Functional properties of recombinant Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein

Azospirillum brasilense glutamate synthase is a complex iron-sulfur flavopr otein that catalyses the NADPH-dependent reductive transfer of glutamine am ide group to the C(2) carbon of 2-oxoglutarate to yield L-glutamate. Its ca talytically active alpha beta protomer is composed of two dissimilar subuni ts tot subunit, 164.2 kDa; beta subunit, 52.3 kDa) and contains one FAD (at Site 1, the pyridine nucleotide site within the beta subunit), one FMN (at Site 2, the 2-oxoglutarate/L-glutamate site in the alpha subunit) and thre e different iron-sulfur clusters tone 3Fe-4S center on the alpha subunit an d two 4Fe-4S clusters of unknown location). A plasmid harboring the gltD an d gltB genes, the genes encoding the glutamate synthase beta and alpha subu nits, respectively, each one under the control of the T7/lac promoter of pE T11a was found to be suitable for the overproduction of glutamate synthase holoenzyme in Escherichia coli BL21(DE3) cells. Recombinant A. brasilense g lutamate synthase could be purified to homogeneity from overproducing E. co li cells by ion exchange chromatography, gel filtration and affinity chroma tography on a 2',5' ADP-Sepharose 4B column. The purified enzyme was indist inguishable from that prepared from Azospirillum cells with respect to cofa ctor content, N-terminal sequence of the subunits, aggregation state, kinet ic and spectroscopic properties. The study of the recombinant holoenzyme al lowed us to establish that the tendency of glutamate synthase to form a sta ble (alpha beta)(4) tetramer at high protein concentrations is a property u nique to the holoenzyme, as the isolated beta subunit does not oligomerize, while the isolated glutamate synthase alpha subunit only forms dimers at h igh protein concentrations. Furthermore, the steady-state kinetic analysis of the glutamate synthase reaction was extended to the study of the effect of adenosine-containing nucleotides. Compounds such as cAMP, AMP, ADP and A TP have no effect on the enzyme activity, while the 2'-phosphorylated analo gs of AMP and NADP(PI) analogs act as inhibitors of the reaction, competiti ve with NADPH. Thus, it can be ruled out that glutamate synthase reaction i s subjected to allosteric modulation by adenosine containing (di)nucleotide s, which may bind to the putative ADP-binding site at the C-terminus of the alpha subunit. At the same time, the strict requirement of a 2'-phosphate group in the pyridine nucleotide for binding to glutamate synthase (GltS) w as established. Finally, by comparing the inhibition constants exhibited by a series of NADP(+) analogs, the contribution to the binding energy of the various parts of the pyridine nucleotide has been determined along with th e effect of substituents on the 3 position of the pyridine ring. With the e xception of thio-NADP(+), which binds the tightest to GltS, it appears that the size of the substituent is the factor that affects the most the intera ction between the NADP(H) analog and the enzyme.