Quaternary structure of Azospirillum brasilense NADPH-dependent glutamate synthase in solution as revealed by synchrotron radiation x-ray scattering.

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.

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.

Properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803. Comparison with the Azospirillum brasilense NADPH-dependent enzyme and its isolated alpha subunit.

The properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803 were determined by means of kinetic and spectroscopic approaches in comparison to those exhibited by the bacterial NADPH-dependent enzyme form. The ferredoxin-dependent enzyme was found to be similar to the bacterial glutamate synthase alpha subunit with respect to cofactor content (one FMN cofactor and one [3Fe-4S] cluster per enzyme subunit), overall absorbance properties, and reactivity of the FMN N(5) position with sulfite, as expected from the similar primary structure of ferredoxin-dependent glutamate synthase and of the bacterial NADPH-dependent glutamate synthase alpha subunit. The ferredoxin- and NADPH-dependent enzymes were found to differ with respect to the apparent midpoint potential values of the FMN cofactor and of the [3Fe-4S] cluster, which are less negative in the ferredoxin-dependent enzyme form. This feature is, at least in part, responsible for the efficient oxidation of L-glutamate catalyzed by this enzyme form, but not by the bacterial NADPH-dependent counterpart. At variance with earlier reports on ferredoxin-dependent glutamate synthase, in the Synechocystis enzyme the [3Fe-4S] cluster is not equipotential with the flavin cofactor. The present studies also demonstrated that binding of reduced ferredoxin to ferredoxin-dependent glutamate synthase is essential in order to activate reaction steps such as glutamine binding, hydrolysis, or ammonia transfer from the glutamine amidotransferase site to the glutamate synthase site of the enzyme. Thus, ferredoxin-dependent glutamate synthase seems to control and coordinate catalytic activities taking place at its subsites by regulating the reactions of the glutamine amidotransferase site. Association with reduced ferredoxin appears to be necessary, but not sufficient, to trigger the required activating conformational changes.

Structural studies on the synchronization of catalytic centers in glutamate synthase.

The complex iron-sulfur flavoprotein glutamate synthase (GltS) plays a prominent role in ammonia assimilation in bacteria, yeasts, and plants. GltS catalyzes the formation of two molecules of l-glutamate from 2-oxoglutarate and l-glutamine via intramolecular channeling of ammonia. GltS has the impressive ability of synchronizing its distinct catalytic centers to avoid wasteful consumption of l-glutamine. We have determined the crystal structure of the ferredoxin-dependent GltS in several ligation and redox states. The structures reveal the crucial elements in the synchronization between the glutaminase site and the 2-iminoglutarate reduction site. The structural data combined with the catalytic properties of GltS indicate that binding of ferredoxin and 2-oxoglutarate to the FMN-binding domain of GltS induce a conformational change in the loop connecting the two catalytic centers. The rearrangement induces a shift in the catalytic elements of the amidotransferase domain, such that it becomes activated. This machinery, over a distance of more than 30 A, controls the ability of the enzyme to bind and hydrolyze the ammonia-donating substrate l-glutamine.