Cloning and sequence analysis of the estA gene encoding enzyme for producing (R)-beta-acetylmercaptoisobutyric acid from Pseudomonas aeruginosa 1001

The estA gene encoding the enzyme that catalyzes the production of (R)-beta -acetylmercaptoisobutyric acid from (R,S)-ester from Pseudomonas aeruginos a 1001, was cloned in Escherichia coli and its nucleotide sequence was dete rmined, revealing the presumed open reading frame encoding a polypeptide of 316 amino acid residues (948 nucleotides). The overall A + T and C + G com positions were 32.59% and 67.41%, respectively. The amino acid sequence of the estA gene product showed a significant similarity with that of the tria cylglycerol lipase from Psychrobacter immobilis (38% identity), triacylglyc erol lipase from Moraxella sp, (36% identity), and two forms of carboxyl es terases from Acinetobacter calcoaceticus (17% and 17% identities). The dedu ced 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.