Cloning and Sequence Analysis of the estA gene encoding enzyme for producing ( R)-β-acetylmercaptoisobutyric acid from Pseudomonas aeruginosa 1001

Abstract

The estA gene encoding the enzyme that catalyzes the production of ( R)-β-acetylmercaptoisobutyric acid from ( R,S)-ester from Pseudomonas aeruginosa 1001, was cloned in Escherichia coli and its nucleotide sequence was determined, revealing the presumed open reading frame encoding a polypeptide of 316 amino acid residues (948 nucleotides). The overall A+T and C+G compositions were 32.59% and 67.41%, respectively. The amino acid sequence of the estA gene product showed a significant similarity with that of the triacylglycerol lipase from Psychrobacter immobilis (38% identity), triacylglycerol lipase from Moraxella sp. (36% identity), and two forms of carboxyl esterases from Acinetobacter calcoaceticus (17% and 17% identities). The deduced amino acid sequences have a pentapeptide consensus sequence, G-X-S-X-G, having an active serine residue, and another active site, dipeptides H-G, located at 70–100 amino acids upstream of the G-X-S-X-G consensus sequence.