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

• Peptide backbone in proteins

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

• Nucleobases in DNA and RNA

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 • Found in several cofactors (NAD, FAD, Biotin)

• Found in many small hormones (epinephrine)

• Found in many neurotransmitters (serotonin)

• Found in many pigments (chlorophyll)

• 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.

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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.
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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), membrane-
bound 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

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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.

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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 FAD-
containing diaphorase and a 95-kDa catalytic subunit with MGD cofactor and a putative N-
terminal [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
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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 nitrate-
respiring 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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WH Freeman and Company(2008) Lehninger Principles of Biochemistry 5th Edition

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