1ofe: Difference between revisions

Glutamate synthases (GltS) are crucial enzymes in ammonia assimilation in, plants and bacteria, where they catalyze the formation of two molecules of, L-glutamate from L-glutamine and 2-oxoglutarate. The plant-type, ferredoxin-dependent GltS and the functionally homologous alpha subunit of, the bacterial NADPH-dependent GltS are complex four-domain monomeric, enzymes of 140-165 kDa belonging to the NH(2)-terminal nucleophile family, of amidotransferases. The enzymes function through the channeling of, ammonia from the N-terminal amidotransferase domain to the FMN-binding, domain. Here, we report the X-ray structure of the Synechocystis, ferredoxin-dependent GltS with the substrate 2-oxoglutarate and the, covalent inhibitor 5-oxo-L-norleucine bound in their physically distinct, active sites solved using a new crystal form. The covalent, Cys1-5-oxo-L-norleucine adduct mimics the glutamyl-thioester intermediate, formed during L-glutamine hydrolysis. Moreover, we determined a high, resolution structure of the GltS:2-oxoglutarate complex. These structures, represent the enzyme in the active conformation. By comparing these, structures with that of GltS alpha subunit and of related enzymes we, propose a mechanism for enzyme self-regulation and ammonia channeling, between the active sites. X-ray small-angle scattering experiments were, performed on solutions containing GltS and its physiological electron, donor ferredoxin (Fd). Using the structure of GltS and the newly, determined crystal structure of Synechocystis Fd, the scattering, experiments clearly showed that GltS forms an equimolar (1:1) complex with, Fd. A fundamental consequence of this result is that two Fd molecules bind, consecutively to Fd-GltS to yield the reduced FMN cofactor during, catalysis.

1OFE is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Synechocystis_sp. Synechocystis sp.] with F3S, FMN, AKG and ONL as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Glutamate_synthase_(ferredoxin) Glutamate synthase (ferredoxin)], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.4.7.1 1.4.7.1] Structure known Active Site: AC1. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1OFE OCA].  

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