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.