Two Distinct Alcohol Dehydrogenases Participate in Butane Metabolism byPseudomonas butanovora

Abstract

The involvement of two primary alcohol dehydrogenases, BDH and BOH, in butane utilization in Pseudomonas butanovora (ATCC 43655) was demonstrated. The genes coding for BOH and BDH were isolated and characterized. The deduced amino acid sequence of BOH suggests a 67-kDa alcohol dehydrogenase containing pyrroloquinoline quinone (PQQ) as cofactor and in the periplasm (29-residue leader sequence). The deduced amino acid sequence of BDH is consistent with a 70.9-kDa, soluble, periplasmic (37-residue leader sequence) alcohol dehydrogenase containing PQQ and heme c as cofactors. BOH and BDH mRNAs were induced whenever the cell's 1-butanol oxidation activity was induced. When induced with butane, the gene for BOH was expressed earlier than the gene for BDH. Insertional disruption of bdh or boh affected adversely, but did not eliminate, butane utilization by P. butanovora. The P. butanovora mutant with both genes boh and bdh inactivated was unable to grow on butane or 1-butanol. These cells, when grown in citrate and incubated in butane, developed butane oxidation capability and accumulated 1-butanol. The enzyme activity of BOH was characterized in cell extracts of the P. butanovora strain with bdh disrupted. Unlike BDH, BOH oxidized 2-butanol. The results support the involvement of two distinct NAD ؉ -independent, PQQ-containing alcohol dehydrogenases, BOH (a quinoprotein) and BDH (a quinohemoprotein), in the butane oxidation pathway of P. butanovora.Pseudomonas butanovora (ATCC 43655) is an aerobic gramnegative 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 C 2 to C 9 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 ␤-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 O 2 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-b...

Enzyme-mediated biodegradation of long-chain n-alkanes (C32 and C40) by thermophilic bacteria

“…The fatty acid alcohols generated by terminal oxidation of n-alkanes were further oxidized to aldehydes by ADHs and similarly as reported from Bacillus sp., P. butanovora and G. thermodenitrificans NG80-2 (Li et al 2008;Vangnai and Arp 2001;Vangnai et al 2002). Thus, the ADHs were essential for growth of microorganisms in the presence of nalkanes.…”

“…Alcohols generated by terminal oxidation of n-alkanes are further oxidized to aldehydes by ADHs, therefore, provide complete biotransformation. Similar to AHy, there are few microbes that can excrete four different ADHs with species specificity, i.e., towards primary and secondary alcohols (Vangnai and Arp 2001;Vangnai et al 2002). Therefore, the degradation efficiency and intermediate products from degradation of long-chain hydrocarbons and n-alkanes rely on the type of isolate used and their enzymatic oxidation properties.…”

Monooxygenases involved in the n-alkanes metabolism by Rhodococcus sp. BCP1: molecular characterization and expression of alkB gene

“…Further metabolism probably proceeds via β-oxidation [128]. Gene disruption experiments demonstrated the involvement of two primary alcohol dehydrogenases (BOH and BDH) in the n-butane utilization [133].…”

Analysis of the promoter activities of the genes encoding three quinoprotein alcohol dehydrogenases in Pseudomonas putida HK5

“…grown on or induced with short-chain-length alcohols, while the induction of ADH-IIG is mainly restricted to 1,2-propanediol (Toyama et al, 1995). P. putida HK5 is one of only two organisms reported so far that expresses more than one type of PQQ-ADH in response to exposure to alcohols (Vangnai et al, 2002). Therefore, it is interesting to examine how the expression of the three enzymes is distinguished and regulated at a molecular level.…”

“…Although considerable research has been carried out on the biochemistry and physiology associated with the qADHs (Anthony & Williams, 2003;Chen et al, 2002;Matsushita et al, 1999;Toyama et al, 2004), much less is known about the transcriptional regulation of these enzymes. Insights into the complexity of the transcriptional regulation of qADHs have been obtained from studies of Pseudomonas aeruginosa (Gliese et al, 2004), Pseudomonas butanovora (Vangnai et al, 2002), Pseudomonas putida (Promden et al, 2008) and Methylobacterium extorquens, in which at least 28 genes have been shown to be involved in the oxidation of methanol to formaldehyde (Lidstrom et al, 1994;Springer et al, 1995). In P. aeruginosa, the transcription of a quinoprotein ethanol dehydrogenase (qEDH) promoter, namely the exaA promoter, is regulated by a two-component system: a histidine sensor kinase (ExaD), which is presumably located in the cytoplasm, and a response regulator (ExaE).…”