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氮的代谢-转载

已有 6185 次阅读 2007-11-22 22:16 |个人分类:微生物生理学专栏|文章来源:转载

Nitrogen cycle

Nitrogen is found in many common forms of nitrogen in the environment:

  • N2 - nitrogen gas (dinitrogen, 78% of the air).
  • NO - nitric oxide (used as a neurotransmitter).
  • NO2 - nitrogen dioxide (toxic brown gas).
  • N2O - nitrous oxide (laughing gas).
  • NO2 - nitrite ion (nitrous acid is HNO2).
  • NO3 - nitrate ion (nitric acid is HNO3).
  • NH3 - ammonia (the only common alkaline gas).
  • NH4+ - ammonium ion.
  • R-NH2 - organic nitrogen, including amino acids, proteins, alkaloid bases, urea (a.k.a. carbamide: (NH2)2CO), etc.

Nitrogen is essential to amino acid (protein), base (DNA) and tetrapyrrole (heme) synthesis. However, it is very difficult to convert gaseous dinitrogen to biologically available forms (ammonia, etc). It takes 930 kJ mol−1 to break the very strong N≡N bond. Although the reaction to form ammonia from nitrogen and hydrogen gas does have a negative ?G0 (−16 kJ mol−1), the activation energy is so huge, that this spontaneous reaction occurs at a completely negligible rate. The Haber process is humans' way of making this feasible, and uses a iron catalyst.

 

Nitrogen is constantly cycled in the environment, both within organisms (plants with nitrogen deficiency will strip nitrogen from their lower leaves), and between organisms (free amino acids in the environment are rapidly taken up by bacteria).

Nitrogen cycle.

The nitrogen cycle consists of five main processes:

  1. Fixation. 2N2 + 3H2 → 2NH3. This is an extremely specialised process, and is performed by a number of diazotrophs, such as Anabaena (a cyanobacterium), and Rhizobium (the symbiotic bacterium found in legume root nodules).
  2. Nitrification. This is the oxidation of ammonia to oxyanions. The initial oxidation to nitrite, 2NH3 + e + 3O2 → 2NO2 + 2 H2O + 2H+, is performed by bacteria such as Nitrosomonas. Oxidation to nitrate, 2NO2 + O2 → 2NO3 is performed by Nitrobacter.
  3. Denitrification. Some bacteria, such as Pseudomonas, are able to use nitrate as a terminal electron acceptor in respiration: 2NO3 + 12H+ + 10e + N2 + 6H2O.
  4. Assimilation. Plants assimilate nitrogen in the form of nitrate and (rarely) ammonium. The nitrate is first reduced to ammonium, then combined into organic form, generally via glutamate. Animals generally assimilate nitrogen by simply breaking protein down into amino acids and rearranging them.
  5. Decay (ammonification) and excretion. When plants and animals decay, putrefying bacteria produce ammonia from the proteins they contain. Animals also produce breakdown products such as ammonia, urea, allantoin and uric acid from excess dietary nitrogen. These compounds are also targets of ammonification by bacteria.

Nitrogen fixation

Enzymes that fix nitrogen are widely distributed in bacteria.

  • Proteobacteria
    • Azotobacter (aerobic).
    • Klebsiella (facultatively anaerobic).
    • Rhodospirillum (photosynthetic, anaerobic).
  • Gram positives.
    • Clostridium (anaerobic).
    • Frankia (actinomycete).
  • Cyanobacteria
    • Anabaena (photosynthetic).

Some nitrogen-fixing bacteria form mutualisms with plants:

  • Rhizobium + Fabaceae (legumes).
  • Frankia + Alnus (alder tree).
  • Anabaena + Azolla (water fern).

Azolla can be useful as a green manure in rice paddy fields, but its rapid growth in the absence of exogenous fixed nitrogen means it is also a very successful weed in water courses in Africa.

The Rhizobium-legume symbiosis is particularly famous. Legumes (peas and beans in the family Fabaceae, old name Leguminosae) and Rhizobium bacteria form a mutually beneficial symbiosis: Rhizobium gets sugars and anaerobic conditions, and the legume gets fixed nitrogen. Nitrogen fixation takes place in nodules on the plants' roots. Inside the root nodule, the rhizobia differentiate into bacteroids, which may remind you somewhat of mitochondria or chloroplasts.

