Molecular Dynamics Simulation‐Assisted Tuning of Key Sites to Alter the Substrate Specificity of the Aldo‐Keto Reductase KmAKR

ABSTRACT Aldo‐keto reductase (AKR) is an important biocatalyst for the synthesis of chiral alcohols; however, its inability to catalyze bulky substrates severely limits its industrial applications. Previously, the T23V/Q213A mutant of AKR from Kluyveromyces marxianus ( Km AKR) exhibited an extended substrate scope, but it still showed poor catalytic activity toward some valuable aliphatic and aromatic ketones. Here, we developed a computer‐assisted strategy to virtually screen a mutation library constructed from residues 23 and 213. Guided by the binding free energy calculated from high‐throughput molecular dynamics simulations, the top ten mutants with the lowest binding energies in each group were selected for testing against the corresponding substrates. It was found that most selected mutants exhibited enhanced catalytic activity, yielding the corresponding pharmaceutically important alcohols with high enantioselectivities. Structural and dynamic analyses indicated that residues 23 and 213 functioned as molecular switches to control the dynamics of the loop regions that constitute the substrate‐binding pocket, thereby influencing the substrate specificity of Km AKR.