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.