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 αβ holoenzyme and its isolated α and β subunits (162 and 52 kDa, respectively) were analyzed using synchrotron radiation x-ray solution scattering. The GltS α subunit and αβ holoenzyme were found to be tetrameric in solution, whereas the β subunit was a mixture of monomers and dimers. Ab initio low resolution shapes restored from the scattering data suggested that the arrangement of a subunits in the (αβ)4 holoenzyme is similar to that in the tetrameric α4 complex and that β subunits occupy the periphery of the holoenzyme. The structure of α4 was further modeled using the available crystallographic coordinates of the monomeric a subunit assuming P222 symmetry. To model the entire αβ holoenzyme, a putative αβ protomer was constructed from the coordinates of the α subunit and those of the N-terminal region of porcine dihydropyrimidine dehydrogenase, which is similar to the β subunit. Rigid body refinement yielded a model of GltS with an arrangement of a subunits similar to that in α4, but displaying contacts also between β subunits belonging to adjacent protomers. The holoenzyme model allows for independent catalytic activity of the αβ protomers, which is consistent with the available biochemical evidence.