Evidence of a Distinctive Enantioselective Binding Mode for the Photoinduced Radical Cyclization of α-Chloroamides in Ene-Reductases

[Image: see text] We demonstrate here through molecular simulations and mutational studies the origin of the enantioselectivity in the photoinduced radical cyclization of α-chloroacetamides catalyzed by ene-reductases, in particular the Gluconobacter oxidans ene-reductase and the Old Yellow Enzyme 1, which show opposite enantioselectivity. Our results reveal that neither the π-facial selectivity model nor a protein-induced selective stabilization of the transition states is able to explain the enantioselectivity of the radical cyclization in the studied flavoenzymes. We propose a new enantioinduction scenario according to which enantioselectivity is indeed controlled by transition-state stability; however, the relative stability of the prochiral transition states is not determined by direct interaction with the protein but is rather dependent on an inherent degree of freedom within the substrate itself. This intrinsic degree of freedom, distinct from the traditional π-facial exposure mode, can be controlled by the substrate conformational selection upon binding to the enzyme.