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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 in certain mutants (e.g.
P94L); and entirely reversed in others (e.g.
V151L) in favor of R-α-tetralol. Here,
we employ efficient 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-Rvs.pro-S
reactive poses (FI) is assessed with
classical molecular dynamics (MD); binding affinity of pro-Rvs.pro-S orientations (FII) is compared using the binding free energy method MM/PBSA; and
differences in reaction barriers towards 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