs. It is most prevalent in sediments and rocks, second in the atmosphere (78%).

Approximately 78% of air is Nitrogen. Nitrogen is important to life because it is a key part of amino and nucleic acids. Also, it is an important part of ATP, which is the basic energy molecule for living things.

Neither plants or animals can obtain nitrogen directly from the atmosphere. Instead, they depend on a process known as nitrogen fixation . Key players in this process are legumes and the symbiotic bacteria which are associated with the legume's root nodules. These bacteria are known as nitrogen-fixing bacteria. These organisms convert nitrogen in the soil to ammonia, which can then be taken up by plants. This process also occurs in aquatic ecosystems, where cyanobacteria participate.

After nitrogen has been fixed, other bacteria convert it into nitrate, in a process known as nitrification . In the first step of this process, Nitrosomonas convert ammonia into nitrite, and in the second step, nitrite is converted into nitrate, by Nitrobacter . This nitrate is then consumed by plants.

The final aspect of the nitrogen cycle is the process of denitrification. This process is performed by a variety of microscopic bacteria, fungi, and other organsims. Nitrates in the soil are broken down by these organsisms, and nitrogen is released into the atmosphere. This complete the cycle.

NITROGEN CYCLE PROCESSES

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Nitrogen Fixation

Nitrogen fixation is an anaerobic (without oxygen) process in which atmospheric nitrogen (N 2 )is reduced to NH 3 . Bacteria are responsible for this process. Bacteria in terrestrial and aquatic(water) environments participate in this process. These organisms must have a special enzyme known as dinitogenase to be able to to this.

Plants cannot use the nitrogen in our atmosphere without the assistance of nitrogen-fixing bacteria. These bacteria reduce atmosphereic nitogen to ammonia, which can be used to make other biological compounds. The plants do not use the ammonia directly, but it's a product of the waste.

Nitrogen is an essential plant nutrient. It is the nutrient that is most commonly deficient, contributing to reduced agricultural yields throughout the world. Molecular nitrogen or dinitrogen (N2) makes up four-fifths of the atmosphere but is metabolically unavailable directly to higher plants or animals. It is available to some species of microorganism through Biological Nitrogen Fixation (BNF) in which atmospheric nitrogen is converted to ammonia by the enzyme dinitrogenase. Microorganisms that fix nitrogen are called diazotrophs.

BNF requires energy. Those microbes that fix nitrogen independent of other organisms are called free living. The free-living diazotrophs require a chemical energy source if nonphotosynthetic, whereas the photosynthetic diazotrophs utilize light energy. The free-living diazotrophs contribute little fixed nitrogen to agricultural crops. Associative nitrogen-fixing microorganisms are those diazotrophs that live in close proximity to plant roots (that is, in the rhizosphere or within plants) and can obtain energy materials from the plants. They may make a modest contribution of fixed nitrogen to agriculture and forestry, but quantification of their potential has not been established. The symbiotic relationship between diazotrophs called rhizobia and legumes (for example, clover and soybean) can provide large amounts of nitrogen to the plant and can have a significant impact on agriculture.

The symbiosis between legumes and the nitrogen-fixing rhizobia occurs within nodules mainly on the root and in a few cases on the stem. A similar symbiosis occurs between a number of woody plant species and the diazotrophic actinomycete Frankia. The plant supplies energy materials to the diazotrophs, which in turn reduce atmospheric nitrogen to ammonia. This ammonia is transferred from the bacteria to the plant to meet the plant's nutritional nitrogen needs for the synthesis of proteins, enzymes, nucleic acids, chlorophyll, and so forth.

Nitrification

Plants receive the components of the "fixed" nitorgen using nitrates in the soil to provide the nutrients they need. Bacteria such as Nitrosomonas, Nitrococcus, and Nitrobacter participate.

Nitrification involves two steps. First, the ammonium ion (NH 4 +) is oxidized into NO 2 -. Then, this compound is further oxidized into NO 3 -. Again, bacteria in the soil participate in both processes.

Assimilation

Plant roots assimilate Nitrogen mainly in the form of nitrates while animals assimilate their nitrogen by eating the plants.

Ammonification

Ammonia is formed in the soil by the decompostion of plants and animals and by the release of animal waste.

Denitrification

This is the reduction of nitrates to gaseous nitrogen. Denitrifying bacteria perform almost the reverse of the nitorgen fixing bacteria.

Study Questions for the Nitrogen Cycle

1. List the three major processes involved in the nitrogen cycle.

2. Starting with nitrogen, draw a map showing the chemical transformations it undergoes throughout the cycle.


Connections of Other Sugars to Glucose Metabolism

Glycogen, a polymer of glucose, is a short-term energy storage molecule in animals. When there is adequate ATP present, excess glucose is converted into glycogen for storage. Glycogen is made and stored in the liver and muscle. Glycogen will be taken out of storage if blood sugar levels drop. The presence of glycogen in muscle cells as a source of glucose allows ATP to be produced for a longer time during exercise.

Sucrose is a disaccharide made from glucose and fructose bonded together. Sucrose is broken down in the small intestine, and the glucose and fructose are absorbed separately. Fructose is one of the three dietary monosaccharides, along with glucose and galactose (which is part of milk sugar, the disaccharide lactose), that are absorbed directly into the bloodstream during digestion. The catabolism of both fructose and galactose produces the same number of ATP molecules as glucose.


