Purification and properties of glutamate synthase from Bacillus licheniformis

Abstract

Glutamate synthase [L-glutamate:NADP+ oxidoreductase (transaminating); EC 1.4.1.13] (GItS) was purified to homogeneity from BaciUus licheniformis A5. The native enzyme had a molecular weight of approximately 220,000 and was composed of two nonidentical subunits (molecular weights,-158,000 and-54,000). The enzyme was found to contain 8.1 ± 1 iron atoms and 8.1 + 1 acid-labile sulfur atoms per 220,000-dalton dimer. Two flavin moieties were found per 220,000-dalton dimer, with a ratio of flavin adenine dinucleotide to flavin mononucleotide of 1.2. The UV-visible spectrum of the enzyme exhibited maxima at 263,380 and 450 nm. The GitS from B. Iicheniformis had a requirement for NADPH, oa-ketoglutarate, and glutamine. Classical hyperbolic kinetics were seen for NADPH affinity, which resulted in an apparent Km value of 13 ,uM. Nonhyperbolic kinetics were obtained for a-ketoglutarate and glutamine affinities, and the reciprocal plots obtained for these substrates were biphasic. The apparent Km values obtained for glutamine were 8 and 100 ,LM, and the apparent Km values obtained for a-ketoglutarate were 6 and 50 ,uM. GltS activity was found to be relatively insensitive to inhibition by amino acids, keto acids, or various nucleotides. L-Methionine-DLsulfoximine, L-methionine sulfone, and DL-methionine sulfoxide were found to be potent inhibitors of GltS activity, yielding 10.5 values of 150, 11, and 250 ,uM, respectively. GltSs were purified from cells grown in the presence of ammonia and nitrate as sole nitrogen sources and were compared. Both yielded identical final specific activities and identical physical (UV-visible spectra, flavin, and iron-sulfur composition) and kinetic characteristics.

Purification and characterization of glutamate synthase from Azospirillum brasilense

“…The amino acid composition of glutamate synthase from A. brasilense was similar to the previously published analyses of the enzyme from Bacillus megaterium (9) and E. coli (16). In this respect, these enzymes showed some differences in amino acid composition compared with that of Bacillus licheniformis (24).…”

“…The present purification procedure was quite rapid and gave a good yield of homogeneous enzyme. A few glutamate synthases have been purified to homogeneity (9,16,24,28). In this respect, the chemical and physical properties of the A. brasilense enzyme were similar to those of glutamate synthase from other bacterial sources.…”

“…licheniformis (24), and T. thioparus (1) are of the type a,. In the latter case, the protomer seems to be the active species, whereas with the a4P4 type of glutamate synthase there is no evidence to indicate which of the oligomeric species is involved in catalysis.…”

“…The molecular weights of the protomer (,B) are 188,000 for the Escherichia coli enzyme (16) and 280,000 for the Thiobacillus thioparus enzyme (1). Intermediate values within these limits have been observed for the protomers from other sources (9,24,28). Some of the kinetic properties of glutamate syrthase have been investigated (23), and a two-site ping-pong (uni-uni, bi-bi) mechanism has been postulated for the reaction, with the flavins and the iron-sulfur clusters functioning as electron carriers between the two sites.…”

“…Glutamate synthase has been found in several bacteria (15,18) as well as in yeasts and plants (26). The enzyme has been purified to a homogeneous form from relatively few bacteria (9,16,24,28). The bacterial enzyme is an iron-sulfur flavoprotein containing both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) and is composed of two dissimilar subunits.…”