Biochemistry_of_ammonia_assimilation (1).doc

A TERM PAPER ON BIOCHEMISTRY OF AMMONIA ASSIMILATION SOIL MICROBIOLOGY (MIC 703)

LECTURER IN CHARGE: DR ADELOWO DEPRTMENT OF MICROBIOLOGY, UNIVERSITY OF IBADAN

COMPILED BY

NAME: JIMOH ABDULLAHI ADEKILEKUN MATRIC NO: 172970 DEPARTMENT: MICROBIOLOGY

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INTRODUCTION Nitrogen assimilation Nitrogen assimilation is the formation of organic nitrogen compounds like amino acids from inorganic nitrogen compounds present in the environment. Organisms like plants, fungi and certain bacteria that cannot fix nitrogen gas (N2) depend on the ability to assimilate nitrate or ammonia for their needs. Other organisms, like animals, depend solely on organic nitrogen from their food. Nitrate in the natural environment is relatively rare. Microbes capable of using alternative nitrogen sources have an advantage and a subset of microbes is capable of obtaining the nitrogen they need from nitrogen gas. Nitrogen gas makes up about 79% of our atmosphere and is easily available. Molecular nitrogen is a stable unreactive gas with a triple bond between the two atoms and the reduction of it to ammonia is an energy expensive process. A large amount of ATP, protons and electrons are required to reduce just one molecule of nitrogen gas. N2 + 8H+ + 8e- + 16ATP

2 NH3 + H2 + 16ADP + 16Pi

Figure 2 - Chemical equation for the reduction of nitrogen gas to ammonia by nitrogenase.

Importance of Nitrogen •

Nitrogen, carbon, hydrogen and oxygen are the main elemental constituents of living organisms

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Peptide backbone in proteins

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Functional side chains (His, Lys, Arg, Trp, Asn, Gln) in proteins

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Nucleobases in DNA and RNA 2

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Found in several cofactors (NAD, FAD, Biotin)

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Found in many small hormones (epinephrine)

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Found in many neurotransmitters (serotonin)

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Found in many pigments (chlorophyll)

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Found in many defense chemicals (amanitin)

NITROGEN CYCLE

Ammonia assimilation in plants Plants absorb nitrogen from the soil in the form of nitrate (NO3-) and ammonia (NH3). In aerobic soils where nitrification can occur, nitrate is usually the predominant form of available nitrogen that is absorbed. However this need not always be the case in grasslands and in flooded, anaerobic soils like rice paddies where ammonia can predominate. Plant roots themselves can affect the abundance of various forms of nitrogen by changing the pH, secreting organic compounds or oxygen. This influences microbial activities like the inter-conversion of various nitrogen species, nitrogen fixation by non-nodule forming bacteria and the release of ammonia from organic matter in the soil. 3

Ammonium ions are absorbed by the plant via ammonia transporters. Nitrate is taken up by several nitrate transporters that use a proton gradient to power the transport. Within the root a small fraction of the nitrate is reduced to ammonia while the rest is usually transported via the xylem to the shoots. However, in some plants, the root can be the major site of nitrate reduction. Ammonia that is absorbed or formed from the reduction of nitrate is incorporated into amino acids via the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway. While nearly all] the ammonia is usually incorporated into amino acids at the root itself, plants may transport significant amounts of ammonium ions in the xylem to be fixed in the shoots. This may help avoid the transport of organic compounds down to the roots just to carry the nitrogen back as amino acids

Nitrate reduction Nitrate reduction is carried out in two steps. Nitrate is first reduced to nitrite (NO2-) in the cytosol by nitrate reductase using NADH or NADPH. Nitrite is then reduced to ammonia in the chloroplasts (plastids in roots) by a ferredoxin dependent nitrite reductase. In photosynthesizing tissues, it uses an isoform of ferredoxin (Fd1) that is reduced by PSI while in the root it uses a form of ferredoxin (Fd3) that has a less negetive midpoint potential and can be reduced easily by NADPH. In non photosynthesizing tissues, NADPH is generated by glycolysis and the pentose phosphate pathway. Under anaerobic conditions, nitrate metabolism occurs in several, competing, microbial processes. Nitrate reduction by microorganisms is a major biogeochemical process. The flux through denitrification in the ocean is estimated to be 450 Tg N y-1 .In addition, the anammox pathway accounts for roughly 50% of fixed nitrogen turnover in marine environments. Nitrate concentrations are also an important issue in agricultural fertilizer use and wastewater treatment. Although plants can use nitrate as a nitrogen source, excess nitrate in bodies of water and in drinking water supplies can cause algal blooms and lead to public health problems, respectively.

