Open AccessMolecular Cloning, Sequencing, and Expression in Escherichia coli of the Gene Encoding a Novel 5-Oxoprolinase without ATP-Hydrolyzing Activity from Alcaligenes faecalis N-38A
Author(s) -
Atsuhisa Nishimura,
Hiroshi Oyama,
Takatoshi Hamada,
Kaoru Nobuoka,
Takashi Shin,
Sawao Murao,
Kōhei Oda
Publication year - 2000
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.66.8.3201-3205.2000
Subject(s) - escherichia coli , alcaligenes faecalis , biochemistry , peptide sequence , amino acid , enzyme , alcaligenes , microbiology and biotechnology , biology , molecular cloning , chemistry , signal peptide , gene , bacteria , genetics , pseudomonas
The gene encoding a novel 5-oxoprolinase without ATP-hydrolyzing activity fromAlcaligenes faecalis N-38A was cloned and characterized. The coding region of this gene is 1,299 bp long. The predicted primary protein is composed of 433 amino acid residues, with a 31-amino-acid signal peptide. The mature 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 significant sequence similarity to any other proteins reported so far. The 5-oxoprolinase gene was expressed inEscherichia coli by using a regulatory expression system with an isopropyl-β-d -thiogalactopyranoside-inducibletac promoter, and its expression level was approximately 16 mg per liter. The purified enzyme has the same characteristics as the authentic enzyme, except for the amino terminus, which has three additional amino acids. The enzyme was markedly inhibited byp -chloromercuribenzoic acid, EDTA,o -phenanthroline, HgCl2 , and CuSO4 . The EDTA-inactivated enzyme was completely restored by the 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-oxoprolinase produced byA. faecalis N-38A is a zinc metalloenzyme.