Selective Catalytic Reduction of 3-phenylpropiolonitrile to Cinnamonitrile by CtOYE: A Biocatalytic Approach to Partial Alkyne Reduction

The recently discovered ene reductase from the cyanobacterium Chroococcidiopsis thermalis (CtOYE)—a member of the old yellow enzyme (OYE) family—exhibits high activity and enantioselectivity toward activated alkenes, but its activity toward alkynes remains unexplored. Using 3-phenylpropiolonitrile as a model substrate, we demonstrated that CtOYE catalyzed the partial reduction of the alkyne 3-phenylpropiolonitrile to yield exclusively (Z)-cinnamonitrile, with no over-reduction to the saturated alkane. Molecular docking revealed a unique substrate binding mode where a tyrosine residue (Y351), rather than the canonical proton donor Y183, was positioned to protonate the α-carbon, rationalizing the observed (Z)-selectivity. Molecular dynamics (MD) simulations suggested higher flexibility of the enzyme-alkyne complex compared to the enzyme-alkene product complex, which might contribute to catalytic performance. Through systematic optimization of reaction conditions (pH, temperature, and concentrations of glucose, NADP⁺, glucose dehydrogenase, and enzyme), we achieved a significant increase in conversion. Our findings highlighted the potential of CtOYE as a versatile biocatalyst for the challenging selective reduction of alkynes to valuable (Z)-alkenes, providing a sustainable alternative to metal-based catalysts.