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.

Is It Reliable to Use Common Molecular Docking Methods for Comparing the Binding Affinities of Enantiomer Pairs for Their Protein Target?

“…They found a small difference between the (1R,2S) and (1S,2R) enantiomers, and concluded that racemic cis -cyclopropylamines can be used for the experimental work based on the lack of enantiomeric differences obtained by using docking. In other work, Chen et al [ 29 ] used docking (AutoDock) to study the interactions between mexiletine enantiomers and the D5 variant of monoamine oxidase from Aspergillus niger (MAO-N-D5). They found that the calculated binding energies of (R)-mexiletine and (S)-mexiletine with MAO-N-D5 were −5.91 and −6.84 kcal/mol, respectively, which indicates that MAO-N-D5 binds and reacts preferentially with (S)-mexiletine.…”

High throughput virtual screening reveals SARS-CoV-2 multi-target binding natural compounds to lead instant therapy for COVID-19 treatment

“…Similar observation was indicated for the two thiocarbazate enantiomers of cathepsin L. Molecular docking studies revealed the docking score of S-enantioner (9.03 kcal/mol) more than the R-enantiomer (7.02 kcal/mol), the difference was also validated in their biological activity; S-enantiomer, with an IC50 of 56 nM and R-enantiomer, with an IC50 of 33 μM [128]. The importance of enantiomerism in the interaction of compounds with their binding sites, evaluated by docking scores, was also mentioned in other works [129,130]. On the basis of the earlier reports, it can be accepted that endoribonuclease has shown a better binding affinity for one enantiomer than the other in the present molecular docking study.…”

Directed Evolution Improves the Enzymatic Synthesis of L-5-Hydroxytryptophan by an Engineered Tryptophan Synthase

“…Directed evolution can promote the enzymatic activity of lipase, xylanase, a-galactosidases, monoamine oxidase and nitrilase [14][15][16][17][18][19]. L-tryptophan and S-phenyl-L-cysteine were synthesized by tryptophan synthase of Escherichia coli k-12, and it was found that tryptophan synthase has the potential to synthesize L-tryptophan and its derivatives [20][21][22].…”