How Ene-Reductases Generate Both (E)- and (Z)-Alkenes From Alkynes

Ene-reductases (ERs) catalyze stereoselective anti-addition to activated alkenes, a mechanism that intrinsically favors trans products and appears incompatible with alkyne semihydrogenation, particularly for the formation of ( Z )-alkenes. Here, we investigate how ERs achieve both ( E )- and ( Z )-selective alkyne reduction within this conserved catalytic framework. Screening of a 51-member ER library reveals distinct kinetic partitioning between alkyne-to-alkene and alkene-to-alkane reduction steps, governed by differential stabilization of the substrate and intermediate within the active site. Guided by this insight, engineering of OYE3 suppresses over-reduction and enables rapid, selective formation of ( E )-alkenes under optimized reaction conditions. Unexpectedly, selected ERs also produce ( Z )-alkenes from cyano-substituted alkynes. Combined mutagenesis and molecular dynamics analyses exclude noncanonical proton donors and further suggest a substrate reorientation mechanism involving a ~180° flip within the active pocket. Together, these findings point to a unified mechanistic framework that may account for how ERs generate both ( E )- and ( Z )-alkenes from alkynes, and provide a working hypothesis for the origin of stereochemical divergence in ER-catalyzed alkyne reduction.