Abstract
The members of the Old Yellow Enzyme (OYE) family are capable of catalyzing the asymmetric reduction of (E/Z)-citral to (R)-citronellal—a key intermediate in the synthesis of L-menthol. The applications of OYE-mediated biotransformation are usually hampered by its insufficient enantioselectivity and low activity. Here, the (R)-enantioselectivity of Old Yellow Enzyme from Saccharomyces cerevisiae CICC1060 (OYE2y) was enhanced through protein engineering. The single mutations of OYE2y revealed that the sites R330 and P76 could act as the enantioselectivity switch of OYE2y. Site-saturation mutagenesis was conducted to generate all possible replacements for the sites R330 and P76, yielding 17 and five variants with improved (R)-enantioselectivity in the (E/Z)-citral reduction, respectively. Among them, the variants R330H and P76C partly reversed the neral derived enantioselectivity from 32.66% e.e. (S) to 71.92% e.e. (R) and 37.50% e.e. (R), respectively. The docking analysis of OYE2y and its variants revealed that the substitutions R330H and P76C enabled neral to bind with a flipped orientation in the active site and thus reverse the enantioselectivity. Remarkably, the double substitutions of R330H/P76M, P76G/R330H, or P76S/R330H further improved (R)-enantioselectivity to >99% e.e. in the reduction of (E)-citral or (E/Z)-citral. The results demonstrated that it was feasible to alter the enantioselectivity of OYEs through engineering key residue distant from active sites, e.g., R330 in OYE2y.
Highlights
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Grain and Oil Products Quality Inspection Center of Zhejiang Province, Hangzhou 310012, China; Academic Editor: Stefano Serra
Based on the strategy of site-saturated mutagenesis, several groups have successfully increased (R)-enantioselectivity in the (E/Z)-citral reduction, and our study further demonstrated that it was feasible to achieve the full (R)-enantioselectivity in the Old Yellow Enzyme (OYE)-mediated (E/Z)-citral reduction through protein engineering
Significant increase of (R)-enantioselectivity in the (E/Z)-citral reduction was achieved by saturation mutagenesis of P76 and R330 in OYE2y
Summary
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Grain and Oil Products Quality Inspection Center of Zhejiang Province, Hangzhou 310012, China; Academic Editor: Stefano Serra. The members of the Old Yellow Enzyme (OYE) family are capable of catalyzing the asymmetric reduction of (E/Z)-citral to (R)-citronellal—a key intermediate in the synthesis of. Site-saturation mutagenesis was conducted to generate all possible replacements for the sites R330 and P76, yielding 17 and five variants with improved (R)-enantioselectivity in the (E/Z)-citral reduction, respectively. The variants R330H and P76C partly reversed the neral derived enantioselectivity from 32.66% e.e. The docking analysis of OYE2y and its variants revealed that the substitutions R330H and P76C enabled neral to bind with a flipped orientation in the active site and reverse the enantioselectivity. The results demonstrated that it was feasible to alter the enantioselectivity of OYEs through engineering key residue distant from active sites, e.g., R330 in OYE2y
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