Redesign of a short-chain dehydrogenase/reductase for asymmetric synthesis of ethyl (R)-2-hydroxy-4-phenylbutanoate based on per-residue free energy decomposition and sequence conservatism analysis

Abstract As an important building block for the synthesis of angiotensin-converting enzyme inhibitors, ethyl (R)-2-hydroxyl-4-phenylbutanoate [(R)-HPBE] has attracted increasing attention. The key to industrial biosynthesis of (R)-HPBE is a biocatalyst that efficiently reduces ethyl 2-oxo-4-phenylbutanoate (OPBE) with high R-enantioselectivity. This paper proposed a strategy for identifying key residues involved in enantioselectivity control based on per-residue free energy decomposition and sequence conservatism analysis. Using this strategy, 4 nonconservative sites with high energy contribution to binding of OPBE were chosen as engineering targets, generating variant Mu27 with 99% conversion and 98% (R) ee value at substrate loading of up to 500 mmol/L. MD simulations suggested the higher stability and formation probability of Mu27-OPBEproR prereaction state as key reasons for the excellent R-enantioselectivity of Mu27 towards OPBE. The success in this study provides a viable approach for rational design of alcohol dehydrogenases with high enantioselectivity towards unnatural substrates.