A Comprehensive Understanding of Enzymatic Catalysis by Hydroxynitrile Lyases with S Stereoselectivity from the α/β-Hydrolase Superfamily: Revised Role of the Active-Site Lysine and Kinetic Behavior of Substrate Delivery and Sequential Product Release

Abstract

The highly homologous hydroxynitrile lyases from Manihot esculent (MeHNL) and Hevea brasiliensis (HbHNL) both belong to the α/β-hydrolase superfamily, and they convert cyanohydrins into the corresponding ketone (aldehyde) and hydrocyanic acid, which is important for biosynthesis for carbon–carbon formation. On the basis of extensive MM and ab initio QM/MM MD simulations, one-dimensional and two-dimensional free energy profiles on the whole enzymatic catalysis by MeHNL have been explored, and the effects of key residues around the channel on the delivery of substrate and product have been discussed. The residue Trp128 plays an important gate-switching role to manipulate the substrate access to the active site and product release. In particular, the release of acetone and HCN has been first detected to follow a stepwise mechanism. The release of HCN is quite facile, while the escape of acetone experiences a barrier of ∼10 kcal/mol. The chemical reaction is an endergonic process with a free energy barrier of...