Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamily

Abstract

The copper membrane monooxygenases (CuMMOs) are an important group of enzymes in environmental science and biotechnology. Areas of relevance include the development of green chemistry for sustainable exploitation of methane (CH 4 ) reserves, remediation of chlorinated hydrocarbon contamination and monitoring human impact in the biogeochemical cycles of CH 4 and nitrogen. Challenges for all these applications are that many aspects of the ecology, physiology and structure-function relationships in the CuMMOs are inadequately understood. Here, we describe genetic and physiological characterization of a novel member of the CuMMO family that has an unusual physiological substrate range (C 2 -C 4 alkanes) and a distinctive bacterial host (Mycobacterium). The Mycobacterial CuMMO genes (designated hmoCAB) were amenable to heterologous expression in M. smegmatis-this is the first example of recombinant expression of a complete and highly active CuMMO enzyme. The apparent specific activity of recombinant cells containing hmoCAB ranged from 2 to 3 nmol min -1 per mg protein on ethane, propane and butane as substrates, and the recombinants could also attack ethene, cis-dichloroethene and 1,2-dichloroethane. No detectable activity of recombinants or wild-type strains was seen with methane. The specific inhibitor allylthiourea strongly inhibited growth of wild-type cells on C 2 -C 4 alkanes, and omission of copper from the medium had a similar effect, confirming the physiological role of the CuMMO for growth on alkanes. The hydrocarbon monooxygenase provides a new model for studying this important enzyme family, and the recombinant expression system will enable biochemical and molecular biological experiments (for example, site-directed mutagenesis) that were previously not possible.

Gammaproteobacterial Methanotrophs Dominate Cold Methane Seeps in Floodplains of West Siberian Rivers

“…Remarkably, sequences related to the alkane monooxygenases of Nocardioides and Mycobacterium were amplified. They are only distantly related to known pmoA and amoA sequences, and the primer sets used in this study did not amplify alkane monooxygenase genes from pure cultures of Nocardioides and Mycobacterium (75,76). The fact that these sequences were obtained from the methane seep samples may be indicative of a high number of alkane oxidizers in these environments.…”

Mutagenesis of the hydrocarbon monooxygenase indicates a metal centre in subunit-C, and not subunit-B, is essential for copper-containing membrane monooxygenase activity

“…The strongly conserved protein sequences across the bacterial CuMMOs, as well as their overlapping substrate ranges and shared inhibitor profiles (Bédard & Knowles, 1989;Culpepper & Rosenzweig, 2012;Holmes et al, 1995;Op den Camp et al, 2009;Semrau et al, 2010), validate the use of the more genetically amenable HMOs as a useful alternative model to investigate CuMMO biochemistry (Coleman et al, 2012). Here we use the HMO expression system as a model to test the biological importance of the subunit-B and -C sites.…”

One millimetre makes the difference: high-resolution analysis of methane-oxidizing bacteria and their specific activity at the oxic–anoxic interface in a flooded paddy soil

“…Numerous sequences are located at an intermediate position between the pmoA gene of MOB and the amoA gene of ammonia oxidizers. The substrate of the enzymes encoded by these sequences remains uncertain, with the exception of a few proven methane (Stoecker et al, 2006;Dunfield et al, 2007) and alkane oxidizers (Sayavedra-Soto et al, 2011;Coleman et al, 2012).…”