The hydrocarbon monooxygenase (HMO) of Mycobacterium NBB4 is a member of the copper-containing membrane monooxygenase (CuMMO) superfamily, which also contains particulate methane monooxygenases (pMMOs) and ammonia monooxygenases (AMOs). CuMMOs have broad applications due to their ability to catalyse the oxidation of difficult substrates of environmental and industrial relevance. Most of our understanding of CuMMO biochemistry is based on pMMOs and AMOs as models. All three available structures are from pMMOs. These share two metal sites: a dicopper centre coordinated by histidine residues in subunit-B and a 'variable-metal' site coordinated by carboxylate and histidine residues from subunit-C. The exact nature and role of these sites is strongly debated. Significant barriers to progress have been the physiologically specialized nature of methanotrophs and autotrophic ammonia-oxidizers, lack of a recombinant expression system for either enzyme and difficulty in purification of active protein. In this study we use the newly developed HMO model system to perform site-directed mutagenesis on the predicted metal-binding residues in the HmoB and HmoC of NBB4 HMO. All mutations of predicted HmoC metal centre ligands abolished enzyme activity. Mutation of a predicted copper-binding residue of HmoB (B-H155V) reduced activity by 81 %. Mutation of a site that shows conservation within physiologically defined subgroups of CuMMOs was shown to reduce relative HMO activity towards larger alkanes. The study demonstrates that the modelled dicopper site of subunit-B is not sufficient for HMO activity and that a metal centre predicted to be coordinated by residues in subunit-C is essential for activity.
© 2014 The Authors.