Characterization of three propane-inducible oxygenases in Mycobacterium sp. strain ENV421

Abstract

Aims: Mycobacterium sp. strain ENV421 has the ability to cometabolize a variety of chemicals following growth on propane as a sole source of carbon and energy. In this study, we used genetic and biochemical approaches to identify and characterize multiple propane-inducible oxygenase genes in ENV421.

Methods and results: Gene clusters encoding a CYP153-type cytochrome P450 oxygenase (P450), an AlkB-type alkane monooxygenase (AlkB) and a soluble diiron monooxygenase were identified and cloned using degenerate PCR primers. Reverse transcriptase PCR showed that all three gene clusters were induced by propane. Substrate specificity studies revealed that despite the fact that ENV421 does not grow on medium length alkanes, cloned versions of both the AlkB and P450 were capable of octane oxidation, forming n-octanol. Additionally, the P450 oxygenase had the ability to oxidize indole, medium-to-long-chain alkylbenzenes and a variety of para-substituted methylalkylbenzenes. Successful cloning and expression of the diiron monooxygenase was not achieved, so its substrate specificity could not be determined.

Conclusions: Three types of short-to-medium-chain alkane oxygenases were induced by propane in ENV421, even though the cloned AlkB and P450 oxygenases did not oxidize propane. Curiously, they both oxidized octane, which is not a growth substrate for ENV421. Furthermore, the P450, typically operating as terminal alkane hydroxylase, exhibited interesting regio- and stereoselectivity, catalysing linear alkanes, alkylbenzenes and indole.

Significance and impact of the study: This study describes the first example of a propane-inducible P450 with a broad substrate specificity, including linear alkanes, alkylbenzenes and a multiring compound. The induction of three distinct oxygenase classes by propane is also an interesting finding because it might explain why propane serves as an effective stimulant that promotes the biodegradation of a various environmental contaminants.