The active conformation of glutamate synthase and its binding to ferredoxin

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 at subunit of the bacterial NADPH-dependent GltS are complex four-domain monomeric enzymes of 140-165 kDa. belonging to the NH2-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 Cysl-5-oxo-L-norleucine adduct mimics the glutamyl-thioester intermediate formed during L-glutamine hydrolysis. Moreover, we determined a high resolution structure of the G;tS: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. (C) 2003 Elsevier Science Ltd. All rights reserved.