Nitrogenase is the enzyme that reduces nitrogen to ammonia. It has two components, a reductase, which is a homodimer containing Fe4S4 clusters, which supplies electrons to nitrogenase from pyruvate; and the nitrogenase proper, which is a heterotetramer containing Fe4S4 and a molybdenum-FeS cluster, which supplies electrons and protons to nitrogen.

Nitrogenase - click for Jmol version
Nitrogenase, showing iron, molybdenum and ADP cofactors.

Nitrogenase is very readily destroyed by oxygen within minutes (t½ = 10 min in air). Legumes reduce [O2] in the vicinity of Rhizobium using leghaemoglobin (Km for O2 = 20 nM, 6 times smaller than haemoglobin). Leghaemoglobin is very similar in shape to animal myoglobin, but has a quite different primary structure.

Myoglobin.   Leghaemoglobin.
Myoglobin and leghaemoglobin are only 10% homologous, but have extremely similar tertiary structures.

An alternative strategy is that of the cyanobacterium Anabaena, which differentiates into heterocysts with an oxygen-impermeable cell wall.

The nitrogenase reaction itself is:

N2 + 6e + 6H+ + 12ATP → 2NH3 + 12ADP + 12Pi

This uses a lot of ATP! Furthermore, (like Rubisco), nitrogenase is prone to an additional hydrogenase reaction:

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

This wastes a certain amount of ATP, but is unavoidable, as nitrogenase just isn't specific enough.

Nitrification

Although ammonia can be used by most living organisms, it is very rapidly oxidized to nitrite and nitrate in soil. Ammonia is only stable under anaerobic conditions (such as waterlogged soils). Nitrifying bacteria oxidise ammonia to nitrites and nitrates. Nitrosomonas and Nitrobacter oxidise ammonium to generate energy for carbon fixation.

NH4+ + 1½O2 → NO2+ H2O + 2H+

NO2 + ½O2 → NO3

Denitrification

The nitrogen cycle is completed by denitrifying bacteria, which convert nitrates back to nitrogen gas. They include bacteria like Pseudomonas, which can use nitrate as terminal electron acceptor for respiration.

2NO3 + 12H+ + 10e → N2 + 6H2O

These two processes can be a problem for farmers: fertilisers are often based on ammonium salts, which bind well to negatively-charged soil particles. However, activity of nitrifying bacteria oxidise the ammonium to nitrate, which is much more readily leached from soil. Denitrifying bacteria worsen this problem by exhaling even this nitrate back into the air as (di)nitrogen gas.

Assimilation

If plants can't get nitrogen, most die from chlorosis. Bogs are very poor in nutrients. Carnivorous plants can get nitrogen from animal protein instead.

Nitrate anion uptake.

Nitrates get into most plants by a proton-nitrate symport, powered by a V-type ATPase. Once inside, nitrate is converted by cytoplasmic nitrate reductase.

NO3 + 2e + 2H+ → NO2 + H2O

Nitrate reductase is a homodimer containing FAD, haem and Mo. It is supplied with electrons from NAD(P)H. Nitrate reductase can also reduce chlorate to toxic chlorite (hence sodium chlorite's use as a weedkiller).

Nitrite is rapidly translocated into plastids, as it is very toxic. Plastids contain nitrite reductase.

NO2 + 6e + 8H+ → NH4+ + 2H2O

The Km for nitrite is very low, at 1 µM, reflecting the toxicity. Nitrite reductase contains Fe4S4 and haem. It is supplied with electrons from ferredoxin.

Once it has been produced, ammonia is assimilated by glutamate synthase into the amino acid glutamine. The NH2 group is then moved onto other carbon skeletons to form other amino acids by transaminases (aminotransferases).

Synthetases (as opposed to synthases) synthesise things using ATP. Glutamine synthetase (GS) makes the amino acid glutamine from glutamate and ammonia. It is the main route by which ammonia enters metabolism in plants.

Glutamine synthetase generates glutamine from glutamate and ammonia.

Glutamate + ammonia → glutamine + water

If we keep doing the GS reaction, we will run out of glutamate rapidly. Glutamate synthase (GOGAT - glutamine 2-oxoglutarate aminotransferase) splits one glutamine molecule into two glutamate molecules. One can be used for further ammonia assimilation. The other is profit for making amino acids with.

Glutamate synthase transfers an amine group from glutamine to alpha-ketoglutarate, generating two molecules of glutamate.

