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Journal of Bacteriology, December 2005, p. 8470-8476, Vol. 187, No. 24
0021-9193/05/$08.00+0     doi:10.1128/JB.187.24.8470-8476.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Stereoselective Esterase from Pseudomonas putida IFO12996 Reveals /ß Hydrolase Folds for D-ß-Acetylthioisobutyric Acid Synthesis

Life Science Group, National Synchrotron Radiation Research Center, Hsinchu 300, Taiwan,¹ Department of Chemistry, National Cheng Kung University, Tainan 700, Taiwan,⁵ Department of Chemistry, Tarbiat Modarres University, Tehran, Iran,² Department of Physics,³ Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, Hsinchu 300, Taiwan⁴

Received 20 June 2005/ Accepted 26 September 2005

Esterase (EST) from Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl DL-ß-acetylthioisobutyrate (DL-MATI) to produce D-ß-acetylthioisobutyric acid (DAT), serving as a key intermediate for the synthesis of angiotensin-converting enzyme inhibitors. The EST gene was cloned and expressed in Escherichia coli; the recombinant protein is a non-disulfide-linked homotrimer with a monomer molecular weight of 33,000 in both solution and crystalline states, indicating that these ESTs function as trimers. EST hydrolyzed DL-MATI to produce DAT with a degree of conversion of 49.5% and an enantiomeric excess value of 97.2% at an optimum pH of about 8 to 10 and an optimum temperature of about 57 to 67°C. The crystal structure of EST has been determined by X-ray diffraction to a resolution of 1.6 Å, confirming that EST is a member of the /ß hydrolase fold superfamily of enzymes and includes a catalytic triad of Ser97, Asp227, and His256. The active site is located approximately in the middle of the molecule at the end of a pocket 12 Å deep. EST can hydrolyze the methyl ester group without affecting the acetylthiol ester moiety in DL-MATI. The examination of substrate specificity of EST toward other linear esters revealed that the enzyme showed specific activity toward methyl esters and that it recognized the configuration at C-2.