title: Virtual screening of mandelate racemase mutants with enhanced activity based on binding energy in the transition state.
abstract: Mandelate racemase (MR) is a promising candidate for the dynamic kinetic resolution of racemates. However, the poor activity of MR towards most of its non-natural substrates limits its widespread application. In this work, a virtual screening method based on the binding energy in the transition state was established to assist in the screening of MR mutants with enhanced catalytic efficiency. Using R-3-chloromandelic acid as a model substrate, a total of 53 mutants were constructed based on rational design in the two rounds of screening. The number of mutants for experimental validation was brought down to 17 by the virtual screening method, among which 14 variants turned out to possess improved catalytic efficiency. The variant V26I/Y54V showed 5.2-fold higher catalytic efficiency (k(cat)/K(m)) towards R-3-chloromandelic acid than that observed for the wild-type enzyme. Using this strategy, mutants were successfully obtained for two other substrates, R-mandelamide and R-2-naphthylglycolate (V26I and V29L, respectively), both with a 2-fold improvement in catalytic efficiency. These results demonstrated that this method could effectively predict the trend of mutational effects on catalysis. Analysis from the energetic and structural assays indicated that the enhanced interactions between the active sites and the substrate in the transition state led to improved catalytic efficiency. It was concluded that this virtual screening method based on the binding energy in the transition state was beneficial in enzyme rational redesign and helped to better understand the catalytic properties of the enzyme.
authors.affiliation: Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Jiali
authors.initials: J
authors.lastname: Gu
authors.affiliation: Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.affiliation: Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Fei
authors.initials: F
authors.lastname: Guo
authors.affiliation: Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Wenping
authors.initials: W
authors.lastname: Xie
authors.affiliation: Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Wenqiang
authors.initials: W
authors.lastname: Lu
authors.affiliation: Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Lidan
authors.initials: L
authors.lastname: Ye
authors.affiliation: Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Zhirong
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Shenfeng
authors.initials: S
authors.lastname: Yuan
authors.affiliation: Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China. Electronic address: yuhongwei@zju.edu.cn.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Yu
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis; D020134:Catalytic Domain; D004926:Escherichia coli; D006016:Glycolates / Q000378:metabolism; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D008333:Mandelic Acids / Q000378:metabolism; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D011487:Protein Conformation; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D019998:Racemases and Epimerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: C553672:3-chloromandelic acid; D001426:Bacterial Proteins; D006016:Glycolates; D008333:Mandelic Acids; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C010413:mandelamide; D019998:Racemases and Epimerases; C021540:mandelate racemase
keywords: Binding energy; Mandelate racemase; Molecular dynamics simulation; Transition state; Virtual screening