Glutamate synthase: Identification of the NADPH-Binding site by site-directed mutagenesis

To contribute to the understanding of glutamate synthase and of beta subuni t-like proteins, which have been detected by sequence analyses, we identifi ed the NADPH-binding site out of the two potential ADP-binding regions foun d in the beta subunit. The substitution of an alanyl residue for G298 of th e beta subunit of Azospirillum brasilense glutamate synthase (the second gl ycine in the GXGXXA fingerprint of the postulated NADPH-binding site) yield ed a protein species in which the flavin environment and properties are una ltered. On the contrary, the binding of the pyridine nucleotide substrate i s significantly perturbed demonstrating that the C-terminal potential ADP-b inding fold of the beta subunit is indeed the NADPH-binding site of the enz yme. The major effect of the G298A substitution in the GltS beta subunit co nsists of an approximately 10-fold decrease of the affinity of the enzyme f or pyridine nucleotides with little or no effect on the rate of the enzyme reduction by NADPH. By combining kinetic measurements and absorbance-monito red equilibrium titrations of the G298A-beta subunit mutant, we conclude th at also the positioning of its nicotinamide portion into the active site is altered thus preventing the formation of a stable charge-transfer complex between reduced FAD and NADP(+). During the course of this work, the Azospi rillum DNA regions flanking the gltD and gltB genes, the genes encoding the GltS beta and alpha subunits, respectively, were sequenced and analyzed. A lthough the Azospirillum GltS is similar to the enzyme of other bacteria, i t appears that the corresponding genes differ with respect to their arrange ment in the chromosome and to the composition of the glt operon: no genes c orresponding to E. coli and Klebsiella aerogenes gltF or to Bacillus subtil is gltC, encoding regulatory proteins, are found in the DNA regions adjacen t to that containing gltD and gltB genes in Azospirillum. Further studies a re needed to determine if these findings also imply differences in the regu lation of the glt genes expression in Azospirillum (a nitrogen-fixing bacte rium) with respect to enteric bacteria.