Characterization of the substrate scope of an alcohol dehydrogenase commonly used as methanol dehydrogenase.

Many alcohol dehydrogenases (ADHs) catalyze oxidation of a broad scope of alcohols. When an NAD-dependent ADH oxidizes methanol, albeit at a poor rate, it may be treated as methanol dehydrogenase (MDH). One ADH from Geobacillus stearothermophilus DSM 2334 (GsADH) has been widely used as MDH, but its actual substrate scope remains less characterized. Here we purified recombinant GsADH from Escherichia coli and determined its crystal structure. We collected kinetics data of this enzyme towards a number of short chain alcohols, and found that isopropanol is by far the most favorable substrate. Moreover, molecular docking analysis suggested that substrate preference is mainly attributed to the conformer energy of the protein-substrate complex. Our data clarified the substrate scope of GsADH and provided structural insights, which may facilitate more efficient cofactor regeneration and rational metabolic engineering.