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

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-OPBE proR 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.