Characterization of the Essential Gene glmM Encoding Phosphoglucosamine Mutase in Escherichia coli

Dominique Mengin-Lecreulx,Jean Van Heijenoort

doi:10.1074/jbc.271.1.32

Abstract

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.

Highlights

UDP-GlcNAc1 is one of the main cytoplasmic precursors of bacterial cell-wall peptidoglycan [1, 2]
The observation that a plasmid carrying the ureC gene from H. pylori fully complemented the GPM83 mutant apparently confirmed this hypothesis. We showed that both glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase activities from E. coli were carried by the glmU gene product which acted as a bifunctional enzyme catalyzing two contiguous steps in this pathway [11, 12]
The actual characterization of a phosphoglucosamine mutase activity in crude extracts of E. coli and the demonstration that it is essential for growth is a final confirmation that the steps leading from GlcN-6-P to UDP-GlcNAc are via GlcN-1-P in bacteria (Fig. 1)

Summary

Introduction

UDP-GlcNAc1 is one of the main cytoplasmic precursors of bacterial cell-wall peptidoglycan [1, 2]. The metabolic route leading to the formation of UDP-GlcNAc has been poorly investigated Considering that it was a potential site for the regulation of the flow of metabolites going through the peptidoglycan and lipopolysaccharide pathways, the latter reaction sequence has recently been investigated in more detail [11, 12]. The first of these reactions is catalyzed by the L-glutamine:D-fructose-6-phosphate amidotransferase ( named glucosamine-6-P synthase) [15] Mutants altered in this activity are characterized by an auxotrophy for GlcN or GlcNAc [16, 17], and the corresponding glmS gene has been located at 84 min on the E. coli map [15, 18]. We describe the partial purification and some properties of the phosphoglucosamine mutase which catalyzes the interconversion of GlcN-6-P and GlcN-1-P isomers, as well as the identification of the corresponding glmM gene on the chromosome of E. coli

Methods

Results

Conclusion