Enantioselective Reduction of Citral Isomers in NCR Ene Reductase: Analysis of an Active‐Site Mutant Library

A deeper understanding of the >99 % S ‐selective reduction of both isomers of citral catalyzed by NCR ene reductase was achieved by active‐site mutational studies and docking simulation. Though structurally similar, the E / Z isomers of citral showed a significantly varying selectivity response to introduced mutations. Although it was possible to invert ( E )‐citral reduction enantioselectivity to ee 46 % ( R ) by introducing mutation W66A, for ( Z )‐citral it remained ≥88 % ( S ) for all single‐residue variants. Residue 66 seems to act as a lever for opposite binding modes. This was underlined by a W66A‐based double‐mutant library that enhanced the ( E )‐citral derived enantioselectivity to 63 % ( R ) and significantly lowered the S selectivity for ( Z )‐citral to 44 % ( S ). Formation of ( R )‐citronellal from an ( E / Z )‐citral mixture is a desire in industrial (−)‐menthol synthesis. Our findings pave the way for a rational enzyme engineering solution.