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.
Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
Nico
N
Kress
Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
Johanna
J
Rapp
Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
Bernhard
B
Hauer
D000080462:Acyclic Monoterpenes; D020134:Catalytic Domain; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D062105:Molecular Docking Simulation; D039821:Monoterpenes / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation; D015202:Protein Engineering; D012440:Saccharomyces / Q000201:enzymology; D013237:Stereoisomerism; D017355:Zymomonas / Q000201:enzymology
D000080462:Acyclic Monoterpenes; D005656:Fungal Proteins; D039821:Monoterpenes; D010088:Oxidoreductases; C007076:citral
NCR ene reductase; citral reduction; isomers; protein engineering; structure-activity relationships