Stabilization, purification, and characterization of glutamate synthase from Clostridium pasteurianum

Abstract

Clostridium pasteurianum possesses a high level of glutamate synthase (EC 1.4.1.14) activity and cell yield when grown on 4 mM ammonium chloride and molasses as the sole nitrogen and carbon sources, respectively. The enzyme activity is stabilized by addition of alpha-ketoglutarate, EDTA, and 2-mercaptoethanol. Ammonium sulfate precipitation and single-step combined gel and ion-exchange chromatography followed by fractional dialysis yield a homogeneous protein with 40% recovery of the glutamate synthase activity. The native enzyme (Mr congruent to 590,000) gives five different subunits (as dimers) upon SDS gel electrophoresis. The enzyme has been characterized for pH and temperature optimum, substrate specificity, Kmapp values, energy of activation, half-life, and thermal stabilization. Metal ions and citric acid cycle metabolites do not affect the enzyme activity. Glutamate synthase shows fluorescence maximum at 370 nm when excited at 280 nm. The fluorescence is quenched upon the addition of NADH. Spectroscopic examination of the enzyme gave absorption maximum at 280 and none at 380 and 440 nm, indicating the absence of iron and flavin. The absence of iron and flavin was also confirmed by atomic absorption, chemical analysis, and fluoroscopy, respectively. The C. pasteurianum enzyme differs from that of other aerobic bacterial sources.

Metabolic thermotolerance: magnetic resonance detected protection of glutamate synthase

“…Analysis of Cell-Free Extracts. To confirm the proposed loss of GluS activity following the 45 °C heat shock, GluS activity was assayed in cell-free extracts (Miflin & Lea, 1975;Singhal et al, 1989). GluS activity extracted from control corn root tips gave an NADH-dependent initial reaction rate of 8.9 ± 0.4 nmol/(min-mg, of protein) using glutamine as a nitrogen source, essentially the same value reported for Pisum (Miflin & Lea, 1975).…”

Purification and partial characterization of glutamate synthase from Rhodospirillum rubrum grown under nitrogen-fixing conditions

“…This is similar to most glutamate synthases from prokaryotes, which have been reported to be composed of two dissimilar subunits, one smaller (50-75 kDa) and one larger (135-175 kDa). Recently, however, the purification of glutamate synthase from Clostridium pasteurianum was reported, and this enzyme is composed of two of each five polypeptides with molecular masses 91, 86, 68, 31 and 17.5 kDa [38].…”

Sequence of the GLT1 gene from Saccharomyces cerevisiae reveals the domain structure of yeast glutamate synthase

“…subunits (Singhal et al, 1989). Most of the bacterial enzymes use NADPH as a cofactor, although two NADH-dependent GOGAT's have been found (Wang and Nicholas, 1985;Singhal et al, 1989).…”

“…Most of the bacterial enzymes use NADPH as a cofactor, although two NADH-dependent GOGAT's have been found (Wang and Nicholas, 1985;Singhal et al, 1989). In some cases, the genes that code for the large and small polypeptides have been cloned and sequenced (Oliver et al, 1987;Vanoni et ul., 1990;Pelanda et al, 1993).…”