Identification and Characterization of a Nicotinamide Adenine Dinucleotide-Dependent p-Hydroxybenzyl Alcohol Dehydrogenase from Rhodopseudomonasacidophila M402

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A nicotinamide adenine dinucleotide-linked p-hydroxybenzyl alcohol dehydrogenase activity was demonstrated in cells of Rhodopseudomonas acidophila M402 grown on p-hydroxybenzyl alcohol as a major carbon source under anaerobic-light conditions. The enzymewas purified to homogeneity on polyacrylamide gel electrophoresis. The molecular weight of the enzymewas estimated to be 27, 000 dalton by gel filtration and the isoelectric point was pH 7.4. This dehydrogenase requires NAD⁺ as an electron acceptor and has broad specificity for aromatic alcohols. This enzyme is also capable of oxidizing aliphatic primary alcohols with NAD⁺.
While this enzyme was induced by p-hydroxybenzyl alcohol, the final preparation showed the highest affinity for vanillyl and cinnamyl alcohols rather than p-hydroxybenzyl or benzyl alcohols. Para-derivatives (methyl, methoxy, nitro or chloro) of benzyl alcohol also underwent the dehydrogenation reaction. Moreover, the enzyme was also active on m-derivatives (hydroxy, methyl, methoxy, nitro or chloro) of benzyl alcohol, 39 to 55% of the activity seen for p-hydroxybenzyl alcohol. The most interesting characteristic of this dehydrogenase is its wide affinity range for aliphatic alcohols: 1-butanol, 1-pentanol, 3-methyl-l-butanol, 1-hexanol, 1-heptanol, 1- octanol and 1-nonanol. This substrate specificity resembles that of dye-linked alcohol dehydrogenase (PMS-linked vanillyl alcohol dehydrogenase) from the same organism, but the two enzymes differ in their mode of induction, electron acceptor requirement, ammoniumrequirement for activity, pH optima, molecular weight and other aspects. A typical NAD⁺-dependent alcohol dehydrogenase oxidizes ethanol and other primary alcohols, and acetaldehyde, but not methanol or aromatic alcohols such as benzyl alcohol, hydroxybenzyl alcohol and vanillyl alcohol. Therefore, the p-hydroxybenzyl alcohol dehydrogenase reported in this paper is a new type of alcohol dehydrogenase which is different from hitherto reported alcohol dehydrogenases.