Engineering Soluble Methane Monooxygenase for Biocatalysis

  • Thomas J. SmithEmail author
  • Tim Nichol


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.


  1. Bailey LJ, Elsen NL, Pierce BS, Fox BG (2008) Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase. Protein Expr Purif 57:9–16CrossRefPubMedGoogle Scholar
  2. Banerjee R, Proshlyakov Y, Lipscomb JD, Proshlyakov DA (2015) Structure of the key species in the enzymatic oxidation of methane to methanol. Nature 518:431–434CrossRefPubMedPubMedCentralGoogle Scholar
  3. Blatny JM, Brautaset T, Winther-Larsen HC, Haugan K, Valla S (1997) Construction and use of a versatile set of broad-host-range cloning and expression vectors based on the RK2 replicon. Appl Environ Microbiol 63:370–379PubMedPubMedCentralGoogle Scholar
  4. Borodina E, Nichol T, Dumont MG, Smith TJ, Murrell JC (2007) Mutagenesis of the “leucine gate” to explore the basis of catalytic versatility in soluble methane monooxygenase. Appl Environ Microbiol 73:6460–6467CrossRefPubMedPubMedCentralGoogle Scholar
  5. Brazeau BJ, Lipscomb JD (2003) Key amino acid residues in the regulation of soluble methane monooxygenase catalysis by component B. Biochemistry 42:5618–5631CrossRefPubMedGoogle Scholar
  6. Brazeau BJ, Wallar BJ, Lipscomb JD (2003) Effector proteins from P450cam and methane monooxygenase: lessons in tuning nature’s powerful reagents. Biochem Biophys Res Commun 312:143–148CrossRefPubMedGoogle Scholar
  7. Brusseau GA, Tsien H-C, Hanson RS, Wackett LP (1990) Optimization of trichloroethylene oxidation by methanotrophs and the use of a colorimetric assay to detect soluble methane mono-oxygenase activity. Biodegradation 1:19–29CrossRefPubMedGoogle Scholar
  8. 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
  9. Callaghan AJ, Smith TJ, Slade SE, Dalton H (2002) Residues near the N-terminus of protein B control autocatalytic proteolysis and the activity of soluble methane monooxygenase. Eur J Biochem 269:1835–1843CrossRefPubMedGoogle Scholar
  10. Chang S-L, Wallar BJ, Lipscomb JD, Mayo KH (1999) Solution structure of component B from methane monooxygenase derived through heteronuclear NMR and molecular modeling. Biochemistry 38:5799–5812CrossRefPubMedGoogle Scholar
  11. Chatwood LL, Müller J, Gross JD, Wagner G, Lippard SJ (2004) NMR Structure of the flavin domain from soluble methane monooxygenase reductase from Methylococcus capsulatus (Bath). Biochemistry 43:11983–11991CrossRefPubMedGoogle Scholar
  12. Colby J, Stirling DI, Dalton H (1977) The soluble methane monooxygenase of Methylococcus capsulatus (Bath): its ability to oxygenate n-alkanes, n-alkenes, ethers, and alicyclic, aromatic and heterocyclic compounds. Biochem J 165:395–402CrossRefPubMedPubMedCentralGoogle Scholar
  13. Coleman NV, Bui NB, Holmes AJ (2006) Soluble di-iron monooxygenase gene diversity in soils, sediments and ethene enrichments. Environ Microbiol 8:1228–1239CrossRefPubMedGoogle Scholar
  14. Coufal DE, Blazyk JL, Whittington DA, Wu WW, Rosenzweig AC, Lippard SJ (2000) Sequencing and analysis of the Methylococcus capsulatus (Bath) soluble methane monooxygenase genes. Eur J Biochem 267:2174–2185CrossRefPubMedGoogle Scholar
  15. Dedysh SN, Knief C, Dunfield PF (2005) Methylocella species are facultatively methanotrophic. J Bacteriol 187:4665–4670CrossRefPubMedPubMedCentralGoogle Scholar
  16. 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
