Purification and characterization of glutamate synthase from Azospirillum brasilense

Abstract

Growth conditions for Azospirilum 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%). Azospirilum 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. K. values for NADPH, 2-oxoglutarate, and L-glutamine were 6.25, 29, snd 450 ,uM, 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.

Structure and Regulation of Ferredoxin‐Dependent Glutamase Synthase from Arabidopsis Thaliana

“…Fd-dependent glutamate synthase has been found in higher plants , algae (Galvin et al, 1984) and cyanobacteria (MarquCs et al, 1992). Pyridine-nucleotide-dependent glutamate synthase is present in higher plants , fungi (Hummlet and Mora, 1980), yeasts (Masters and Meister, 1982;Cogoni et al, 1995), algae (Calvin et al, 1984) and bacteria (Miller and Standtman, 1972;Ratti et al, 1985 ;Carlberg and Nordlund, 1991). Fd-dependent glutamate synthase is a monomeric protein of 130-170 kDa containing flavin and an iron-sulfur centre in higher plants (Hirasawa et al, 1996), algae (Galvin et al, 1984) and cyanobacteria (MarquCs et al, 1992).…”

“…In Succharomyces cerevisiae, the enzyme is apparently a monomer (Cogoni et al, 1995) although it was initially thought to be a heterodimer composed of large (169 kDa) and small (61 kDa) subunits (Masters and Meister, 1982). NADPH-dependent glutamate synthase has been extensively studied in bacteria, in which the enzyme is an oligomeric iron-sulfur flavoprotein composed of four dimers of large and small subunits with molecular masses of 135-175 kDa and 51-55 kDa, respectively (Miller and Standtman, 1972;Trotta et al, 1974;Hemmila and Mantsala, 1978;Ratti et al, 1985).…”

Influence of amino acids on nitrogen fixation ability and growth of Azospirillum spp

“…requires a detailed knowledge of the kinetic and regulatory properties of the enzymes that are involved. GDH and GOGAT of A. brasiliense have been obtained in pure form; some of their properties have been studied, and some data on the metabolite control of these enzymes has been reported (27,37). The regulatory properties of GS, GOGAT, and ammonium permease (21) in strains with an effective associative nitrogen fixation remain to be studied.…”

Biochemical properties of glutamate synthase of salt-tolerant Bradyrhizobium sp. strain WR1001

“…The enzyme has an alkaline pH optimum of 8.3. If addition of NaC1 to a cell culture induces H ÷ extrusion from the bacteria and resuits in higher intracellular pH, the increases in pH may maximize the catalytic activity of gluta- Azospirillum brasilense 450 29 [21] Bacillus megaterium 300 40 [20] Rhodospirillum rubrum 130 35 [22] Escherichia coli mate synthase and hence result in high concentrations of intracellular glutamate. In addition, the enzyme is stable in the presence of glutamate.…”