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

Abstract

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 .

Revealing Additional Stereocomplementary Pairs of Old Yellow Enzymes by Rational Transfer of Engineered Residues

“…Further, the same pattern of stereocomplementarity for the glycine/threonine and aspartate/threonine variants was observed for the reduction of 3 , allowing access to (2 S ,5 S )‐ 4 or (2 R ,5 S )‐ 4 with excellent diastereoselectivities of up to 92 or 96 % de , respectively. This is the first report of group 2 members producing (2 S ,5 S )‐ 4 and—other than OYE1 W116X variants from group 1—they represent the only other set of variants so far for this reaction . All our other new variants led to (2 R ,5 S )‐ 4 , even the hotspot position III homologues NCR W100I and TsER A102I.…”

“…In addition to the above studies, a few single‐residue saturation mutagenesis, rational design or directed evolution studies that allow extraction of engineered residues exist. Taken together, several hotspot positions have been identified, and positions I, II, and III (Figure ) in particular were found to control facial selectivity, activity and substrate scope in OYE1, OYE2.6, PETNR, KYE and YqjM …”

Old Yellow Enzyme-Catalysed Asymmetric Hydrogenation: Linking Family Roots with Improved Catalysis

“…However, the group of Pietruszka showed that the stereoselectivity of an uncharacterised OYE could not be predicted solely based on the primary sequence pattern of the surface loop β2 [126]. Stereoselectivity was also investigated by the group of Stewart, who determined how subtle changes in the residues control the orientation of substrate binding in OYEs [127,128], and showed that mutations can lead to inverted enantioselectivity [129,130]. The stereochemical outcome was influenced by mutagenesis of Trp116 in OYE1.…”

Stereodivergent Synthesis of Carveol and Dihydrocarveol through Ketoreductases/Ene‐Reductases Catalyzed Asymmetric Reduction

“…Thus, combining ene‐reductases with ketoreductases was performed to create dihydrocarveol stereoisomers. The biocatalytic reduction of activated C=C bond of carvone ( 1 ) to give dihydrocarvone ( 3 ) has been well studied by several ene‐reductases ,,,. For example, (1 R ,4 R )‐ 3 and (1 R ,4 S )‐ 3 could be obtained through asymmetric reduction of corresponding (4 R )‐ 1 and (4 S )‐ 1 by various ene‐reductases, such as OYE1,, PETNR, and so on .…”

“…The biocatalytic reduction of activated C=C bond of carvone ( 1 ) to give dihydrocarvone ( 3 ) has been well studied by several ene‐reductases ,,,. For example, (1 R ,4 R )‐ 3 and (1 R ,4 S )‐ 3 could be obtained through asymmetric reduction of corresponding (4 R )‐ 1 and (4 S )‐ 1 by various ene‐reductases, such as OYE1,, PETNR, and so on . However, only a few ene‐reductase variants, such as OYEW116A/V and TsER C25G/I67T, displayed switched stereoselectivity to yield dihydrocarvone with S configuration at C‐1 position.…”