<p>Once NO₃⁻ is internalised, it is then reduced to NO₂⁻ by assimilatory nitrate reductase (Nas). This enzyme is NADH-dependent and has two subunits: a large catalytic subunit, which contains the essential active site for the reduction of NO₃⁻, and a small NADH oxidoreductase subunit, which facilitates the transfer of electrons to the active site (Lin & Stewart, 1997; Moreno-Vivián & Flores, 2007). In the following step, NO₂⁻ is further reduced to NH₄⁺ by the monomeric nitrite reductase (Nir). Afterwards, the produced NH₄⁺ is incorporated into amino acids, specifically glutamine and glutamate, through the GS-GOGAT and GDH pathways (Moreno-Vivián & Flores, 2007; van Heeswijk et al., 2013).</p>
<p>The GS-GOGAT pathway consists of two key steps. First, the enzyme glutamine synthetase (GS) catalyses an ATP-dependent reaction that converts glutamate to glutamine by incorporating an ammonium ion. Following this, glutamate synthase (GOGAT) transfers the amide group from glutamine to 2-oxoglutarate, resulting in the production of two glutamate molecules. In contrast, the GDH pathway employs a more direct approach. The enzyme glutamate dehydrogenase (GDH) catalyses the incorporation of an ammonium ion (NH₄⁺) directly into 2-oxoglutarate, forming glutamate in a single step. The resulting amino acids, glutamate and glutamine, undergo further transamidation and transamination, yielding various amino acids, which then serve as building blocks for the biosynthesis of proteins during translation (van Heeswijk et al., 2013).</p>
Figure X. The pathways that result in the biosynthesis of glutamine and glutamate. The GDH pathway is shown in the left panel. The GS-GOGAT pathway is shown in the right panel.
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