title: Semi-rational Directed Evolution of Monoamine Oxidase for Kinetic Resolution of rac-Mexiletine.
abstract: Semi-rational directed evolution was applied to the D5 variant of monoamine oxidase from Aspergillus niger (MAO-N-D5) with the aim of deriving the more desirable (R)-mexiletine through the kinetic resolution of mexiletine enantiomers. Although MAO-N-D5 shows no activity towards rac-mexiletine, theoretical molecular docking studies revealed the potential binding conformations of both mexiletine enantiomers and MAO-N-D5. The key factors affecting the catalytic activity and specificity were identified. Based on the docking results, six residues in the binding pocket and along the binding pathway were selected as key sites for saturation mutagenesis of MAO-N-D5. Through several rounds of screening and combinatorial experiments, two active MAO variants with high enantioselectivities towards (S)-mexiletine evolved, namely A-1 (F210V/L213C, E = 101) and AC-1 (F210V/I367T, E = 69). Molecular simulation experiments indicated that the introduced activity of these variants may be due to the reduced steric hindrance in the binding pocket of the relatively small-sized amino acid residues, a synergetic effect of the entrance residue mutation, and the formation of a new disulfide bond.
authors.affiliation: Laboratory of Biocatalysis, College of Life & Environmental Sciences, Hangzhou Normal University, Hangzhou, 311121, Zhejiang, China.
authors.forename: Zhenming
authors.initials: Z
authors.lastname: Chen
authors.forename: Yuanhui
authors.initials: Y
authors.lastname: Ma
authors.forename: Mengyan
authors.initials: M
authors.lastname: He
authors.forename: Hongyang
authors.initials: H
authors.lastname: Ren
authors.forename: Shuo
authors.initials: S
authors.lastname: Zhou
authors.forename: Dunyue
authors.initials: D
authors.lastname: Lai
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Wang
authors.forename: Linshu
authors.initials: L
authors.lastname: Jiang
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology; D019020:Directed Molecular Evolution / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008801:Mexiletine / Q000737:chemistry / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D008995:Monoamine Oxidase / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D013237:Stereoisomerism
chemical_list: D008801:Mexiletine; D008995:Monoamine Oxidase