Stereoselective esterase from Pseudomonas putida IFO12996 reveals alpha/beta hydrolase folds for D-beta-acetylthioisobutyric acid synthesis.
Esterase (EST) from Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl dl-beta-acetylthioisobutyrate (dl-MATI) to produce d-beta-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 degrees C. The crystal structure of EST has been determined by X-ray diffraction to a resolution of 1.6 A, confirming that EST is a member of the alpha/beta 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 approximately 12 A 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.
Elmi
Fatemeh
F
Life Science Group, National Synchrotron Radiation Research Center, Hsinchu 300, Taiwan.
Lee
Hsin-Tai
HT
Huang
Jen-Yeng
JY
Hsieh
Yin-Cheng
YC
Wang
Yu-Ling
YL
Chen
Yu-Jen
YJ
Shaw
Shyh-Yu
SY
Chen
Chun-Jung
CJ
D001426:Bacterial Proteins; D001665:Binding Sites; D002087:Butyrates / Q000378:metabolism*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D016958:Pseudomonas putida / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D002087:Butyrates; D004269:DNA, Bacterial; D021122:Protein Subunits; C090217:3-acetylthioisobutyric acid; D004950:Esterases