title: Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding.
abstract: 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 RhEst1A147I/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. 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.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Zheng-Jiao
authors.initials: ZJ
authors.lastname: Luan
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Hui-Lei
authors.initials: HL
authors.lastname: Yu
authors.affiliation: UT/ORNL Center for Molecular Biophysics, Oak Ridge National Laboratory, Oak Ridge, TN, United States; Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN, United States. Electronic address: chengx@ornl.gov.
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Cheng
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China; Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai, China. Electronic address: jianhexu@ecust.edu.cn.
authors.forename: Jian-He
authors.initials: JH
authors.lastname: Xu
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D055162:Biocatalysis; D043182:Carboxylesterase / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D006860:Hydrogen Bonding; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012240:Rhodococcus / Q000201:enzymology; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D043182:Carboxylesterase
keywords: Carboxylic esterase; Catalytic efficiency; Molecular dynamics simulation; Rational design; Structural mechanism