Quantum chemical study of mechanism and stereoselectivity of secondary alcohol dehydrogenase

Secondary alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) is a Zn- and NADP-dependent enzyme that catalyses the reversible transformation of secondary alcohols into ketones. It is of potential biocatalytic interest as it can be used in the synthesis of chiral alcohols by asymmetric reduction of ketones. In this paper, density functional theory calculations are employed to elucidate the origins of the enantioselectivity of TbSADH using a large model of the active site and considering two different substrates, 2-butanol and 3-hexanol. For these two substrates the enzyme has experimentally been shown to have the opposite enantioselectivity. The energy profiles for the reactions are calculated and the stationary points along the reaction path are characterised. The calculations first confirm that the general mechanism proposed for other alcohol dehydrogenases is energetically viable. In this mechanism, a proton is first transferred from the substrate to a histidine residue at the surface, followed by a hydride transfer to the NADP cofactor. The calculated overall energy barrier is consistent with the measured rate constant. Very importantly, the calculations are able to reproduce and rationalise the enantioselectivity of the enzyme for both substrates. The detailed characterisation of the energies and geometries of the involved transition states will be valuable in the rational engineering of TbSADH to expand its utility in biocatalysis.