Engineering an alcohol dehydrogenase for nalancing kinetics in NADPH regeneration with 1,4-butanediol as a cosubstrate

Abstract

\u3cp\u3eCofactor regeneration using diols as smart cosubstrates is one of the most promising approaches, due to the thermodynamic preference and 0.5-equiv requirement. In order to establish an efficient NADPH regeneration system with 1,4-butanediol (1,4-BD), a NADP\u3csup\u3e+\u3c/sup\u3e-dependent alcohol dehydrogenase from Kluyveromyces polysporus (KpADH) was engineered to solve the kinetic imbalance. Several hotspots were identified through molecular dynamic simulation and subjected to saturation and combinatorial mutagenesis. Variant KpADH\u3csub\u3eV84I/Y127M\u3c/sub\u3e exhibited a lower K\u3csub\u3eM\u3c/sub\u3e of 15.1 mM and a higher k\u3csub\u3ecat\u3c/sub\u3e of 30.1 min\u3csup\u3e-1\u3c/sup\u3e than \u3csup\u3eWT\u3c/sup\u3eKpADH. The oxidation of 1,4-BD to 4-hydroxybutanal was found to be the rate-limiting step, for which the k\u3csub\u3ecat\u3c/sub\u3e/K\u3csub\u3eM\u3c/sub\u3e value of double mutant KpADH\u3csub\u3eV84I/Y127M\u3c/sub\u3e was 2.00 min\u3csup\u3e-1\u3c/sup\u3e·mM\u3csup\u3e-1\u3c/sup\u3e, 11.6-fold higher than that of \u3csup\u3eWT\u3c/sup\u3eKpADH. KpADH\u3csub\u3eV84I/Y127M\u3c/sub\u3e preferred diols with a longer chain length (C5-C6). The ratio of k\u3csub\u3ecat\u3c/sub\u3e/K\u3csub\u3eM\u3c/sub\u3e toward 2-hydroxytetrahydrofuran (2-HTHF), in comparison to 1,4-BD, in KpADH\u3csub\u3eV84I/Y127M\u3c/sub\u3e was dramatically reduced by almost 100-fold compared to \u3csup\u3eWT\u3c/sup\u3eKpADH, which was advantageous for NADPH regeneration. As much as 100 mM phenylpyruvic acid could be reduced into d-phenylalanine with 99.2% conversion in 6 h using merely 0.5 equiv of 1,4-BD. Both the improved catalytic efficiency toward 1,4-BD and the balanced k\u3csub\u3ecat\u3c/sub\u3e/K\u3csub\u3eM\u3c/sub\u3e between 1,4-BD and 2-HTHF contributed to the higher NADPH regeneration efficiency. This study provides guidance for engineering alcohol dehydrogenases for cosubstrate specificity toward diols and its application in NADPH regeneration for the preparation of chiral compounds of pharmaceutical relevance.\u3c/p\u3