Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis. A novel iron-sulfur protein.

158

115

L-Glutamine:D-fructose-6-phosphate amidotransferase (glucosamine synthetase) has been purified to homogeneity from Escherichia coli. A subunit molecular weight of 70,800 was estimated by gel electrophoresis in sodium dodecyl sulfate. Pure glucosamine synthetase did not exhibit detectable NH3-dependent activity and did not catalyze the reverse reaction, as reported for more impure preparations [Gosh, S., Blumenthal, H. J., Davidson, E., & Roseman, S. (1960) J. Biol. Chem. 235, 1265]. The enzyme has a Km of 2 mM for fructose 6-phosphate, a Km of 0.4 mM for glutamine, and a turnover number of 1140 min-1. The amino-terminal sequence confirmed the identification of residues 2-26 of the translated E. coli glmS sequence [Walker, J. E., Gay, J., Saraste, M., & Eberle, N. (1984) Biochem. J. 224, 799]. Methionine-1 is therefore removed by processing in vivo, leaving cysteine as the NH2-terminal residue. The enzyme was inactivated by the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) and by iodoacetamide. Glucosamine synthetase exhibited half-of-the-sites reactivity when incubated with DON in the absence of fructose 6-phosphate. In its presence, inactivation with [6-14C]DON was accompanied by incorporation of 1 equiv of inhibitor per enzyme subunit. From this behavior, a dimeric structure was tentatively assigned to the native enzyme. The site of reaction with DON was the NH2-terminal cysteine residue as shown by Edman degradation.

Section: Discussionmentioning

confidence: 94%

Glucosamine synthetase from Escherichia coli: purification, properties, and glutamine-utilizing site location

Badet¹,

Vermoote²,

Haumont³

et al. 1987

158

115

“…This has prompted us to consider the possibility that ColV might be a metalloprotein. A candidate metal ion is iron, which is known to result in a reddish-brown color when bound to proteins (Wong et al, 1977). This is intriguing because the ColV genes are under iron regulation, and ColV uptake is mediated by Cir.…”

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of Colicin V from Escherichia coli Culture Supernatants

Fath¹,

Zhang²,

Rush³

et al. 1994

The peptide antibiotic, colicin V (ColV), has been purified and characterized from Escherichia coli culture supernatants by precipitation with trichloroacetic acid (TCA) and high-performance liquid chromatography (HPLC). Polyacrylamide gel electrophoresis (PAGE) and Western analysis identifies ColV as a polypeptide with an apparent molecular mass of 5.8 kDa. The protein identified remains biologically active after purification and SDS-PAGE. A mutant form of ColV, ColV-1, removes the carboxy-terminal 21 amino acids and replaces them with eight heterologous residues. The ColV-1 mutant is also secreted into the extracellular medium, demonstrating that the carboxy-terminal 21 amino acids are not required for secretion by the dedicated ColV export system, CvaAB/TolC. N-Terminal amino acid sequencing shows that the primary translation product of cvaC, the ColV structural gene, is processed to remove the N-terminal 15 amino acids. The cleavage site is preceded by the sequence Ser-Gly-Gly, making it a potential substrate for leader peptidase. The ColV leader sequence has many characteristics in common with the amino-terminal leader sequences of the lactococcins, lactacins, and pediocins from Gram-positive bacteria. Mass spectroscopy of purified ColV shows that it has a mass of 8741.0 amu, consistent with the mass of the unmodified 88 amino acid polypeptide. The purification scheme provides a rapid and simple way to obtain ColV for further biochemical analysis.

“…The conductivity of the clear yellow supernatant fraction from the protamine 6 Based on the assay of Lowry et al (1951) after precipitation of the protein with trichloroacetic acid and washing of the precipitate with ethanol. c Based on an absorbance of 278 nm and an extinction coefficient determined from the dry weight (Wong et al, 1977). d This preparation contained 3.1 atoms of Fe per subunit.…”

Section: Resultsmentioning

confidence: 99%

“…During purification of amidotransferase, protein was assayed after precipitation with trichloroacetic acid by the method of Lowry et al (1951) with bovine serum albumin as a standard. The concentration of purified amidotransferase was determined from the absorbance at 278 nm and the extinction coefficient based on dry weight (Wong et al, 1977). Iron and S2~were analyzed as previously described (Averill et al, 1980).…”

Section: Methodsmentioning

confidence: 99%

“…The enzyme is also oxygen labile in vitro (Turnbough & Switzer, 1975b). The site of reaction with oxygen in vitro was found to be a 4Fe-4S cluster covalently bound to the enzyme (Wong et al, 1977;Averill et al, 1980). The enzyme has thus become an object of considerable interest, from the perspective both of novel regulatory mechanisms and of novel functions 0006-2960/81/0420-5669S01.25/0 © 1981 American Chemical Society for iron-sulfur centers in enzymes.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Purification and properties of glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis

Wong¹,

Bernlohr²,

Turnbough³

et al. 1981

A procedure for the rapid and efficient purification of glutamine phosphoribosylpyrophosphate amidotransferase to better than 98% homogeneity from depressed Bacillus subtilis cells is described. The molecular weight of the subunit was estimated to be about 50 000. The purified enzyme exhibits microheterogeneity on electrophoresis on highly resolving polyacrylamide gels; it is suggested that this heterogeneity results from limited proteolytic modification of the native subunit. The native enzyme exists in equilibrium among tetrameric, dimeric, and monomeric forms. The influence of enzyme concentration and the presence of substrates and allosteric inhibitors on this equilibrium are described. There is no simple correlation between allosteric inhibition and stabilization of dimeric or tetrameric states. The amino acid composition of the amidotransferase is reported; presence of a 4Fe-4S center in the enzyme was described previously. Preparation of inactive apoprotein by treatment with 1,10-phenanthroline and general characteristics of the apoprotein are presented.