Characterization of the essential gene glmM encoding phosphoglucosamine mutase in Escherichia coli.
Two different approaches to identify the gene encoding the phosphoglucosamine mutase in Escherichia coli were used: (i) the purification to near homogeneity of this enzyme from a wild type strain and the determination of its N-terminal amino acid sequence; (ii) the search in data bases of an E. coli protein of unknown function showing sequence similarities with other hexosephosphate mutase activities. Both investigations revealed the same open reading frame named yhbF located within the leuU-dacB region at 69.5 min on the chromosome (Dallas, W. S., Dev, I. K., and Ray, P. H. (1993) J. Bacteriol. 175, 7743-7744). The predicted 445-residue protein with a calculated mass of 47.5 kDa contained in particular a short region GIVISASHNP with high similarity to the putative active site of hexosephosphate mutases. In vitro assays showed that the overexpression of this gene in E. coli cells led to a significant overproduction (from 15- to 50-fold) of phosphoglucosamine mutase activity. A hexose 1,6-diphosphate-dependent phosphorylation of the enzyme, which probably involves the serine residue at position 102, is apparently required for its catalytic action. As expected, the inactivation of this gene, which is essential for bacterial growth, led to the progressive depletion of the pools of precursors located downstream from glucosamine 1-phosphate in the pathway for peptidoglycan synthesis. This was followed by various alterations of cell shape and finally cells were lysed when their peptidoglycan content decreased to a critical value corresponding to about 60% of its normal level. The gene for this enzyme, which is essential for peptidoglycan and lipopolysaccharide biosyntheses, has been designated glmM.
Mengin-Lecreulx
D
D
Laboratoire des Enveloppes Bactériennes et des Peptides, Unité de Recherche Associée 1131 du CNRS, Université Paris-Sud, Orsay, France.
van Heijenoort
J
J
D000595:Amino Acid Sequence; D002384:Catalysis; D002876:Chromosomes, Bacterial; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005944:Glucosamine / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives*; D005958:Glucosephosphates / Q000096:biosynthesis / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010733:Phosphoglucomutase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids
D005958:Glucosephosphates; C038515:glucosamine 1-phosphate; C001293:glucosamine 6-phosphate; D019298:Glucose-6-Phosphate; C097631:phosphoglucosamine mutase; D010733:Phosphoglucomutase; D005944:Glucosamine