Glutamine + α-ketoglutarate → glutamate + glutamate

Amino acids may be synthesised by reversible transamination of NH2 from glutamate to other carbon skeletons, or by irreversible modification of the carbon skeleton, or both. Transaminases (also termed aminotransferases) can transfer amino (-NH2) groups from amino acids onto α-keto acids like oxaloacetate. They are used to generate synthesise amino acids from Krebs cycle intermediates. They contain pyridoxal phosphate (vitamin B6), which carries the amine group during catalysis (forming a Schiff base).

Pyridoxal phosphate is the cofactor in aminotransferases (transaminases).

Most transaminases have a Keq close to 1. Whether they run backwards or forwards is largely determined by the relative concentrations of substrates, so they can be used both anabolically to make amino acids, and catabolically, to degrade them.

Transaminases swap amine groups between amino acids and α-keto acids.

Glutamate + oxaloacetate → α-ketoglutarate + aspartate

Autotrophs synthesise all their amino acids themselves, but heterotrophs often lack some of the necessary transaminases. This means that they need to get certain essential amino acids from their diet.

Amino acids may be classified according to the parent compounds they are derived from:

  • Ketoglutarate: Glutamine, glutamate, proline, arginine.
  • Oxaloacetate: Aspartate, asparagine, methionine, threonine, lysine, isoleucine
  • Phosphoenol pyruvate and erythrose (shikimate pathway): tryptophan, phenylalanine, tyrosine.
  • Ribose-5-phosphate: histidine
  • 3-phosphoglycerate: serine, glycine, cysteine
  • Pyruvate: alanine, valine, leucine.

A simple example is the synthesis of serine from 3-phosphoglycerate.

Serine synthesis.

  1. Dehydrogenase removes 2H with NAD from PGA to form 3-phospho hydroxypyruvate.
  2. Transaminase adds amine from glutamate: phosphoserine.
  3. Phosphatase removes P group to form serine.

Serine can also be formed from glycine by serine hydroxymethyl transferase. This enzyme uses a methylene-tetrahydrofolate cofactor to modify the carbon skeleton Folate (a B vitamin) and B12 are used to transfer small carbon units (like CH3- and -CH2-) in the C1 metabolic pathways, which is very important for nucleotide synthesis too.

Glutamine synthetase (GS) uses ATP so it is negatively allosterically regulated by many nitrogenous metabolites, including tryptophan, histidine, alanine, glycine and carbamoyl phosphate. It is also regulated by a complex interaction with an adenyl transferase enzyme:

Glutamine synthetase is regulated by adenylation.

When there is a lot of ATP, but little glutamine, adenyl transferase adenylates glutamine synthetase and this activates it to make glutamine from ammonia.

If ATP levels fall, and glutamine is present in excess, uridyl transferase uridylates PII, inactivating adenyl transferase. The adenyl group from GS is cleaved off, and the enzyme becomes inactive.

There are many inherited diseases caused by amino-acid metabolism mutations. Phenylketonuria suffers lack phenylalanine hydroxylase (phe→tyr), so accumulate phenylalanine degradation products, which cause brain damage. Albinism sufferers lack tyrosine monoxygenase, so cannot make melanin from tyrosine.

Nitrogen excretion

Autotrophs make their own nitrogen and rarely need to excrete it. Heterotrophs get their nitrogen from digested protein and nucleic acid, and this is often in excess, so nitrogenous waste is produced and must be excreted. Different animals have different ways of doing this.

Amino acids are deaminated by transaminases, and the carbon skeletons (keto acids, like pyruvate and oxaloacetate) feed back into Krebs.

Amino acid + α-ketoglutarate → keto acid + glutamate.

The nitrogen ends up in glutamate, which is itself deaminated by glutamate dehydrogenase (GDH).

Glutamate + NADP → α-ketoglutarate + ammonia + NADPH

Note that running this backward will assimilate ammonia. Some bacteria can use this pathway to assimilate ammonia rather than excrete it; however, most organisms cannot because the Km is too high, and toxic concentrations of ammonia must be present for it to work.

Ammoniotelic excretion

Ammonia is extremely toxic. Fish can get away with excreting it through their gills because there is a lot of water around to dilute it in.

Ureotelic excretion

Mammals aren't so lucky. They produce the less toxic, highly soluble urea (via carbamoyl phosphate and the urea cycle), which can be excreted in urine.

