Dr. Riddhi Datta

• Global nitrogen supply is generally distributed between 3 major pools:
• Atmospheric pool
• Soil and associated ground water pool
• Nitrogen contained within biomass
• The complex pattern of nitrogen exchange between thee 3 pools is known as nitrogen cycle.

Nitrogen cycle

Atmospheric nitrogen

Biological nitrogen fixation

Industrial nitrogen fixation

Electrical nitrogen fixation

Denitrification

Ammonia Nitrite Nitrate

Soil nitrogen pool

Ammonification

Uptake

Decaying biomass

Plant biomass

Animal biomass

Death

Death

Food

Dr. Riddhi Datta

• Ammonification: In the process of decomposition, organic nitrogen is converted to ammonia by a variety of microorganisms. This process of release of ammonia and its formation of ammonium ions is known as ammonification.
• Example: Bacillus vulgaris, Bacillus ramosus

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• Nitrification: The process of converting ammonia to nitrate by a variety of microorganisms is known as nitrification. The first step is the oxidation of ammonia to nitrite by Nitrosomonas or Nitrococcus bacteria. Nitrite is then further oxidized to nitrate by Nitrobacter. These two groups of bacteria are chemoautotrophs and are called nitrifying bacteria.

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• Denitrification: The process which involves conversion of nitrates and nitrites to dinitrogen by a group of microorganisms (called denitrifiers) is called denitrification.
• Example: Thiobacillus denitrificans

Dr. Riddhi Datta

Nitrate Assimilation

Plant roots actively absorb nitrate from the soil solution via several low- and high-affinity nitrate–proton co-transporters. Plants eventually assimilate most of this nitrate into organic nitrogen compounds.

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Step 1:

• Reduction of nitrate to nitrite in the cytosol that involves the transfer of two electrons.
• Nitrate reductase (NR), a complex metalloenzyme, catalyzes this reaction:

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NO₃^- + NAD(P)H + H⁺ + 2e^- NO₂^- + NAD(P)⁺ + H₂O

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• Electron donor in the reaction:
• Most cases NADH
• In nongreen tissues NADH or NADPH

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Step 2:

• Reduction of nitrite to ammonia in the chloroplast (or plastid) that involves transfer of six electrons.
• Nitrite reductase (NiR) catalyzes the reaction:

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NO₂^- + 6 Fd_red + 8H⁺ + 6e^- NH₄⁺ + 6Fd_ox + 2H₂O

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• Electron donor in the reaction:
• Reduced ferredoxin

Dr. Riddhi Datta

The nitrate reductases of higher plants are composed of two identical subunits, each containing three prosthetic groups:

• flavin adenine dinucleotide (FAD),
• heme,
• a molybdenum ion complexed to an organic molecule called a pterin.

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Nitrate reductase is the main molybdenum-containing protein in vegetative tissues.

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One symptom of molybdenum deficiency is the accumulation of nitrate that results from diminished nitrate reductase activity.

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FAD binding domain accepts 2 electrons from NAD(P)H, passes them to heme domain and then to the molybdenum complex where they are transferred to nitrate.

Nitrate reductase

Dr. Riddhi Datta

• NR activity coordinates N and C assimilation.
• NR synthesis is regulated at the transcription and translation levels by:
• Nitrate
• Light
• Carbohydrates
• NR is subject to posttranslational modification involving a reversible phosphorylation

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• Light, carbohydrate levels, and other environmental factors stimulate a protein phosphatase that dephosphorylates a key serine residue in the hinge 1 region of NR and activates the enzyme.

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• Darkness and Mg²⁺ stimulate a protein kinase that phosphorylates the same serine residues, which then interact with a 14-3-3 inhibitor protein, and thereby inactivate NR.

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• Regulation of NR activity through phosphorylation and dephosphorylation provides rapid control than through synthesis or degradation of the enzyme (minutes versus hours).

Regulation of Nitrate reductase

Dr. Riddhi Datta

Regulation of Nitrate reductase

Dr. Riddhi Datta

Nitrite reductase

• Nitrite is a highly reactive, potentially toxic ion and is immediately transported from the cytosol into chloroplasts (or plastids). There nitrite reductase reduces nitrite to ammonium.

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• NiR consists of a single polypeptide containing two prosthetic groups:
• an iron–sulfur cluster (Fe₄S₄)
• a specialized heme

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• The electrons flow from ferredoxin, through Fe₄S₄ and heme, finally to nitrite.