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The first step of the nitrate degradation pathway is the two electron reduction of nitrate to nitrite, which is accomplished by three classes of nitrate reductases in bacteria and archaea. Assimilatory nitrate reductases (NAS) are usually cytoplasmic and enable microbes to use environmental nitrate as a nitrogen source. Periplasmic nitrate reductases (NAP) perform redox balancing, scavenge nitrate in nitrate-limited environments, and serve in aerobic or anaerobic denitrification. Found in the membrane, respiratory nitrate reductases (NAR) enable the use of nitrate as a terminal electron sink in DNRA and in anaerobic denitrification. Subsequently, nitrite can be reduced directly to ammonia, the most negative oxidation state of nitrogen. This reaction is catalyzed by cytochrome c nitrite reductase (NrfA) or octoheme cytochrome c nitrite reductase in DNRA and by NAD(P)H-dependent nitrite reductase (NirB) in nitrite assimilation. In denitrification, nitrite is reduced to nitric oxide in a reaction catalyzed by copper nitrite reductase (NirK). The resulting nitric oxide is then reduced to nitrous oxide by nitric oxide reductase. Next, nitrous oxide is reduced to N2 by nitrous oxide reductase. Note that the release to the environment of nitrous oxide, a potent greenhouse gas and ozone layer depleter, can occur before the final reduction step to N2. Finally, N2 from denitrification can be reduced to ammonia by nitrogenase in the energy intensive process of nitrogen fixation, catalyzed by nitrogenase. The following is a text-format nitrate (anaerobic) pathway map. One of the organisms that can initiate the pathway is given, but other organisms can also carry out later steps. Follow the links for more information on compounds or reactions.

Nitrate

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Paracoccus denitrificans | nitrate reductase | nitrate reductase (NADH) | (cytochrome) | ferredoxin- | nitrate reductase nitrate reductase | | | cytochrome cd1 V nitrite reductase Nitrite-------------------------------+ Escherichia coli | | V | Nitric oxide | Parococcus Kuenenia | denitrificans stuttgartiensis | / \ | / \ | nitric oxide / \ hydrazine NAD(P)H | reductase / \ synthase nitrite reductase | / \ | V V cytochrome c | Nitrous oxide Hydrazine nitrite reductase | \ / | \ / | \ / | nitrous oxide \ / hydrazine | reductase \ / dehydrogenase | \ / | \ / | V V | Nitrogen | Bradyrhizobium japonicum | | V nitrogenase | Ammonia <------------------------------+

Assimilatory nitrate reductase Assimilatory nitrate reductase is an enzyme of the assimilative metabolism involved in reduction of nitrate to nitrite. The nitrite is immediately reduced to ammonia (probably via hydroxylamine) by the activity of nitrite reductase. The term assimilatory refers to the fact that the product of the enzymatic activity remains in the organism. In this case, the product is ammonia which has an inhibitive effect on assimilatory nitrate reductase, thus ensuring that the organism produces the ammonia according to its requirements.

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Ammonia assimilation is a vital process controlling plant growth and development.

