Activity and Enantioselectivity of Hydroxynitrile Lyase from Granulicella tundricula: Computational Insights

Hydroxynitrile lyase from Granulicella tundricola (GtHNL) is a Mn(II)-dependent enzyme that catalyzes the R-selective synthesis of cyanohydrins. Mn(II) is pentacoordinated by the enzyme in a distorted octahedral 17 electron complex. The aldehyde coordinates to the vacant site of Mn(II), and together with H106 as a base, the Lewis acid catalyzes the enantioselective synthesis. This study performed in the framework of density functional theory aims to elucidate the reaction mechanism examining the source of enantioselectivity of GtHNL in the reaction between benzaldehyde and HCN to obtain R-mandelonitrile. Starting from a model built from the available crystallographic structure with its distorted octahedral Mn(II), different binding modes of the substrates are first evaluated, followed by the calculation of the mechanistic pathway (transition states and intermediates) from each complex obtained. The effect of amino acid mutations at the active site on the enzyme reactivity was further discussed. The calculated energies are consistent with experimental observations, and the analysis of transition-state geometries provides insights into the origins of enantioselectivity of the enzyme.