Engineering the hydrogen transfer pathway of an alcohol dehydrogenase to increase activity by rational enzyme design

Abstract

The reduction catalyzed by an alcohol dehydrogenase (ADH) consists of two steps, hydride transfer (HT) from the NAD(P)H cofactor to the substrate followed by proton transfer (PT) originating from the aqueous solvent. We report a rational enzyme design strategy in the quest to improve the catalytic efficiency of an ADH by targeting appropriately chosen remote residues that function in PT. An ADH from Thermoanaerobacter brockii (TbSADH) was selected as the catalyst involving the enantioselective reduction of a bulky prochiral ketone (4-chlorophenyl)(pyridin-2-yl)-methanone (CPMK). Previous studies demonstrated that the protonated histidine residue H42 in TbSADH plays a key role in PT. In order to explore any change of the HT mechanism possibly affecting enzyme activity, the residue H42 was rationally mutated to other amino acids. After experimental validation, the substitution H42T was found to confer enhanced reduction ability in the transformation of CPMK to the corresponding (S)-configured product with a high activity (kcat/Km = 787.38 s−1 mM−1), a 9-fold increase relative to that of the starting enzyme. We performed molecular dynamics (MD) simulations together with quantum mechanical (QM) calculations which shed light on the origin of the improved activity. It revealed that the enlarged volume of the substrate-binding pocket brought by mutation H42T appears to be the main reason for the increase of activity. QM calculations displayed that the water-linked hydrogen bond network through H42T lead to lower activate energy. Our study indicates that the key residues participating in the catalytic proton transfer process may also serve as hotspots for engineering the activity of ADHs. The detailed information that we have learned about the mechanism can be exploited in the study of other NAD(P)H-dependent dehydrogenases.