22.1: Overview of Nitrogen Metabolism - Biology

Different Nitrogen States

For Nitrogen to be used by different life forms on Earth, it must change into different states. Nitrogen in the atmosphere, or air, is N2. Other important states of nitrogen include Nitrates (N03), Nitrites (NO2), and Ammonium (NH4).

This picture shows the flow of the nitrogen cycle. The most important part of the cycle is bacteria. Bacteria help the nitrogen change between states so it can be used. When nitrogen is absorbed by the soil, different bacteria help it to change states so it can be absorbed by plants. Animals then get their nitrogen from the plants.

  • Fixation - Fixation is the first step in the process of making nitrogen usable by plants. Here bacteria change nitrogen into ammonium.
  • Nitrification - This is the process by which ammonium gets changed into nitrates by bacteria. Nitrates are what the plants can then absorb.
  • Assimilation - This is how plants get nitrogen. They absorb nitrates from the soil into their roots. Then the nitrogen gets used in amino acids, nucleic acids, and chlorophyll.
  • Ammonification - This is part of the decaying process. When a plant or animal dies, decomposers like fungi and bacteria turn the nitrogen back into ammonium so it can reenter the nitrogen cycle.
  • Denitrification - Extra nitrogen in the soil gets put back out into the air. There are special bacteria that perform this task as well.

Why is nitrogen important to life?

Plants and animals could not live without nitrogen. It is an important part of many cells and processes such as amino acids, proteins, and even our DNA. It is also needed to make chlorophyll in plants, which plants use in photosynthesis to make their food and energy.

How have humans altered the nitrogen cycle?

Unfortunately, human activity has altered the cycle. We do this by adding nitrogen into the soil with fertilizer as well as other activities that put more nitrous oxide gas into the atmosphere. This adds in more nitrogen than is needed by normal cycle and upsets the cycle's balance.


Methods

Plant growth conditions

Matsumae, a major rice cultivar in north China, was chosen as the test organism. Matsumae as salt tolerance rice cultivar can grow in the moderate-salinized field of northeast China. Seeds were germinated and grown in petri dishes for 6 d in a growth cabinet (29°C during the day and 25°C during the night, 16/8 h photoperiod at 250 μmol m –2 s –1 ). Seedlings were then transferred to buckets containing 2000 mL of sterile nutrient solution for solution culture. The nutrient solution was replaced daily. The buckets were placed in a growth chamber that was maintained at 27.0 ± 1.5°C during the day and 22.0 ± 1.5°C during the night, under a 16/8 h photoperiod at 250 μmol m –2 s –1 . The nutrient solution used in this work accorded to the components described by the International Rice Research Institute [17], and contained 1.44 mM NH4NO3, 0.32 mM NaH2PO4, 0.6 mM K2SO4, 1.0 mM CaCl2, 1.6 mM MgSO4, 0.072 mM Fe-EDTA, 0.2 mM Na2SiO3, 9.1 μM MnCl2, 0.154 μM ZnSO4, 0.156 μM CuSO4, 18.5 μM H3BO3 and 0.526 μM H2MoO4 at pH 5.2.

Stress treatment

After 22 days of growth in hydroponic medium, rice plants were subjected to salt stress (100 mM NaCl) by transferring them to another barrel containing 2000 mL of the treatment solution amended with the above nutrients and 100 mM NaCl. A bucket including 20 seedlings represented one replicate, and there were four replicates per treatment. 8 buckets of seedlings were randomly divided into 2 sets, four buckets per set. Each bucket was considered as one replicate with four replicates per set, one set was used as control, and another set was treated with salt stress. Treatment solutions were replaced daily. The nutrient solution without stress salts was used as control. The 20 seedlings in each bucket were harvested after treatment for 6 d.

Measurements of physiological indices

Old leaf was defined as second leaf at bottom, and young leaf as newly emerged leaf. The young and old leaves of 10 seedlings in each bucket were separated and mixed, then immediately frozen in liquid nitrogen and then stored at −70°C for RNA isolation. Another 10 seedlings in each bucket were washed with distilled water, after which the old and young leaves were separated and freeze-dried. Dry samples of plant material were treated with 10 mL deionized water at 100°C for 1 h, and the extract used to determine the contents of free inorganic ions. The contents of NO3 – , Cl – , H2PO4 – , and SO4 2– were determined by ion chromatography (DX-300 ion chromatographic system AS4A-SC ion-exchange column, CD M-II electrical conductivity detector, mobile phase: Na2CO3/NaHCO3 = 1.7/1.8 mM DIONEX, Sunnyvale, USA). Ammoniacal nitrogen was measured by ninhydrin colourimetry methods [18]. A flame photometer was used to determine K + and Na + contents.

Quantitative real time PCR analysis

We extracted the total RNA from the young and old leaves of seedlings grown under stress or non-stress conditions using TRIzol reagent (Invitrogen). The RNA was treated with DNaseI (Invitrogen), reverse-transcribed using SuperScriptTM RNase H-Reverse Transcriptase (Invitrogen), and then subjected to real-time PCR analysis using gene-specific primers. The gene-specific primers and corresponding references are listed in Additional file 1. PCR amplification was conducted with an initial step at 95°C for 1 min followed by 45 cycles of 5 s at 95°C, 10 s at 60°C and 30 s at 72°C. Amplification of the target gene was monitored every cycle by SYBR Green. Amplification of the rice UBQ5 (GenBank Accession AK061988) mRNA was used as an internal quantitative control [19–21]. The relative expression of the target genes was calculated using the △ Ct method [22]. We optimized PCR reaction system, after which the amplification efficiencies of each target gene and reference gene were approximately equal.


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