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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 {alpha}/ß Hydrolase Folds for D-ß-Acetylthioisobutyric Acid Synthesis

Fatemeh Elmi,1,2 Hsin-Tai Lee,3 Jen-Yeng Huang,1 Yin-Cheng Hsieh,4 Yu-Ling Wang,4 Yu-Jen Chen,5 Shyh-Yu Shaw,5* and Chun-Jung Chen1,3*

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 {alpha}/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.

* Corresponding author. Mailing address for Chun-Jung Chen: Life Science Group, National Synchrotron Radiation Research Center, Hsinchu 300, Taiwan. Phone: 886-3-578-0281, ext. 7330. Fax: 886-3-578-3813. E-mail: cjchen{at}nsrrc.org.tw. Mailing address for Shyh-Yu Shaw: Department of Chemistry, National Cheng Kung University, Tainan 700, Taiwan. Phone: 886-6-208-0476. Fax: 886-6-274-0552. E-mail: syshaw{at}mail.ncku.edu.tw.

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.





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