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 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.
van den Heuvel
Robert H H
RH
Department of Genetics and Microbiology, University of Pavia, via Abbiategrasso 207, Italy.
Svergun
Dmitri I
DI
Petoukhov
Maxim V
MV
Coda
Alessandro
A
Curti
Bruno
B
Ravasio
Sergio
S
Vanoni
Maria A
MA
Mattevi
Andrea
A
D018360:Crystallography, X-Ray; D000458:Cyanobacteria / Q000737:chemistry; D003980:Diazooxonorleucine / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005288:Ferredoxins / Q000737:chemistry* / Q000378:metabolism*; D005970:Glutamate Synthase / Q000737:chemistry* / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D012542:Scattering, Radiation
D004791:Enzyme Inhibitors; D005288:Ferredoxins; D007656:Ketoglutaric Acids; D000644:Quaternary Ammonium Compounds; D003980:Diazooxonorleucine; D005970:Glutamate Synthase