Copurification of glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase activities of Escherichia coli: characterization of the glmU gene product as a bifunctional enzyme catalyzing two subsequent steps in the pathway for UDP-N-acetylglucosamine synthesis.
The glmU gene product of Escherichia coli was recently identified as the N-acetylglucosamine-1-phosphate uridyltransferase activity which catalyzes the formation of UDP-N-acetylglucosamine, an essential precursor for cell wall peptidoglycan and lipopolysaccharide biosyntheses (D. Mengin-Lecreulx and J. van Heijenoort, J. Bacteriol. 175:6150-6157, 1993). Evidence that the purified GlmU protein is in fact a bifunctional enzyme which also catalyzes acetylation of glucosamine-1-phosphate, the preceding step in the same pathway, is now provided. Kinetic parameters of both reactions were investigated, indicating in particular that the acetyltransferase activity of the enzyme is fivefold higher than its uridyltransferase activity. In contrast to the uridyltransferase activity, which is quite stable and insensitive to thiol reagents, the acetyltransferase activity was rapidly lost when the enzyme was stored in the absence of reducing thiols or acetyl coenzyme A or was treated with thiol-alkylating agents, suggesting the presence of at least one essential cysteine residue in or near the active site. The acetyltransferase activity is greatly inhibited by its reaction product N-acetylglucosamine-1-phosphate and, interestingly, also by UDP-N-acetylmuramic acid, which is one of the first precursors specific for the peptidoglycan pathway. The detection in crude cell extracts of a phosphoglucosamine mutase activity finally confirms that the route from glucosamine-6-phosphate to UDP-N-acetylglucosamine occurs via glucosamine-1-phosphate in bacteria.
Mengin-Lecreulx
D
D
Laboratoire des Enveloppes Bactériennes et des Peptides, Unité de Recherche, Associée 1131 du Centre National de la Recherche Scientifique, Université Paris-Sud, Orsay, France.
van Heijenoort
J
J
D000123:Acetyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D000478:Alkylation; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology*; D005798:Genes, Bacterial / Q000502:physiology; D007700:Kinetics; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D017875:Phosphotransferases (Phosphomutases) / Q000378:metabolism; D013439:Sulfhydryl Reagents; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000096:biosynthesis*
D013439:Sulfhydryl Reagents; D014537:Uridine Diphosphate N-Acetylglucosamine; D000123:Acetyltransferases; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase; D017875:Phosphotransferases (Phosphomutases)