  17. Elango N, Radmakrishnan R, Froland WA, Wallar BJ, Earhart CA, Lipscomb JD, Ohlendorf DH (1997) Crystal structure of the hydroxylase component of methane monooxygenase from Methylosinus trichosporium OB3b. Protein Sci 6:556–568CrossRefPubMedPubMedCentralGoogle Scholar
  18. Fox BG, Liu Y, Dege JE, Lipscomb JD (1991) Complex formation between the protein components of methane monooxygenase from Methylosinus trichosporium OB3b. J Biol Chem 266:540–550PubMedGoogle Scholar
  19. George AR, Wilkins PC, Dalton H (1996) A computational investigation of the possible substrate binding sites in the hydroxylase of soluble methane monooxygenase. J Molec Catal B 2:103–113CrossRefGoogle Scholar
  20. Green J, Dalton H (1989) Substrate specificity of soluble methane monooxygenase – mechanistic implications. J Biol Chem 264:17698–17703PubMedGoogle Scholar
  21. Hakemian AS, Rosenzweig AC (2007) The biochemistry of methane oxidation. Ann Rev Biochem 76:223–241CrossRefPubMedGoogle Scholar
  22. Jahng D, Wood TK (1994) Trichloroethylene and chloroform degradation by a recombinant pseudomonad expressing soluble methane monooxygenase from Methylosinus trichosporium OB3b. Appl Environ Microbiol 60:2473–2482PubMedPubMedCentralGoogle Scholar
  23. Jahng D, Kim CS, Hanson RS, Wood TK (1996) Optimization of trichloroethylene degradation using soluble methane monooxygenase of Methylosinus trichosporium OB3b expressed in recombinant bacteria. Biotechnol Bioeng 51:349–359CrossRefPubMedGoogle Scholar
  24. Jiang H, Chen Y, Jiang P, Zhang C, Smith TJ, Murrell JC, Xing X-H (2010) Methanotrophs: multifunctional bacteria with promising applications in environmental bioengineering. Biochem Eng J 49:277–288CrossRefGoogle Scholar
  25. Johnson GR, Olsen RH (1995) Nucleotide-sequence analysis of genes encoding a toluene benzene-2-monooxygenase from Pseudomonas sp. strain JS150. Appl Environ Microbiol 61:3336–3346PubMedPubMedCentralGoogle Scholar
  26. Leahy JG, Batchelor PJ, Morcomb SM (2003) Evolution of the soluble diiron monooxygenases. FEMS Microbiol Rev 27:449–479CrossRefPubMedGoogle Scholar
  27. Lee SJ, McCormick MS, Lippard SJ, Cho US (2013) Control of substrate access to the active site in methane monooxygenase. Nature 494:380–384CrossRefPubMedPubMedCentralGoogle Scholar
  28. Lewis JC, Coelho PS, Arnold FH (2011) Enzymatic functionalization of carbon–hydrogen bonds. Chem Soc Rev 40:2003–2021CrossRefPubMedGoogle Scholar
  29. Lindner AS, Adriaens P, Semrau JD (2000) Transformation of ortho-substituted biphenyls by Methylosinus trichosporium OB3b: substituent effects on oxidation kinetics and product formation. Arch Microbiol 174:35–41CrossRefPubMedGoogle Scholar
  30. Lipscomb JD (1994) Biochemistry of the soluble methane monooxygenase. Ann Rev Microbiol 48:371–399CrossRefGoogle Scholar
  31. Lloyd JS, Bhambra A, Murrell JC, Dalton H (1997) Inactivation of the regulatory protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath) by proteolysis can be overcome by a Gly to Gln modification. Eur J Biochem 248:72–79CrossRefPubMedGoogle Scholar
  32. Lloyd JS, DeMarco P, Dalton H, Murrell JC (1999a) Heterologous expression of soluble methane monooxygenase genes in methanotrophs containing only particulate methane monooxygenase. Arch Microbiol 171:364–370CrossRefPubMedGoogle Scholar
  33. Lloyd JS, Finch R, Dalton H, Murrell JC (1999b) Homologous expression of soluble methane monooxygenase genes in Methylosinus trichosporium OB3b. Microbiology 145:461–470CrossRefPubMedGoogle Scholar
  34. 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.
