The UDP- N-acetylglucosamine 1-carboxyvinyl-transferase of Enterobacter cloacae Molecular cloning, sequencing of the gene and overexpression of the enzyme

Abstract

The UDP- N-acetylglucosamine 1-carboyyvinyltransferase ( enol-pyruvyltransferase, EC 2.5,1.7) which catalyses the first committed step in the biosynthesis of the bacterial cell-wall peptidoglycan was purified to near homogeneity from Enterobacter cloacae and the NH 2-terminal amino-acid sequence determined. Using the polymerase chain reaction a 53-bp DNA fragment was synthesized; this fragment encodes the NH 2-terminal sequence of the enzyme. A clone was then isolated which contained an open reading frame of 1257 bp coding for a protein of 419 amino acids. This protein was overexpressed 100-fold in transformed Escherichia coli cells and shown to possess the enolpyruvyltransferase activity. The overall amino-acid sequence of the enolpyruvyltransferase is significantly similar to that of the 5- enolpyruvylshikimate 3-phosphate synthase, the only other enzyme known to catalyse the transfer of the enolpyruvate moiety of phospho enolpyruvate to a substrate.