Unpicking the Cause of Stereoselectivity in Actinorhodin Ketoreductase Variants with Atomistic Simulations

Ketoreductase enzymes (KRs) with a high degree of regio- and stereoselectivity are useful biocatalysts for the production of small, specific chiral alcohols from achiral ketones. Actinorhodin KR (actKR), part of a type II polyketide synthase involved in the biosynthesis of the antibiotic actinorhodin, can also turn over small ketones. In vitro studies assessing stereocontrol in actKR have found that, in the "reverse" direction, the wild-type (WT) enzyme's mild preference for S-α-tetralol is enhanced by certain mutations (e.g., P94L) and entirely reversed by others (e.g., V151L) in favor of R-α-tetralol. Here, we employ computationally cost-effective atomistic simulations to rationalize these trends in WT, P94L, and V151L actKR using trans-1-decalone (1) as the model substrate. Three potential factors (FI-FIII) are investigated: frequency of pro-R vs pro-S reactive poses (FI) is assessed with classical molecular dynamics (MD), binding affinity of pro-R vs pro-S orientations (FII) is compared using the binding free energy method MM/PBSA, and differences in reaction barriers toward trans-1-decalol (FIII) are assessed by hybrid semiempirical quantum/classical (QM/MM) MD simulations with umbrella sampling, benchmarked with density functional theory. No single factor is found to dominate stereocontrol: FI largely determines the selectivity of V151L actKR, whereas FIII is more dominant in the case of P94L. It is also found that formation of S-trans-1-decalol or R-trans-1-decalol mainly arises from the reduction of the trans-1-decalone enantiomers (4aS,8aR)-1 or (4aR,8aS)-1, respectively. Our work highlights the complexity of enzyme stereoselectivity as well as the usefulness of atomistic simulations to aid the design of stereoselective biocatalysts.