  35. Martin H, Murrell JC (1995) Methane monooxygenase mutants of Methylosinus trichosporium constructed by marker-exchange mutagenesis. FEMS Microbiol Lett 127:243–248CrossRefGoogle Scholar
  36. Merkx M, Lippard SJ (2002) Why OrfY? Characterization of MmoD, a long overlooked component of the soluble methane monooxygenase from Methylococcus capsulatus (Bath). J Biol Chem 277:5858–5865CrossRefPubMedGoogle Scholar
  37. Nordlund P, Dalton H, Ecklund H (1992) The active-site structure of methane monooxygenase is closely related to the binuclear iron center of ribonucleotide reductase. FEBS Lett 307:257–262CrossRefPubMedGoogle Scholar
  38. Pikus JD, Studts JM, McClay K, Steffan RJ, Fox BG (1997) Changes in the regiospecificity of aromatic hydroxylation produced by active site engineering in the diiron enzyme toluene 4-monooxygenase. Biochemistry 36:9283–9289CrossRefPubMedGoogle Scholar
  39. Richards AO, Stanley SH, Suzuki M, Dalton H (1994) The biotransformation of propylene to propylene oxide by Methylococcus capsulatus (Bath): 3. Reactivation of inactivated whole cells to give a high productivity system. Biocatalysis 8:253–267CrossRefGoogle Scholar
  40. Rosenzweig AC, Frederick CA, Lippard SJ, Nordlund P (1993) Crystal structure of a bacterial nonheme iron hydroxylase that catalyzes the biological oxidation of methane. Nature 366:537–543CrossRefPubMedGoogle Scholar
  41. Rosenzweig AC, Nordlund P, Takahara PM, Frederick CA, Lippard SJ (1995) Geometry of the soluble methane monooxygenase catalytic diiron center in two oxidation states. Chem Biol 2:409–418CrossRefPubMedGoogle Scholar
  42. Rosenzweig AC, Brandstetter H, Whittington DA, Nordlund P, Lippard SJ, Frederick CA (1997) Crystal structure of the methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): implications for substrate gating and component interactions. Proteins 29:141–152CrossRefPubMedGoogle Scholar
  43. Saeki H, Furuhashi K (1994) Cloning and characterisation of the Nocardia corallina B-276 gene cluster encoding alkene monooxygenase. J Ferment Bioeng 78:399–406CrossRefGoogle Scholar
  44. Semrau JD, Jagadevan S, DiSpirito AA, Khalifa A, Scanlan J, Bergman BH, Freemeier BC, Baral BS, Bandow NL, Vorobev A, Haft DH, Vuilleumier S, Murrell JC (2013) Methanobactin and MmoD work in concert to act as the ‘copper switch’ in methanotrophs. Environ Microbiol 15:3077–3086PubMedGoogle Scholar
  45. Shu L, Nesheim JC, Kauffmann K, Münck E, Lipscomb JD, Que L (1997) An FeIV2O2 diamond core structure for the key intermediate Q of methane monooxygenase. Science 275:515–517CrossRefPubMedGoogle Scholar
  46. Sjöberg B-M (1997) Ribonucleotide reducatases – a group of enzymes with different metallosites and a similar reaction mechanism. Struct Bond 88:139–173CrossRefGoogle Scholar
  47. Smith TJ, Dalton H (2004) Biocatalysis by methane monooxygenase and its implications for the petroleum industry. Stud Surface Sci Catal 151:177–192CrossRefGoogle Scholar
  48. Smith TJ, Murrell JC (2009) Methanotrophy/methane oxidation. In: Schaechter M (ed) Encyclopedia of microbiology, vol 3. Elsevier, San Diego, CA, pp 293–298CrossRefGoogle Scholar
  49. Smith TJ, Murrell JC (2011) Mutagenesis of soluble methane monooxygenase. Methods Enzymol 495:135–147CrossRefPubMedGoogle Scholar
  50. Smith TJ, Slade SE, Burton NP, Murrell JC, Dalton H (2002) Improved system for protein engineering of the hydroxylase component of soluble methane monooxygenase. Appl Environ Microbiol 68:5265–5273CrossRefPubMedPubMedCentralGoogle Scholar
  51. Stafford GP, Scanlan J, McDonald IR, Murrell JC (2003) rpoN, mmoR and mmoG, genes involved in regulating the expression of soluble methane monooxygenase in Methylosinus trichosporium OB3b. Microbiology 149:1771–1784CrossRefPubMedGoogle Scholar
  52. Stanley SH, Prior SD, Leak DJ, Dalton H (1983) Copper stress underlies the fundamental change in intracellular location of methane monooxygenase in methane-oxidizing organisms – studies in batch and continuous cultures. Biotechnol Lett 5:487–492CrossRefGoogle Scholar
  53. Wang W, Lippard SJ (2014) Diiron oxidation state control of substrate access to the active site of soluble methane monooxygenase mediated by the regulatory component. J Am Chem Soc 136:2244–2247CrossRefPubMedPubMedCentralGoogle Scholar
  54. West CA, Salmond GPC, Dalton H, Murrell JC (1992) Functional expression in Escherichia coli of protein B and protein C from soluble methane monooxygenase of Methylococcus capsulatus (Bath). J Gen Microbiol 138:1301–1307CrossRefPubMedGoogle Scholar
  55. Whittington DA, Rosenzweig AC, Frederick CA, Lippard SJ (2001) Xenon and halogenated alkanes track putative substrate binding cavities in the soluble methane monooxygenase hydroxylase. Biochemistry 40:3476–3482CrossRefPubMedGoogle Scholar
  56. Zhou NY, Jenkins A, Chion CKNCK, Leak DJ (1999) The alkene monooxygenase from Xanthobacter strain Py2 is closely related to aromatic monooxygenases and catalyzes aromatic monohydroxylation of benzene, toluene, and phenol. Appl Environ Microbiol 65:1589–1595PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Biomolecular Sciences Research CentreSheffield Hallam UniversitySheffieldUK

Personalised recommendations