Engineering Soluble Methane Monooxygenase for Biocatalysis
Soluble methane monooxygenase (sMMO) has more than 100 known substrates in addition to its natural substrate methane. It is one of the most versatile and powerful biological oxidation catalysts, although regioselectivity and enantioselectivity with the wild-type enzyme are generally low. Protein engineering of sMMO has presented a major challenge because attempts to express the active site-containing hydroxylase component of the enzyme in Escherichia coli have to date been unsuccessful. Use of a homologous expression system, in which the enzyme is expressed in a methane-oxidising bacterium where the chromosomal copy of the sMMO genes is deleted, has allowed construction, expression and purification of active mutant enzymes. This work has given the first indications for the roles of specific amino acids in the hydroxylase component of sMMO in substrate oxidation and control of regioselectivity. Most recently, an enzyme with significantly improved activity and regioselectivity with a diaromatic substrate has been prepared. It is hoped that future work will produce recombinant sMMO derivatives developed for the production of high-value fine and bulk chemicals.
TJS gratefully acknowledges funding for work on expression and mutagenesis of sMMO from the Biotechnology and Biological Sciences Research Council and the Biomolecular Sciences Research Centre at Sheffield Hallam University.
- Burrows KJ, Cornish A, Scott D, Higgins IJ (1984) Substrate specificities of the soluble and particulate methane mono-oxygenases of Methylosinus trichosporium OB3b. J Gen Microbiol 130:3327–3333Google Scholar
- DeWitt JG, Bentsen JG, Rosenzweig AC, Hedman B, Green J, Pilkington S, Papaefthymiou GC, Dalton H, Hodgson KO, Lippard SJ (1991) X-ray absorption, Mössbauer, and EPR studies of the dinuclear iron center in the hydroxylase component of methane monooxygenase. J Am Chem Soc 113:9219–9235CrossRefGoogle Scholar
- Lock M, Nichol T, Murrell JC, Smith TJ (2017) Mutagenesis and expression of methane monooxygenase to alter regioselectivity with aromatic substrates. FEMS Microbiol Lett 364. https://doi.org/10.1093/femsle/fnx137