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 wildtype 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 wildtype 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 wildtype 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 H 2 .

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BaylisÀHillman adducts are highly useful synthetic intermediates; to enhance their value further, we sought enantiocomplementary alkene reductases to introduce chirality. Two solutions emerged: (1) a wild-type protein from Pichia stipitis (OYE 2.6), whose performance significantly outstrips that of the standard enzyme (Saccharomyces pastorianus OYE1), and (2) a series of OYE1 mutants at position 116 (Trp in the wild-type enzyme). To understand how mutations could lead to inverted enantioselectivity, we solved the X-ray crystal structure of the Trp116Ile OYE1 variant complexed with a cyclopentenone substrate. This revealed key proteinÀligand interactions that control the orientation of substrate binding above the FMN cofactor.

A systematic saturation mutagenesis campaign was carried out on an alkene reductase from Pichia stipitis (OYE 2.6) to develop variants with reversed stereoselectivities. Wild-type OYE 2.6 reduces three representative Baylis–Hillman adducts to the corresponding S products with almost complete stereoselectivities and good catalytic efficiencies. We created and screened 13 first-generation, site-saturation mutagenesis libraries, targeting residues found near the bound substrate. One variant (Tyr78Trp) showed high R selectivity toward one of the three substrates, but no change (cyclohexenone derivative) and no catalytic activity (acrylate derivative) for the other two. Subsequent rounds of mutagenesis retained the Tyr78Trp mutation and explored other residues that impacted stereoselectivity when altered in a wild-type background. These efforts yielded double and triple mutants that possessed inverted stereoselectivities for two of the three substrates (conversions >99% and at least 91% ee (R)). To understand the reasons underlying the stereochemical changes, we solved crystal structures of two key mutants: Tyr78Trp and Tyr78Trp/Ile113Cys, the latter with substrate partially occupying the active site. By combining these experimental data with modeling studies, we have proposed a rationale that explains the impacts of the most useful mutations.

We have probed Pichia stipitis CBS 6054 Old Yellow Enzyme 2.6 (OYE 2.6) by several strategies including X‐ray crystallography, ligand binding and catalytic assays using the wild‐type as well as libraries of site‐saturation mutants. The alkene reductase crystallized in space group P 63 2 2 with unit cell dimensions of 127.1×123.4 Å and its structure was solved to 1.5 Å resolution by molecular replacement. The protein environment surrounding the flavin mononucleotide (FMN) cofactor was very similar to those of other OYE superfamily members; however, differences in the putative substrate binding site were also observed. Substrate analog complexes were analyzed by both UV‐Vis titration and X‐ray crystallography to provide information on possible substrate binding interactions. In addition, four active site residues were targeted for site saturation mutagenesis (Thr 35, Ile 113, His 188, His 191) and each library was tested against three representative Baylis–Hillman adducts. Thr 35 could be replaced by Ser with no change in activity; other amino acids (Ala, Cys, Leu, Met, Gln and Val) resulted in diminished catalytic efficiency. The Ile 113 replacement library yielded a range of catalytic activities, but had very little impact on stereoselectivity. Finally, the two His residues (188 and 191) were essentially intolerant of substitutions with the exception of the His 191 Asn mutant, which did show significant catalytic ability. Structural comparisons between OYE 2.6 and Saccharomyces pastorianus OYE1 suggest that the key interactions between the substrate hydroxymethyl groups and the side‐chain of Thr 35 and/or Tyr 78 play an important role in making OYE 2.6 an (S)‐selective alkene reductase.