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

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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 NH2‐terminal amino‐acid sequence determined. Using the polymerase chain reaction a 53‐bp DNA fragment was synthesized; this fragment encodes the NH2‐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 phosphoenolpyruvate to a substrate.