Molecular cloning, sequencing, and expression in Escherichia coli of the gene encoding a novel 5-oxoprolinase without ATP-hydrolyzing activity from Alcaligenes faecalis N-38A

The gene encoding a novel 5-oxoprolinase without ATP-hydrolyzing activity f rom Alcaligenes faecalis N-38A was cloned and characterized. The coding reg ion of this gene is 1,299 bp long. The predicted primary protein is compose d of 433 amino acid residues, with a 31-amino-acid signal peptide. The matu re protein is composed of 402 amino acid residues with a molecular mass of 46,163 Da, The derived amino acid sequence of the enzyme showed no signific ant sequence similarity to any other proteins reported so far. The 5-oxopro linase gene was expressed in Escherichia coli by using a regulatory express ion system with an isopropyl-beta-D-thiogalactopyranoside-inducible tac pro moter, and its expression level was approximately 16 mg per Liter. The puri fied enzyme has the same characteristics as the authentic enzyme, except fo r the amino terminus, which has three additional amino acids. The enzyme wa s markedly inhibited by p-chloromercuribenzoic acid, EDTA o-phenanthroline, HgCl2, and CuSO4. The EDTA-inactivated enzyme was completely restored by t he addition of Zn2+ or Co2+. In addition, the enzyme was found to contain 1 g-atom of zinc per mol of protein. These results suggest that the 5-oxopro linase produced by A. faecalis N-38A is a zinc metalloenzyme.