Stereoselective esterase from Pseudomonas putida IFO12996 reveals alpha/beta hydrolase folds for D-beta-acetylthioisobutyric acid synthesis

J Bacteriol. 2005 Dec;187(24):8470-6. doi: 10.1128/JB.187.24.8470-8476.2005.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins
  • Binding Sites
  • Butyrates / metabolism*
  • Cloning, Molecular
  • Crystallography, X-Ray
  • DNA, Bacterial / chemistry
  • Enzyme Stability
  • Escherichia coli
  • Esterases / chemistry*
  • Esterases / genetics
  • Esterases / metabolism*
  • Hydrogen-Ion Concentration
  • Models, Molecular
  • Molecular Sequence Data
  • Molecular Weight
  • Protein Conformation
  • Protein Folding
  • Protein Structure, Tertiary
  • Protein Subunits
  • Pseudomonas putida / enzymology*
  • Sequence Analysis, DNA
  • Stereoisomerism
  • Substrate Specificity
  • Temperature


  • Bacterial Proteins
  • Butyrates
  • DNA, Bacterial
  • Protein Subunits
  • 3-acetylthioisobutyric acid
  • Esterases

Associated data

  • GENBANK/DQ187948