title: Quaternary structure of Azospirillum brasilense NADPH-dependent glutamate synthase in solution as revealed by synchrotron radiation x-ray scattering.
abstract: Azospirillum brasilense glutamate synthase (GltS) is the prototype of bacterial NADPH-dependent enzymes, a class of complex iron-sulfur flavoproteins essential in ammonia assimilation processes. The catalytically active GltS alpha beta holoenzyme and its isolated alpha and beta subunits (162 and 52 kDa, respectively) were analyzed using synchrotron radiation x-ray solution scattering. The GltS alpha subunit and alpha beta holoenzyme were found to be tetrameric in solution, whereas the beta subunit was a mixture of monomers and dimers. Ab initio low resolution shapes restored from the scattering data suggested that the arrangement of alpha subunits in the (alpha beta)4 holoenzyme is similar to that in the tetrameric alpha 4 complex and that beta subunits occupy the periphery of the holoenzyme. The structure of alpha 4 was further modeled using the available crystallographic coordinates of the monomeric alpha subunit assuming P222 symmetry. To model the entire alpha beta holoenzyme, a putative alpha beta protomer was constructed from the coordinates of the alpha subunit and those of the N-terminal region of porcine dihydropyrimidine dehydrogenase, which is similar to the beta subunit. Rigid body refinement yielded a model of GltS with an arrangement of alpha subunits similar to that in alpha 4, but displaying contacts also between beta subunits belonging to adjacent protomers. The holoenzyme model allows for independent catalytic activity of the alpha beta protomers, which is consistent with the available biochemical evidence.
authors.affiliation: European Molecular Biology Laboratory, Hamburg Outstation, Notkestrasse 85, D-22603 Hamburg, Germany.
authors.forename: Maxim V
authors.initials: MV
authors.lastname: Petoukhov
authors.forename: Dmitri I
authors.initials: DI
authors.lastname: Svergun
authors.forename: Peter V
authors.initials: PV
authors.lastname: Konarev
authors.forename: Sergio
authors.initials: S
authors.lastname: Ravasio
authors.forename: Robert H H
authors.initials: RH
authors.lastname: van den Heuvel
authors.forename: Bruno
authors.initials: B
authors.lastname: Curti
authors.forename: Maria A
authors.initials: MA
authors.lastname: Vanoni
mesh_terms: D000818:Animals; D015806:Azospirillum brasilense / Q000201:enzymology*; D002384:Catalysis; D018360:Crystallography, X-Ray; D042943:Dihydrouracil Dehydrogenase (NADP); D019281:Dimerization; D005970:Glutamate Synthase / Q000737:chemistry*; D008954:Models, Biological; D008958:Models, Molecular; D009249:NADP / Q000737:chemistry*; D010088:Oxidoreductases / Q000737:chemistry; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D013552:Swine; D017356:Synchrotrons; D014965:X-Rays
chemical_list: D009249:NADP; D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP); D005970:Glutamate Synthase