Nitrogen Metabolism

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Nitrogen Cycle

Nitrogen metabolism is the complex process of how organisms convert and utilize nitrogen. The nitrogen cycle describes the exchange of nitrogen between three main pools: the atmosphere, soil and groundwater, and biomass.

The nitrogen cycle involves several key processes:

Nitrogen Fixation: This is the conversion of atmospheric nitrogen gas (N2​) into a usable form like ammonia (NH3​). It can occur through biological, industrial, or electrical means.

Ammonification: In this decomposition process, microorganisms convert organic nitrogen from decaying biomass into ammonia (NH3​) and ammonium ions (NH4+​). Example: Bacillus vulgaris and Bacillus ramosus

Nitrification: This two-step process converts ammonia to nitrate (NO3−​). First, bacteria like Nitrosomonas or Nitrococcus oxidize ammonia to nitrite (NO2−​). Next, Nitrobacter oxidizes the nitrite to nitrate. These bacteria are chemoautotrophs and are known as nitrifying bacteria.

Denitrification: This process converts nitrates and nitrites back into dinitrogen gas (N2​) and is carried out by microorganisms called denitrifiers, such as Thiobacillus denitrificans.

Nitrate Assimilation: Plants actively absorb nitrate from the soil and convert it into organic nitrogen compounds.

Nitrate Assimilation in Plants

Nitrate assimilation is a two-step process in plants:

Step 1: Reduction of Nitrate to Nitrite 

This reaction occurs in the cytosol and is catalyzed by the enzyme nitrate reductase (NR). It involves the transfer of two electrons from an electron donor, typically NADH in most cases or NADPH in nongreen tissues.

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

• flavin adenine dinucleotide (FAD)
• heme
• a molybdenum ion complexed to a pterin molecule

FAD accepts two electrons from NAD(P)H, which are then passed to the heme domain and finally to the molybdenum complex to be transferred to nitrate. Molybdenum is crucial for this enzyme's function, and a deficiency in it can lead to nitrate accumulation.

Regulation of Nitrate Reductase

NR synthesis is regulated at transcriptional and translational levels by several factors, including nitrate, light, and carbohydrate accumulation. The enzyme is also subject to posttranslational modification through reversible phosphorylation.

• Activation: Light, carbohydrates, and other environmental factors stimulate a protein phosphatase that dephosphorylates a key serine residue on the hinge 1 region of NR enzyme, activating it.

• Deactivation: In darkness, a protein kinase stimulated by Mg2+ phosphorylates the same serine residue. This phosphorylation allows the enzyme to interact with a 14-3-3 inhibitor protein, rendering it inactive.

This phosphorylation-based regulation provides a rapid way to control NR activity, happening in minutes compared to the hours it takes for enzyme synthesis or degradation.

Step 2: Reduction of Nitrite to Ammonium 

Nitrite is a highly reactive and potentially toxic ion, so it's immediately transported from the cytosol to chloroplasts or plastids where it is reduced to ammonium (NH4+​) by the enzyme nitrite reductase (NiR). This process requires the transfer of six electrons. The electrons are donated by reduced ferredoxin (Fred​). 

NiR consists of a single polypeptide with two prosthetic groups: 

• an iron-sulfur cluster (Fe4​S4​) 
• a specialized heme

Electrons flow from ferredoxin, through the iron-sulfur cluster and heme, to nitrite. Reduced ferredoxin is derived from the photosynthetic electron transport chain or from NADPH generated in oxidative pentose phosphate pathway in nongreen tissue.

Regulation of Nitrite Reductase

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

• Deactivation: The accumulation of end products, such as asparagine and glutamine, represses this induction.

Ammonia Assimilation

Plant cells quickly convert the ammonium generated from nitrate assimilation into amino acids to prevent toxicity. The main pathway for this conversion is the GS-GOGAT pathway, which involves two key enzymes: 

• glutamine synthetase (GS) 
• glutamate synthase (GOGAT)

Step 1: Glutamine Synthetase (GS) 

GS combines ammonium with glutamate to form glutamine. This reaction requires ATP and a divalent cation like Mg2+, Mn2+,or Co2+ as a cofactor.

Plants have three classes of GS with different functions and locations:

• Cytosolic GS: Found in germinating seeds and vascular bundles, producing glutamine for intercellular nitrogen transport. Its expression isn't regulated by light or carbohydrates.

• Plastidal GS: Located in roots, generating amide nitrogen for local consumption. Its expression is regulated by light and carbohydrate levels.

• Chloroplastic GS: Found in shoots, where it reassimilates photorespiratory ammonium (NH4+​). Its expression is also regulated by light and carbohydrate levels.

Step 2: Glutamate Synthase (GOGAT) 

GOGAT transfers the amide group from glutamine to 2-oxoglutarate, producing two molecules of glutamate. There are two types of GOGAT in plants:

• NADH-GOGAT: Accepts electrons from NADH and is found in the plastids of non-photosynthetic tissues like roots or vascular bundles. It assimilates ammonium absorbed from the soil or glutamine translocated from roots or senescing leaves.

• Fd-GOGAT: Accepts electrons from ferredoxin and is found in chloroplasts, where it functions in photorespiratory nitrogen metabolism.

Overall reaction in GS-GOGAT pathway

Alternate Ammonia Assimilation Pathways

Plants can also assimilate ammonia through several alternative pathways.

Glutamate Dehydrogenase (GDH)

This enzyme catalyzes a reversible reaction that can either synthesize or deaminate glutamate. An NADH-dependent form is found in mitochondria, while an NADPH-dependent form is in the chloroplasts of photosynthetic organs.

Transamination

This process incorporates nitrogen from glutamine and glutamate into other amino acids. It involves the transfer of an amino group from one amino acid to a keto acid, producing a new amino acid and a new keto acid. This is a reversible process requiring pyridoxal phosphate (vitamin B6) as a cofactor. 

Examples:

Asparagine Synthetase (AS)

AS transfers the amide nitrogen from glutamine to aspartate, producing asparagine and glutamate. It is found in the cytosol of leaves, roots, and nitrogen-fixing nodules.

Regulation of Ammonium Metabolism

• The regulation of ammonium metabolism is vital for maintaining the nitrogen-to-carbon (N:C) ratio in plants.

• High light and carbohydrate levels: Under these conditions, AS is inhibited, and plastid GS and Fd-GOGAT are activated. This favors the production of carbon-rich compounds like glutamine and glutamate, which are used for synthesizing new plant materials.

• Energy-limited conditions: In these situations, the expression of plastid GS and Fd-GOGAT is reduced, and AS is activated. This leads to more asparagine, an amide that helps in long-distance transport or long-term nitrogen storage.

Amino acid biosynthesis

Humans and most animals cannot synthesize certain essential amino acids and must obtain them from their diet. In contrast, plants can synthesize all 20 standard amino acids. The nitrogen-containing amino group is derived from transamination reactions with glutamine or glutamate. The carbon skeletons for amino acids are derived from intermediates of glycolysis (3-phosphoglycerate, phosphoenolpyruvate, or pyruvate) or the citric acid cycle (2-oxoglutarate or oxaloacetate).