PropaneMonooxygenase and NAD ⁺ -Dependent Secondary AlcoholDehydrogenase in Propane Metabolism by Gordonia sp.StrainTY-5

Abstract

A new isolate, Gordonia sp. strain TY-5, is capable of growth on propane and n-alkanes with C 13 to C 22 carbon chains as the sole source of carbon. In whole-cell reactions, significant propane oxidation to 2-propanol was detected. A gene cluster designated prmABCD, which encodes the components of a putative dinucleariron-containing multicomponent monooxygenase, including the large and small subunits of the hydroxylase, an NADH-dependent acceptor oxidoreductase, and a coupling protein, was cloned and sequenced. A mutant with prmB disrupted (prmB::Kan r ) lost the ability to grow on propane, and Northern blot analysis revealed that polycistronic transcription of the prm genes was induced during its growth on propane. These results indicate that the prmABCD gene products play an essential role in propane oxidation by the bacterium. Downstream of the prm genes, an open reading frame (adh1) encoding an NAD ؉ -dependent secondary alcohol dehydrogenase was identified, and the protein was purified and characterized. The Northern blot analysis results and growth properties of a disrupted mutant (adh1::Kan r ) indicate that Adh1 plays a major role in propane metabolism. Two additional NAD ؉ -dependent secondary alcohol dehydrogenases (Adh2 and Adh3) were also found to be involved in 2-propanol oxidation. On the basis of these results, we conclude that Gordonia sp. strain TY-5 oxidizes propane by monooxygenase-mediated subterminal oxidation via 2-propanol.Gaseous n-alkanes ranging from C 2 to C 5 are recognized as components of nonmethane hydrocarbons, and the increased concentrations of these gases in the atmosphere threaten to destabilize ecosystems through a variety of mechanisms (48). Although these gases are produced as natural intermediates of bacterial, plant, and mammalian metabolism, the main sources of pollution are natural oil seepage and oil spills (42). From a biotechnological perspective, gaseous alkanes are inexpensive carbon sources for microbial cultivation, and the enzymes participating in the oxidation pathway promise to be versatile biocatalysts.A number of microorganisms have been isolated for their ability to use gaseous n-alkanes as a sole carbon source. In the case of bacteria, these abilities have been found in some Pseudomonas strains (57) and many strains belonging to the order Actinomycetales, such as those of the genera Rhodococcus, Mycobacterium, Corynebacterium, Nocardia, and Pseudonocardia (3,15). Some of the bacteria are known to degrade various environmental pollutants (trichloroethylene, chloroform, methyl ethers, etc.) through cometabolism with gaseous alkanes (13, 52).The pathways for the oxidation of gaseous alkanes have received little attention compared with those for the microbial oxidation of methane (34) and liquid n-alkanes (24). Recently, the terminal oxidation pathway of butane (butane 3 1-butanol 3 butyraldehyde 3 butyrate) by "Pseudomonas butanovora" has been confirmed through enzymological and genetic approaches (2, 14). The first reaction is catalyzed by a soluble butane m...

Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly ‘Pseudomonas butanovora’

“…TY-6 and Pseudonocardia sp. TY-7 have suggested that they are similar to sBMO in that they are specific for short-chain C 2 -C 6 alkanes but poorly suited for methane oxidation (Kotani et al, 2003(Kotani et al, , 2006.…”

Bioremediation Approaches for Treating Low Concentrations of N-Nitrosodimethylamine in Groundwater

“…Cloning and DNA sequence analysis of the fragment expressed by strain ENV425 following growth on propane identified the gene as a homologue of the PMO present in Gordonia sp. Strain TY-5 (Kotani et al, 2003) and its putative homologues in Rhodococcus sp. Strain RHA1 (McLeod et al, 2006) and Mycobacterium smegmatis MC2 155 (Genbank number CP000480) ( Figure 22).…”

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

“…Gaseous n-alkanes ranging from C 2 to C 5 are recognized as components of non-methane hydrocarbons, and their increased concentrations in the atmosphere threaten to destabilize ecosystems through a variety of mechanisms [118,119]. Although these gases are produced as natural intermediates of bacterial, plant, and mammalian metabolism, the main sources of pollution is represented by natural oil seepages and oil spills [20,119].…”

“…Although these gases are produced as natural intermediates of bacterial, plant, and mammalian metabolism, the main sources of pollution is represented by natural oil seepages and oil spills [20,119]. From a biotechnological perspective, gaseous alkanes are inexpensive carbon sources for microbial and increases the rates of substrate turnover [25,122,123].…”

“…The TY-5 PrMO confers to this Gram-positive strain the ability to use propane as only carbon and energy source. Similarly to bmo genes and mmo genes, the prmABCD gene cluster is polycistronically transcribed and it codes for three components, a hydroxylase (large and small subunits), an oxidoreductase (PrmB) and a coupling protein (PrmD) [119]. It has been defined narrow-substrate-range propane monooxygenase because, by contrast to most of gaseous n-alkanes monooxygeanses, it cannot oxidize gaseous n-alkanes other than propane.…”