Abstract
Methane monooxygenase (MMO), which exists in particulate (pMMO) or soluble forms (sMMO) in methanotrophic bacteria, is an industrially promising enzyme that catalyses oxidation of low-reactive methane and other carbon feedstocks into methanol and their corresponding oxidation products. However, the simple, fast and high-yield production of functionally active MMO, which has so far been unsuccessful despite diverse approaches based on either native methanotroph culture or recombinant expression systems, remains a major challenge for its industrial applications. Here we developed pMMO-mimetic catalytic protein constructs by genetically encoding the beneficial reassembly of catalytic domains of pMMO on apoferritin as a biosynthetic scaffold. This approach resulted in high-yield synthesis of stable and soluble protein constructs in Escherichia coli, which successfully retain enzymatic activity for methanol production with a turnover number comparable to that of native pMMO.
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Data availability
The data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.
References
Elliott, S. J. et al. Regio- and stereoselectivity of particulate methane monooxygenase from Methylococcus capsulatus (Bath). J. Am. Chem. Soc. 119, 9949–9955 (1997).
Merkx, M. et al. Dioxygen activation and methane hydroxylation by soluble methane monooxygenase: a tale of two irons and three proteins. Angew. Chem. Int. Ed. 40, 2782–2807 (2001).
Park, D. & Lee, J. Biological conversion of methane to methanol. Korean J. Chem. Eng. 30, 977–987 (2013).
Wang, V. C.-C. et al. Alkane oxidation: methane monooxygenase, related enzymes, and their biomimetics. Chem. Rev. 117, 8574–8621 (2017).
Chan, S. I. & Yu, S. S.-F. Controlled oxidation of hydrocarbons by the membrane-bound methane monooxygenase: the case for a tricopper cluster. Acc. Chem. Res. 41, 969–979 (2008).
Danciu, T. et al. Direct synthesis of propylene oxide with CO2 as the solvent. Angew. Chem. Int. Ed. 42, 1140–1142 (2003).
Hua, Q. et al. Crystal-plane-controlled selectivity of Cu2O catalysts in propylene oxidation with molecular oxygen. Angew. Chem. Int. Ed. 53, 4856–4861 (2014).
Clomburg, J. M., Crumbley, A. M. & Gonzalez, R. Industrial biomanufacturing: the future of chemical production. Science 355, aao0804 (2017).
Hakemian, S. & Rosenzweig, A. C. The biochemistry of methane oxidation. Annu. Rev. Biochem. 76, 223–241 (2007).
Lieberman, R. L. et al. Purified particulate methane monooxygenase from Methylococcus capsulatus (Bath) is a dimer with both mononuclear copper and a copper-containing cluster. Proc. Natl Acad. Sci. USA 100, 3820–3825 (2003).
Krause, S. M. et al. Lanthanide-dependent cross-feeding of methane-derived carbon is linked by microbial community interaction. Proc. Natl Acad. Sci. USA 114, 358–363 (2017).
Gilman, A. et al. Bioreactor performance parameters for an industrially-promising methanotroph Methylomicrobium buryatense 5GB1. Microb. Cell Fact. 14, 182 (2015).
Islam, R. S., Tisi, D., Levy, M. S. & Lye, G. J. Scale-up of Escherichia coli growth and recombinant protein expression conditions from microwell to laboratory and pilot scale based on matched k L a. Biotechnol. Bioeng. 99, 1128–1139 (2008).
Jahng, D. et al. Optimization of trichloroethylene degradation using soluble methane monooxygenase of Methylosinus trichosporium OB3b expressed in recombinant bacteria. Biotechnol. Bioeng. 51, 349–359 (1996).
Balasubramanian, R. et al. Oxidation of methane by a biological dicopper centre. Nature 465, 115–119 (2010).
Gau, Z. et al. Functional expression of the particulate methane mono-oxygenase gene in recombinant Rhodococcus erythropolis. FEMS Microbiol. Lett. 263, 136–141 (2006).
Afiah, T. S. N. & Rusmana, I. Cuperedoxin domain of particulate methane monooxygenase (pMMO) gene expression in recombinant Escherichia coli. Malays. J. Microbiol. 12, 56–61 (2016).
Tie-nam, Z. et al. Heterologous expression of pmoB in Escherichia coli and catalytic oxygenation of methane to methanol. J. Mol. Catal. 30, 177–181 (2016).
Chan, S. I. et al. Redox potentiometry studies of particulate methane monooxygenase: support for a trinuclear copper cluster active site. Angew. Chem. Int. Ed. 46, 1992–1994 (2007).
Piku, B., Katterle, B. & Andersson, K. K. The membrane-associated form of methane mono-oxygenase from Methylococcus capsulatus (Bath) is a copper/iron protein. Biochem. J. 369, 417–427 (2003).
Yoshizwa, K. & Shiota, Y. Conversion of methane to methanol at the mononuclear and dinuclear copper sites of particulate methane monooxygenase (pMMO): a DFT and QM.MM study. J. Am. Chem. Soc. 128, 9873–9881 (2006).
Lieberman, R. L. & Rosenzweig, A. C. Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane. Nature 434, 177–182 (2005).
