Novel alcohol dehydrogenase activation by the choline component of Deep Eutectic Solvents

Alcohol dehydrogenases (ADHs) are important enzymes for biocatalytic applications, both in the enantioselective reduction of ketones and the oxidation of alcohols to generate aldehydes or carboxylic acids. Their application has been hindered by their incompatibility with organic solvents. Deep Eutectic Solvents (DESs) are a neoteric solvent medium that can stabilise both ADHs and their NAD(P)⁺ cofactors. The stabilising effect of DES allows wider application of ADHs in biocatalytic synthesis. The behaviour of enzymes in DES is not fully understood; some are inhibited while others become activated, depending on the DES composition. Herein, we explored the effect of choline chloride (ChCl)/glycerol (Gly) based DES solutions on Horse Liver ADH over an extended concentration range. This revealed a hitherto unreported range where ADH was activated by 40–50%. Examination of DES components showed that ChCl alone was responsible for this activation. Structure activity studies revealed that the activation was due solely to the quaternary nitrogen moiety of ChCl. Other alkylammonium salts showed even greater activation (ca. two-fold for tetrabutylammonium chloride), depending on their alkyl substituents. This work reports, for the first time, non-covalent activation of an ADH by ChCl, an endogenous cellular compound. Molecular modelling indicated that activation was due to alkylammonium ions binding to the protein at a location centered around aspartate-273. These findings show that HLADH activation in DES solutions is not a bulk solvent effect but rather a net result of the activatory or inhibitory effects of individual DES components. The biocatalytic and physiological relevance of ADH activation is discussed.