Two Distinct Alcohol Dehydrogenases Participate in Butane Metabolism by Pseudomonas butanovora

Pseudomonas butanovora (ATCC 43655) is an aerobic gram-negative proteobacterium closely related to the genera Thauera and Azoarcus as shown by analysis of its 16S rRNA (1). This organism has been classified in the genus Pseudomonas based on its morphology, physiology, and biochemistry (39, 40). P. butanovora was isolated from activated sludge from an oil-refining company for the purpose of generating biomass from n-alkanes (39, 40). P. butanovora can derive energy for growth from C2 to C9 n-alkanes and any of their oxidation products as well as from a variety of other carbon sources (39, 40). Butane-grown P. butanovora can oxidize some chlorinated hydrocarbons by cometabolism through the action of a monooxygenase (18) and thus may have applications in bioremediation schemes. The pathway for the oxidation of butane in P. butanovora proceeds primarily from butane to 1-butanol, to butyraldehyde, to butyrate (2), and then probably to the β-oxidation pathway of fatty acid oxidation. As in other alkane utilizers (3, 27, 36), in P. butanovora the oxidation of the alkane (butane) is initiated by the action of a monooxygenase (19). Each intermediate in the pathway accumulated in the presence of appropriate inhibitors, supported cell growth, and stimulated O2 consumption (2). The presence of a terminal butane oxidation pathway (i.e., production of 1-butanol) in P. butanovora was indicated (2). Although butane-grown cells consumed 2-butanol, 2-butanol production (indicative of a subterminal oxidation pathway) was not demonstrated, even in the presence of appropriate inhibitors of 2-butanol consumption. For P. butanovora four different alcohol dehydrogenases (ADHs) with different specificities towards primary and secondary alcohols were identified on native gels stained for activity (45). Among these ADHs, 1-butanol dehydrogenase (BDH) was characterized biochemically (45). BDH enzyme activity was detected in butane- and 1-butanol-grown cells but not lactate-grown cells. BDH is a soluble, periplasmic, type II NAD+-independent quinohemoprotein that contains 1.0 mol of pyrroloquinoline quinone (PQQ) and 0.25 mol of ratio heme c as prosthetic groups and exists as a monomer with an apparent molecular mass of 67 kDa (45). The liquid-alkane metabolisms of other gram-negative proteobacteria such as Pseudomonas oleovorans and Pseudomonas putida (6, 10, 11) and Acinetobacter sp. (14, 23, 28, 29) have been studied. From these studies and from our research with the gaseous alkane utilizer P. butanovora, several differences among the enzymes involved in the metabolism of alkanes are starting to emerge. First, the essential enzymes in the utilization of the alkane differ in cellular location among these proteobacteria and P. butanovora. For example, the oxidation of octane in P. oleovorans and Acinetobacter sp. strain ADP1 proceeds through membrane-bound monooxygenases. In P. butanovora the oxidation of butane proceeds via a soluble alkane monooxygenase (D. Arp Laboratory, unpublished results). Second, the oxidation of the resulting alcohol in n-alkane metabolism proceeds via diverse enzymes depending on the bacterium. In P. oleovorans there is an inducible ADH which is a flavin-containing enzyme (43), while for P. butanovora BDH, an inducible PQQ- and heme c-containing ADH, has been described (45). Constitutive ADH activity was not detected in P. butanovora (33), while Acinetobacter sp. strain HO1-N has at least one constitutive ADH activity (35). Third, these alkane-utilizing proteobacteria have different gene arrangements. The genes coding for the enzymes in alkane metabolism in P. oleovorans are clustered in an operon (alk) (43), and in Acinetobacter sp. strain ADP1, the genes for alkane metabolism are spread through its chromosome (28, 29). The genetic arrangement of the genes for alkane metabolism in P. butanovora has not yet been determined. However, in P. butanovora, the genes for alkane metabolism may be arranged in different operons as in Acinetobacter sp. strain ADP1, since each enzyme activity in the butane oxidation pathway is induced independently by the substrate being oxidized (33). This study suggests that the NAD+-independent PQQ alcohol dehydrogenase BOH (a quinoprotein) is linked to butane metabolism in conjunction with the previously characterized BDH (a quinohemoprotein [45]). The inferred amino acid sequence of the gene coding for BOH (boh) showed a polypeptide similar to periplasmic PQQ-containing ADHs in other bacteria. The expression of boh was compared to the expression of bdh (encoding BDH). Inactivation of each gene coding for BOH or BDH decreased the rate of growth on butane, and inactivation of both genes eliminated the growth of P. butanovora on butane and on 1-butanol.