Purification and characterization of glutamate synthase from Azospirillum brasilense

Author:

Ratti S,Curti B,Zanetti G,Galli E

Abstract

Growth conditions for Azospirillum brasilense Sp6 were devised for maximal expression of glutamate synthase. The enzyme levels were largely affected by the type and concentration of the nitrogen source. A 10-fold increase in the synthesis of the enzyme was observed at a limiting concentration of ammonia. The enzyme was purified to homogeneity by a procedure which was fairly rapid and allowed a good recovery of enzyme (30%). Azospirillum glutamate synthase is a complex iron-sulfur flavoprotein with a stoichiometry of 1 flavin adenine dinucleotide:1 flavin mononucleotide:8 Fe:8 S per protomer with a molecular weight of 185,000. The protomer is composed of two dissimilar subunits with molecular weights of 135,000 and 50,000. Kinetic parameters were determined. Km values for NADPH, 2-oxoglutarate, and L-glutamine were 6.25, 29, and 450 microM, respectively. The optimum pH was about 7.5. Complete reduction of the enzyme under anaerobic conditions was obtained either by NADPH (in the presence of a regenerating system) or dithionite or by photochemical reduction (in the presence of EDTA and 5-deazariboflavin). No stable long-wavelength intermediates were observed.

Publisher

American Society for Microbiology

Subject

Molecular Biology,Microbiology

Reference31 articles.

1. Purification and properties of glutamate synthase from Thiobacillus thioparus;Adachi K.;J. Bacteriol.,1977

2. Isolation and characterization of glutamate synthase mutants of Azospirillum brasilense;Bani D.;J. Gen. Microbiol.,1980

3. Semi-micro methods for analysis of labile sulfide and of labile sulfide plus sulfane sulfur in unusually stable iron-sulfur proteins;Beinert H.;Anal. Biochem.,1983

4. Chemical modification and ligand binding studies with Escherichia coli glutamate synthase;Bower S.;Biochemistry,1983

5. Simplified methods for the estimation of iron in mitochondria and submitochondrial fractions;Doeg K. A.;Arch. Biochem. Biophys.,1962

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