Specificity and other properties of an alcohol dehydrogenase purified from Comamonas terrigena

Abstract

An NAD-dependent alcohol dehydrogenase (alcohol : NAD⁺ oxidoreductase, EC 1.1.1.1) active towards l-octan-2-ol but not towards the corresponding d-isomer was purified to homogeneity from the soil bacterium Comamonas terrigena. The enzyme is a tetramer (molecular weight 125 000–141 000) and is most active at pH 8.5–9.9. Preferred alcohol substrates are l-alkan-2-ols, activity towards which was inhibited by EDTA, 1,10-phenanthroline and 2,2′-bipyridine. The enzyme exhibits much weaker activity towards primary alcohols, symmetrical secondary alcohols and asymmetric secondary alcohols in which the hydroxyl moiety is located at positions other than C-2, and little or no activity towards d-alkan-2-ols. For l-alkan-2-ols, symmetrical secondary alcohols and primary alcohols, log K_m values decrease linearly with increase in the number of carbon atoms in the alkyl chain. A plot of standard free-energy of binding (ΔG⁰′) of substrates against the number of carbon atoms in the alkyl chain (primary alcohols) or the longer of the two portions of the alkyl chain (secondary alcohols) gives a single straight-line relationship, suggesting that hydrophobic interactions make an important contribution to substrate binding. The observed specificity was interpreted in terms of a model in which secondary alcohols interact with the enzyme through the hydrogen and hydroxyl group that participate in NAD⁺ reduction, and one of the two alkyl segments. The size of the unbound alkyl segment markedly affects V, the optimum being a single methyl unit. This specificity was correlated with that of the CS2 secondary alkylsulphohydrolase that catalyses the production of l-alkan-2-ols from d-alkan-2-yl sulphate surfactants.