Urea.

Ammonia is added to hydrogencarbonate to form carbamoyl phosphate by carbamoyl-P synthetase.

Carbamoyl phosphate.

NH3 + HCO3 + 2ATP → NH2CO-Pi + 2ADP.

Carbamoyl-P is an intermediate in nucleotide base synthesis (including uric acid), and also feeds into the urea cycle. The urea cycle is closely linked to the Krebs cycle (and was also discovered by Hans Krebs), via oxaloacetate and fumarate.

Simplified urea cycle.

Urea is costly to produce: it requires 4 ATP per turn. Urea contains two nitrogens, so urea costs 2 ATP per ammonia to excrete, and it also needs lots of water to get rid of it in urine.

Uricotelic excretion

Birds and reptiles aren't even as lucky as mammals, because urea would accumulate in eggs and kill them. They excrete solid uric acid, which wastes no water at all, but is even more expensive to make. It is synthesised from glutamine, aspartate and glycine, via a complex and ATP-costly route.

Uric acid is a purine (like adenine and guanine). Bacterial and insect action on bat faeces (guano) produces many purines, including guanine (this is where the name comes from).

Uric acid.

Base excretion

Excess pyrimidines and purines from food must also be excreted. Pyrimidines can be partly respired (via acetyl-CoA) and the nitrogen lost as ammonia or urea.

Purines are oxidised to uric acid, then (in mammals) one ring is opened to form allantoin, which is lost in urine; however, primates can't do this. If you consume too many purines (e.g. from beer) you get uric acid crystals in your joints, which is the exceedingly painful condition called gout.

Diagrams

You may find these two diagrams useful:

Test yourself

  1. What is the purpose of leghaemoglobin in legume root nodules?
  2. Why can't plants use nitrogen from the air for growth?
  3. Glutamine synthetase is a dodecameric protein. Why should this come as no surprise to you?
  4. Glutamate dehydrogenase is does not use ATP, and can assimilate ammonia. So why is glutamine synthetase used instead by most organisms for this job?
  5. In what ways is nitrogen metabolism linked intimately to Krebs cycle?

Answers

  1. Leghaemoglobin (as the name implies) sequesters oxygen in root nodules. This is important as Rhizobium bacteroids cannot fix nitrogen in the presence of oxygen, because nitrogenase is rapidly destroyed by oxygen.
  2. Nitrogen gas consists of N2 molecules. Although fixation is exergonic (−ve ?G) the triple bond between the N atoms is extremely strong, and the activation energy is enormous. Plants lack a suitable catalyst (nitrogenase), except those in symbiosis with nitrogen-fixing bacteria.
  3. Glutamine synthetase is allosterically regulated, so it must be multimeric.
  4. GDH has a much higher Km for ammonia than GS. Most organisms would die if exposed to a sufficient ammonia concentrations to make GDH run at a significant rate.
  5. Links between urea and Krebs cycle.
    • Assimilation: α-ketoglutarate supplied by Krebs to glutamate synthase.
    • Transamination: keto acids supplies by Krebs to transaminases.
    • Degradation: urea cycle requires aspartate (from oxaloacetate) and produces fumarate. These are Krebs intermediates.

Bibliography

  • Berg, J. M., Tymoczko, J. L. and Stryer, L. (2006). Biochemistry. 6th edition. W. H. Freeman and Company, New York. 649-678. "Protein turnover and amino acid catabolism".
  • Berg, J. M., Tymoczko, J. L. and Stryer, L. (2006). Biochemistry. 6th edition. W. H. Freeman and Company, New York. 679-708. "The biosynthesis of amino acids".
  • Madigan, M. T. and Martinko, J. M. (2006). Biology of microorganisms. 11th edition. Pearson Prentice Hall, Upper Saddle River, New Jersey, USA. 555-557. "Nitrification and anammox".
  • Madigan, M. T. and Martinko, J. M. (2006). Biology of microorganisms. 11th edition. Pearson Prentice Hall, Upper Saddle River, New Jersey, USA. 558-560. "Nitrate reduction and denitrification".
  • Taiz, L. and Zeiger, E. (2006). Plant Physiology. 4th edition. Sinauer Associates Incorporated, Sunderland, Massachusetts. 289-313. "Assimilation of mineral nutrients".

(转载自:http://www.steve.gb.com/science/nitrogen_metabolism.html#diagrams



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