InterPro domain: IPR012220

This group represents the eukaryotic type of glutamate synthase (NADH-GOGAT, 1.4.1.14 ). This pyridine-linked form is found in both photosynthetic and nonphotosynthetic eukaryotes. It displays a single-subunit structure corresponding to the fusion of the small and the large bacterial subunits.

Glutamate synthase (GOGAT, GltS) is a complex iron-sulphur flavoprotein that catalyses the reductive synthesis of L-glutamate from 2-oxoglutarate and L-glutamine via intramolecular channelling of ammonia, a reaction in the bacterial, yeast and plant pathways for ammonia assimilation [ 1 ]. GOGAT is a multifunctional enzyme that functions through three distinct active centres carrying out multiple reaction steps: L-glutamine hydrolysis, conversion of 2-oxoglutarate into L-glutamate, and electron uptake from an electron donor [ 2 ]. The small subunit functions as a FAD-dependent NADPH oxidoreductase, which serves to transfer reducing equivalents to the site of glutamate synthesis on the large subunit through the enzyme [3Fe-4S] cluster (on the large subunit) and at least one of its [4Fe-4S] centres [ 3 , 4 ]. The large subunit contains the GltS L-glutamine amidotransferase (GAT) site where L-Gln binds and is hydrolysed to yield L-Glu and ammonia. The latter is transferred through the intramolecular ammonia tunnel [ 5 ] to the glutamate synthase site where 2-OG binds, is converted to the iminoglutarate (2-IG) intermediate, and reduced to L-Glu by receiving reducing equivalents from the reduced FMN cofactor at this site [ 5 ].

There are four classes of GOGAT [ 6 , 6 ]:

1. Bacterial NADPH-dependent GOGAT (NADPH-GOGAT, 1.4.1.13 ). This standard bacterial NADPH-GOGAT is composed of a large (alpha, GltB) subunit and a small (beta, GltD) subunit.

2. Ferredoxin-dependent form in cyanobacteria and plants (Fd-GOGAT from photosynthetic cells, 1.4.7.1 ) displays a single-subunit structure corresponding to the large bacterial subunit.

3. Pyridine-linked form in both photosynthetic and nonphotosynthetic eukaryotes (eukaryotic GOGAT or NADH-GOGAT, 1.4.1.14 ) displays a single-subunit structure corresponding to the fusion of the small and the large bacterial subunits.

4. The archaeal type with stand-alone proteins corresponding to the N-terminal, FMN-binding, and the C-terminal domains of the large subunit [ 7 , 7 ] ( IPR012375 , IPR012061 ), and to the small subunit.

The large subunit of GOGAT consists of three domains: N-terminal domain (amidotransferase domain IPR017932 ); central (consisting of IPR006982 and the FMN-binding domain IPR002932 ), and the C-terminal domain ( IPR002489 ).

The N-terminal amidotransferase domain is characterised by a four layer alpha/beta/beta/alpha architecture and is similar to other Ntn-amidotransferases [ 7 ]. It contains the typical catalytic centre of Ntn-amidotransferases, and the N-terminal Cys-1 catalyses the hydrolysis of L-glutamine generating ammonia and the first molecule of L-glutamate [ 7 ].

The second (central) domain consists of IPR006982 and IPR002932 . IPR006982 connects the amidotransferase domain with the FMN-binding domain and has an alpha/beta overall topology [ 7 ]. The FMN-binding domain ( IPR002932 ) has a classic beta/alpha barrel topology. In this domain, the 2-iminoglutarate intermediate, formed upon the addition of ammonia onto 2-oxoglutarate, is reduced by the FMN cofactor producing the second molecule of L-glutamate [ 7 ]. This domain also contains the enzyme 3Fe-4S cluster [ 7 ].

The C-terminal, or GXGXG structural domain, has a right-handed beta-helix topology composing seven beta-helical turns. This domain does not have a direct function in glutamate synthase activity but rather a structural function through extensive interactions with the amidotransferase and FMN-binding domains [ 7 , 7 ].

The structural data combined with the catalytic properties of GltS indicate that binding of ferredoxin and 2-oxoglutarate to the FMN-binding domain of GltS induce a conformational change in the loop connecting the two catalytic centres. The rearrangement induces a shift in the catalytic elements of the amidotransferase domain, such that it becomes activated [ 7 ].

For additional information please see [ 7 ].

1. Cross-talk and ammonia channeling between active centers in the unexpected domain arrangement of glutamate synthase. Structure 8, 1299-308
2. Structural studies on the synchronization of catalytic centers in glutamate synthase. J. Biol. Chem. 277, 24579-83
3. Glutamate synthase: a complex iron-sulfur flavoprotein. Cell. Mol. Life Sci. 55, 617-38
4. Evolutionary analyses of the small subunit of glutamate synthase: gene order conservation, gene fusions, and prokaryote-to-eukaryote lateral gene transfers. Eukaryotic Cell 1, 304-10
5. Properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803. Comparison with the Azospirillum brasilense NADPH-dependent enzyme and its isolated alpha subunit. Biochemistry 41, 8120-33
6. Phylogenetic analyses of two "archaeal" genes in thermotoga maritima reveal multiple transfers between archaea and bacteria. Mol. Biol. Evol. 18, 362-75
7. Saccharomyces cerevisiae has a single glutamate synthase gene coding for a plant-like high-molecular-weight polypeptide. J. Bacteriol. 177, 792-8