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• A small percentage (0.02–0.2%) of the nitrite reduced is released as nitrous oxide (N₂O).

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• Reduced ferredoxin is derived from photosynthetic electron transport in the chloroplasts and from NADPH generated by the oxidative pentose phosphate pathway in nongreen tissues.

Dr. Riddhi Datta

Regulation of nitrite reductase

• NiR is encoded in the nucleus and synthesized in the cytoplasm with an N-terminal transit peptide that targets it to the plastids.

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• Activation: High nitrate or exposure to light induce the transcription of NiR mRNA.

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• Deactivation: Accumulation of the end products—asparagine and glutamine—represses this induction.

Dr. Riddhi Datta

• Plant cells avoid ammonium toxicity by rapidly converting the ammonium generated from nitrate assimilation or photorespiration into amino acids.

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• The primary pathway for this conversion involves the sequential actions of:
• glutamine synthetase
• glutamate synthase

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Step 1: Glutamine synthetase (GS)

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• Glutamine synthetase (GS) combines ammonium with glutamate to form glutamine:

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Glutamate + NH₄⁺ + ATP → glutamine + ADP + Pi

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• Involves a divalent cation such as Mg²⁺, Mn²⁺, or Co²⁺ as a cofactor

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Ammonia assimilation

Dr. Riddhi Datta

Plants contain three classes of GS:

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• Cytosolic GS
• Expressed in germinating seeds or vascular bundles
• Produce glutamine for intercellular nitrogen transport
• Light and carbohydrate levels have no regulatory effect

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• Pastidal GS
• Expressed in roots
• Generates amide nitrogen for local consumption
• Light and carbohydrate levels regulate expression

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• Chloroplastic GS
• Expressed in shoot
• Reassimilates photorespiratory NH₄⁺
• Light and carbohydrate levels regulate expression

Ammonia assimilation

Dr. Riddhi Datta

Ammonia assimilation

Step 2: Glutamate synthase (or glutamine:2-oxoglutarate aminotransferase, or GOGAT)

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• GOGAT transfers the amide group of glutamine to 2-oxoglutarate, yielding two molecules of glutamate

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• Plants contain two types of GOGAT:

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• NADH-GOGAT
• Accepts electrons from NADH
• Located in plastids of non-photosynthetic tissues such as roots or the vascular bundles of developing leaves
• Assimilates of NH₄⁺ absorbed from the rhizosphere or glutamine translocated from roots or senescing leaves

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Glutamine + 2-oxoglutarate + NADH + H⁺ → 2 glutamate + NAD⁺

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• Fd-GOGAT
• Accepts electrons from ferredoxin
• Found in chloroplasts
• Serves in photorespiratory nitrogen metabolism

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Glutamine + 2-oxoglutarate + Fd_red → 2 glutamate + Fd_ox

Dr. Riddhi Datta

The GS-GOGAT pathway that forms glutamine and glutamate. A reduced cofactor is required for the reaction:

• ferredoxin (Fd) in green leaves
• NADH in nonphotosynthetic tissue

Dr. Riddhi Datta

Ammonia assimilation via alternate pathway

GDH (Glutamate dehydrogenase)

• Catalyzes a reversible reaction that synthesizes or deaminates glutamate.

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• An NADH-dependent form of GDH is found in mitochondria, and an NADPH-dependent form is localized in the chloroplasts of photosynthetic organs.

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Dr. Riddhi Datta

Ammonia assimilation via alternate pathway

Transamination

• Once assimilated into glutamine and glutamate, nitrogen is incorporated into other amino acids via transamination reactions that are catalyzed by aminotransferases.

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• Involves transfer of an amino group from one amino acid (except lysine, proline, and threonine) to a keto acid (without an amine group), producing a new amino acid and a corresponding new keto acid.

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• It is thus a reversible amination and deamination.

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• All transamination reactions require pyridoxal phosphate (vitamin B6) as a cofactor.

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Dr. Riddhi Datta

Ammonia assimilation via alternate pathway

Transamination

• Transamination occurs in the mitochondria and cytoplasm of eukaryotic cells

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Dr. Riddhi Datta

Ammonia assimilation via alternate pathway

AS (Asparagine synthetase)

• AS transfer of the amide nitrogen from glutamine to aspartate.

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• found in the cytosol of leaves and roots and in nitrogen-fixing nodules

Ammonia assimilation via alternate pathway

AS (Asparagine synthetase)

Regulation of ammonium metabolism: critical for maintaining N:C ratio