X‑ray Crystallography Reveals How Subtle Changes Control the Orientation of Substrate Binding in an Alkene Reductase

Reductions of (S)- and (R)-carvone by wild-type Saccharomyces pastorianus Old Yellow Enzyme (OYE 1) and a systematic collection of Trp 116 variants revealed that, for (S)-carvone, six Trp 116 mutants displayed inverted diastereoselectivity compared to the wild-type. For example, Ile and Val showed inverted stereoselectivity, but Leu and Phe maintained the wild-type stereopreference. For (R)-carvone, only two Trp 116 mutants (Ala and Val) reduced this alkene with reversed selectivity; all other catalytically active variants including Leu and Ile retained the wild-type diastereoselectivity. The same set of mutant enzymes was also used to catalyze the dehydrogenation of (S)- and (R)-carvone under aerobic conditions. To understand how small changes to the active site structure of OYE 1 could significantly influence its catalytic properties, we solved X-ray crystal structures of the wild-type as well as six key Trp 116 variants after individually soaking with both (S)- and (R)-carvone. In many cases, pseudo-Michaelis complexes formed in crystallo, and these revealed the details of protein–substrate interactions. Taken together, our results showed that the wild-type OYE 1 reduces carvone from a less preferred substrate binding orientation. The indole ring of Trp 116 physically blocks access to a hydrophobic active site pocket. Relieving the steric congestion by mutating Trp 116 allows entry of the isopropenyl side-chain of carvone into this hydrophobic pocket and also makes the opposite face of the π system accessible to hydride addition, thereby yielding the opposite diastereomer after net trans-addition of H2.