COMPLEXITY OF NITRATE REDUCTION PATHWAYS Nitrogen is a basic element for life because it is a component of the two preeminent biological macromolecules: proteins and nucleic acids. Nitrogen exists in the biosphere in several oxidation states, from N(V) to N(−III). Interconversions of these nitrogen species constitute the global biogeochemical nitrogen cycle, which is sustained by biological processes, with bacteria playing a predominant role. 7

Briefly, inorganic nitrogen is converted to a biologically useful form by dinitrogen fixation or nitrate assimilation and the further incorporation of ammonia into C skeletons. Nitrogen is removed from the environment by both nitrification, the oxidative conversion of ammonia to nitrate, and denitrification, a respiratory process whereby nitrate is successively reduced to nitrite, N oxides (NO and N2O), and dinitrogen (N2). Nitrate reduction plays a key role in the nitrogen cycle and has important agricultural, environmental, and public health implications. Assimilatory nitrate reduction, performed by bacteria, fungi, algae, and higher plants, is one of the most fundamental biological processes, accounting for more than 104 megatons of inorganic nitrogen transformed each year (38). However, there is worldwide concern over the excessive use of fertilizers in agricultural activities, leading to nitrate accumulation in groundwater. Consumption of drinking water with high nitrate levels has been associated with methemoglobinemia and gastric cancer due to endogenous formation of genotoxic N-nitroso compounds by bacteria in the gastrointestinal tract. The main threat to the environment comes from eutrophication of aquatic ecosystems. Nitrogen oxides generated by denitrification are also associated with the greenhouse effect and the depletion of stratospheric ozone. Nitrate reduction can be performed with three different purposes: the utilization of nitrate as a nitrogen source for growth (nitrate assimilation), the generation of metabolic energy by using nitrate as a terminal electron acceptor (nitrate respiration), and the dissipation of excess reducing power for redox balancing (nitrate dissimilation). Four types of nitrate reductases catalyze the two-electron reduction of nitrate to nitrite: the eukaryotic assimilatory nitrate reductases and three distinct bacterial enzymes, comprising the cytoplasmic assimilatory (Nas), membranebound respiratory (Nar), and periplasmic dissimilatory (Nap) nitrate reductases. All eukaryotic and bacterial nitrate reductases contain a molybdenum cofactor at their active sites. The basic structure of the eukaryotic cofactor is molybdopterin, a 6-alkyl pterin derivative with a phosphorylated C4 chain with two thiol groups binding the Mo atom. By contrast, the cofactor found in bacterial nitrate reductases and some molybdoenzymes is thebis-molybdopterin guanine dinucleotide (MGD) form. Nitrite oxidase of nitrifying bacteria also shows nitrate reductase activity. This membrane-bound enzyme, which contains MGD and shows a high sequence similarity to the membrane-bound Nar, catalyzes nitrite oxidation to nitrate to allow 8

chemoautotrophic growth, but it can also catalyze the reverse reaction. As nitrite oxidase is not a proper nitrate reductase, we will not consider it further. Eukaryotic assimilatory nitrate reductases are cytosolic homodimeric enzymes that use pyridine nucleotides as electron donors. Each monomer is composed of a 100- to 120-kDa polypeptide with three prosthetic groups, flavin adenine dinucleotide (FAD), cytochromeb557 , and Mo cofactor, which are located in three functional domains highly conserved among eukaryotic species. The Mo cofactor domain is located at the N-terminal end, the heme region corresponds to the middle domain, and the FAD-NAD(P)H domain is present at the C-terminal end. Structural genes coding for nitrate and nitrite reductases and for high-affinity nitrate and nitrite transporters have been cloned in several eukaryotes (Fig.1). Biochemistry and molecular genetics of eukaryotic nitrate reduction have been investigated intensively during the last decades. However, eukaryotic and prokaryotic assimilatory nitrate reductases share no sequence similarity and have little in common beyond their physiological function.

Fig. 1. 9

Nitrate assimilation pathway in the eukaryotic green alga Chlamydomonas reinhardtii.

