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Title: Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding

A new carboxylic esterase RhEst1 which catalyzes the hydrolysis of (S)-(+)-2,2-dimethylcyclopropanecarboxylate (S-DmCpCe), the key chiral building block of cilastatin, was identified and subsequently crystallized in our previous work. Mutant RhEst 1A147I/V148F/G254A was found to show a 5-fold increase in the catalytic activity. In this work, molecular dynamic simulations were performed to elucidate the molecular determinant of the enzyme activity. Our simulations show that the substrate binds much more strongly in the A147I/V148F/G254A mutant than in wild type, with more hydrogen bonds formed between the substrate and the catalytic triad and the oxyanion hole. The OH group of the catalytic residue Ser101 in the mutant is better positioned to initiate the nucleophilic attack on S-DmCpCe. Interestingly, the "170-179" loop which is involved in shaping the catalytic sites and facilitating the product release shows remarkable dynamic differences in the two systems. Based on the simulation results, six residues were identified as potential "hot-spots" for further experimental testing. Consequently, the G126S and R133L mutants show higher catalytic efficiency as compared with the wild type. In conclusion, this work provides molecular-level insights into the substrate binding mechanism of carboxylic esterase RhEst1, facilitating future experimental efforts toward developing more efficient RhEst1 variants for industrial applications.
 [1] ;  [1] ;  [1] ;  [2] ;  [1]
  1. East China Univ. of Science and Technology, Shanghai (China)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
Publication Date:
Grant/Contract Number:
AC05-00OR22725; De-AC05-00OR22725
Accepted Manuscript
Journal Name:
Journal of Molecular Graphics and Modelling
Additional Journal Information:
Journal Volume: 62; Journal Issue: C; Journal ID: ISSN 1093-3263
Research Org:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org:
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES; carboxylic esterase; molecular dynamics simulation; rational design; structural mechanism; catalytic efficiency
OSTI Identifier:
Alternate Identifier(s):
OSTI ID: 1432567