Cyclic Alcohol Dehydrogenase Secondary Alcohol Dehydrogenase Membrane Bound

Cyclic alcohol ^ Cyclic ketone

Aliphatic secondary alcohol ^ Aliphatic ketone

Numerous investigations on microbial and enzymatic oxidation of cyclic alcohols have been carried out with various bacteria. Most of them are related to NAD-dependent cyclohexanol dehydrogenase (EC 1.1.1.245) from pseudomonads and Acinetobacter sp. [23-26]. Microbial cyclopentanol oxidation was also studied in Pseudomonas sp. and NAD-dependent cyclopentanol dehydrogenase (EC 1.1.1.163) was indicated as the responsible enzyme in the first step of the oxidative metabolism of cyclic alcohol [27]. Cyclohexanol oxidation was also observed by a secondary alcohol oxidase from Pseudomonas sp. in which hydrogen peroxide-producing flavin-dependent enzyme is functional [28-30]. The microorganisms described in the abovementioned papers do not accumulate oxidation products outside the cells, because most enzymes dealt with are cytoplasmic enzymes. Nothing has been reported, therefore, about membrane-bound cyclic alcohol dehydrogenase located on the outer surface of the cytoplasmic membrane allowing accumulation of the oxidation products outside the cells.

A quinoprotein catalyzing oxidation of cyclic alcohols was found in the membrane fraction of acetic acid bacteria. After extensive screening, Gluconobacter frateurii CHM 9 was selected. EDTA treatment with the membrane fraction indicated that the membrane-bound cyclic alcohol dehydrogenase is a PQQ-dependent dehydrogenase. From the membrane fraction, PQQ-dependent cyclic alcohol dehydrogenase was purified [31]. In contrast, from the cytoplasmic fraction of the same organism, an NAD-dependent cyclic alcohol dehydrogenase was purified and crystallized. The substrate specificities of the two differently localized enzymes showed an interesting contrast with each other, suggestive of their different physiological roles in the organisms. Unlike the already known cytosolic NAD(P)H-dependent alcohol-aldehyde or alcohol-ketone oxidoreductases, the PQQ-dependent enzyme is unable to catalyze reduction of cyclic ketones or aliphatic ketones to cyclic alcohols or aliphatic secondary alcohols.

PQQ-dependent cyclic alcohol dehydrogenase was solubilized from the membrane fraction by differential solubilization to eliminate the predominantly existing ADH III and the two subsequent steps of column chromatography gave a homogeneous enzyme preparation. The purified PQQ-dependent enzyme has a molecular mass of 83 kDa in SDS-PAGE. A wide variety of cyclic alcohols are oxidized with the PQQ-dependent enzyme. Once the enzyme was solubilized, only the PMS-DCIP assay was valid, because the enzyme does not contain any heme c component. When the PQQ-dependent enzyme was compared with the cytosolic NAD-dependent enzyme, the reaction rate and the total enzyme activity found in cyclic alcohol oxidation with the PQQ-dependent enzyme was more than 100 times higher than that with the cytosolic NAD-dependent enzyme.

The NAD-dependent enzyme is unfavorable for oxidation reactions, but is favorable for the reduction of cyclic ketones to cyclic alcohols or aliphatic ketones to aliphatic secondary alcohols. Thus, the NAD-dependent enzyme makes no contribution to cyclic alcohol oxidation, and reduction of cyclic ketones and aliphatic ketones to their corresponding alcohols is favored in the presence of NADH.

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