Unpicking the Cause of Stereoselectivity in Actinorhodin Ketoreductase Variants with Atomistic Simulations
2018-12-04T13:44:53Z (GMT) by
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 (<i>act</i>KR), part of a type II polyketide synthase involved in the biosynthesis of the antibiotic actinorhodin, can also turn over small ketones. <i>In vitro</i> studies assessing stereocontrol in <i>act</i>KR have found that, in the “reverse” direction, the wild-type (WT) enzyme’s mild preference for <i>S-α</i>-tetralol is enhanced in certain mutants (<i>e.g.</i> P94L); and entirely reversed in others (<i>e.g.</i> V151L) in favor of <i>R-α</i>-tetralol. Here, we employ efficient atomistic simulations to rationalize these trends in WT, P94L, and V151L <i>act</i>KR, using <i>trans-</i>1-decalone (<b>1</b>) as the model substrate. Three potential factors (<b>FI</b>-<b>FIII</b>) are investigated: frequency of <i>pro-R</i> <i>vs.</i> <i>pro-S</i> reactive poses (<b>FI</b>) is assessed with classical molecular dynamics (MD); binding affinity of <i>pro-R</i> <i>vs.</i> <i>pro-S</i> orientations (<b>FII</b>) is compared using the binding free energy method MM/PBSA; and differences in reaction barriers towards <i>trans-</i>1-decalol (<b>FIII</b>) 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: <b>FI</b> largely determines the selectivity of V151L <i>act</i>KR, whereas <b>FIII</b> is more dominant in the case of P94L. It is also found that formation of <i>S-trans-</i>1-decalol or <i>R-trans-</i>1-decalol mainly arises from the reduction of the <i>trans</i>-1-decalone enantiomers <i>(4aS,8aR)</i>-<b>1</b> or <i>(4aR,8aS)</i>-<b>1</b>, respectively. Our work highlights the complexity of enzyme stereoselectivity as well as the usefulness of atomistic simulations to aid the design of stereoselective biocatalysts.