BACTERIAL ASSIMILATORY NITRATE REDUCTASES (NAS) Nitrate assimilation has been studied at the biochemical or genetic level in several phototrophic and heterotrophic bacteria. Two classes of assimilatory nitrate reductases are found in bacteria: the ferredoxin- or flavodoxin-dependent Nas and the NADH-dependent enzyme. Both types of Nas contain MGD cofactor and one N-terminal iron-sulfur cluster but are devoid of heme groups, in contrast to eukaryotic and other bacterial nitrate reductases. The cyanobacterial ferredoxin-Nas is a single subunit of 75 to 85 kDa, whereas the flavodoxin-Nas of Azotobacter vinelandii is a polypeptide of 105 kDa. The purified Nas proteins of A. vinelandii and Plectonema boryanum contain one Mo, four Fe, and four acid-labile S atoms per moleculE. Amino acid sequence analysis reveals the presence of a Cys motif in the N-terminal end of the proteins, probably binding one [4Fe-4S] or [3Fe-4S] center. Ferredoxin-Nas is also present in Azotobacter chroococcum, Clostridium perfringens, and Ectothiorhodospira shaposhnikovii. On the other hand, the NADH-Nas proteins of Klebsiella pneumoniae and Rhodobacter capsulatus are heterodimers of a 45-kDa FADcontaining diaphorase and a 95-kDa catalytic subunit with MGD cofactor and a putative Nterminal [4Fe-4S] center. This NADH-Nas, as deduced by the Klebsiella nasA gene sequence, probably contains an additional [2Fe-2S] center linked to a C-terminal Cys cluster that is similar to a sequence of the NifU protein. This region is absent from the ferredoxin-Nas and could act as a ferredoxin-like electron transfer domain. The Bacillus subtilis NADH-Nas does not contain the NifU-like domain in the catalytic subunit but has two tandem NifU-like modules in a central region of the FAD-containing diaphorase.

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Fig. 2.

Nitrate assimilation and comparison of the organization of the nitrate assimilation gene clusters in bacteria. The scheme shows the cyanobacterial ferredoxin (Fd)-dependent assimilatory nitrate and nitrite reductases (right) and the NADH-dependent nitrate and nitrite reductases from Klebsiella and Rhodobacter(left). The organization of the Synechococcus,Synechocystis, and K. oxytoca(pneumoniae) nitrate assimilation gene clusters is shown beneath the corresponding proteins. Genes are drawn approximately to scale, and arrows show the direction of transcription. The genes and their products are shown in the same color. Regulatory genes are indicated by black arrows with white vertical lines. The product of the white gene is not shown. Although all nitrate reductases can use reduced viologens as electron donors, the ability to use bromophenol blue as an artificial reductant is a characteristic of both eukaryotic and prokaryotic assimilatory enzymes. In R. capsulatus, Nas is inhibited by cyanide and azide but is unaffected by cyanate and chlorate. NADH also inactivates Nas under aerobic conditions by formation of 11

superoxide anion at the diaphorase flavin center, and this activity is protected by superoxide dismutase. Organization of the genes coding for the assimilatory nitrate reductases.The genes coding for the assimilatory nitrate-reducing system are normally clustered and have been cloned in several bacterial species. These gene clusters include regulatory and structural genes coding for proteins required for uptake and reduction of both nitrate and nitrite. Nomenclature of these genes is confusing because different names have been given to homologous genes in different bacteria. In our opinion, the nas gene designation inK. pneumoniae is more appropriate. In this bacterium, the nasR gene encoding a transcription antiterminator is linked to the nasFEDCBA operon. The nasFED genes code for a multicomponent nitrate or nitrite transport system, the nasBgene encodes a siroheme-dependent assimilatory nitrite reductase, and the NADH-nitrate reductase is encoded by the nasC(diaphorase) and nasA (catalytic subunit) genes. RESPITATORY MEMBRANE-BOUND NITRATE REDUCTASES (NAR) Structure and biochemical properties of membrane-bound nitrate reductases.Membrane-bound nitrate reductases are associated with denitrification and anaerobic nitrate respiration. Although the most exhaustive biochemistry and genetic studies have been performed in E. coli and Paracoccus denitrificans, Nar enzymes have been purified from several denitrifying and nitraterespiring bacteria. A thermophilic Nar protein with an optimal temperature of 80°C has also been found in Thermus thermophilus. In E. coli, there are two different membrane-bound isoenzymes: NRA, which is expressed under anaerobiosis in the presence of nitrate and represents 90% of total activity, and NRZ, which is expressed constitutively.

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Fig. 3.

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