Smith, S. M. et al. Crystal structure and characterization of particulate methane monooxygenase from Methylocystis species strain M. Biochemistry 50, 10231–10240 (2011).
Cao, L., Caldararu, O., Rosenzweig, A. C. & Ryde, U. Quantum refinement does not support dinuclear copper sites in crystal structure of particulate methane monooxygenase. Angew. Chem. Int. Ed. 57, 162–166 (2017).
Sirajuddin, S. et al. Effects of zinc on particulate methane monooxygenase activity and structure. J. Biol. Chem. 289, 21782–21794 (2014).
Culpepper, M. A. & Rosenzweig, A. C. Architecture and active site of particulate methane monooxygenase. Crit. Rev. Biochem. Mol. Biol. 47, 483–492 (2012).
Luzzago, A. & Cesareni, G. Isolation of point mutations that affect the folding of the H chain of human ferritin in E. coli. EMBO J. 8, 569–576 (1989).
Wang, L. et al. Structure-based design of ferritin nanoparticle immunogens displaying antigenic loops of Neisseria gonorrhoeae. FEBS Open Bio 7, 1196–1207 (2017).
Duwe, S. et al. Expression-enhanced fluorescent proteins based on enhanced green fluorescent protein for super-resolution microscopy. ACS Nano 9, 9528–9541 (2015).
Lee, S. H. et al. A novel approach to ultrasensitive diagnosis using supramolecular protein nanoparticles. FASEB J. 21, 1324–1334 (2007).
Lee, B. R. et al. Engineered human ferritin nanoparticles for direct delivery of tumor antigens to lymph node and cancerimmunotherapy. Sci. Rep. 6, 35182 (2016).
Kwon, K. C. et al. Enhanced in vivo tumor detection by active tumor cell targeting using multiple tumor receptor-binding peptides presented on genetically engineered human ferritin nanoparticles. Small 12, 4241–4253 (2016).
Lee, J. H. et al. Multiplex diagnosis of viral infectious diseases (AIDS, hepatitis C, and hepatitis A) based on point of care lateral flow assay using engineered proteinticles. Biosens. Bioelectron. 69, 213–225 (2015).
Lee, J. H. et al. Proteinticle engineering for accurate 3D diagnosis. ACS Nano 7, 10879–10886 (2013).
Cornell, T. A. et al. The crystal structure of a maxi/mini-ferritin chimera reveals guiding principles for the assembly of protein cages. Biochemistry 56, 3894–3899 (2017).
Jetz, G. et al. Ferritin: a versatile building block for bionanotechnology. Chem. Rev. 115, 1653–1701 (2015).
Lee, E. J. et al. A novel bioassay platform using ferritin based nanoprobe hydrogel. Adv. Mater. 24, 4739–4744 (2012).
Masuda, T., Gota, F., Yoshifara, T. & Mikami, B. The universal mechanism for iron translocation to the ferroxidase site in ferritin, which is mediated by the well conserved transit site. Biochem. Biophys. Res. Commun. 400, 94–99 (2010).
Lontoh, S. et al. Differential inhibition in vivo of ammonia monooxygenase, soluble methane monooxygenase and membrane-associated methane monooxygenase by phenylacetylene. Environ. Microbiol. 2, 485–494 (2000).
Lee, S. W. et al. Effect of nutrient and selective inhibitor amendments on methane oxidation, nitrous oxide production, and key gene presence and expression in landfill cover soils: characterization of the role of methanotrophs, nitrifiers, and denitrifiers. Environ. Microbiol. 85, 389–403 (2009).
Paszczynski, A. J. et al. Proteomic and targeted qPCR analyses of subsurface microbial communities for presence of methane monooxygenase. Biodegradation 22, 1045–1059 (2011).
Sirajuddin, S. & Rosenzweig, A. C. Enzymatic oxidation of methane. Biochemistry 54, 2283–2294 (2015).
Choi, D. W. et al. The membrane-associated methane monooxygenase (pMMO) and pMMO-NADH: quinone oxidoreductase complex form Methylococcus capsulatus Bath. J. Bacteriol. 185, 5755–5764 (2003).
Yu, S. S. F. et al. Production of high-quality particulate methane monooxygenase in high yields from Methylococcus capsulatus (Bath) with a hollow-fiber membrane bioreactor. J. Bacteriol. 185, 5915–5924 (2003).
Martinho, M. et al. Mössbauer studies of the membrane-associated methane monooxygenase from Methylococcus capsulatus Bath: evidence for a diiron center. J. Am. Chem. Soc. 129, 15783–15785 (2007).
Zahn, J. & DiSpiriop, A. A. Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath). J. Bacteriol. 178, 1018–1029 (1996).
Lieberman, R. L. & Rosenzweig, A. C. Biological methane oxidation: regulation, biochemistry, and active site structure of particulate methane monooxygenase. Crit. Rev. Biochem. Mol. Biol. 39, 147–164 (2004).
Tonge, G. M., Harrison, D. E. F. & Higgins, I. J. Purification and properties of the methane mono-oxygenase enzyme system from Methylosinus trichosporium OB3b. Biochem. J. 161, 333–344 (1977).
Lee, D. S. et al. A protein nanofiber hydrogel for sensitive immunoassays. Analyst 138, 4786–4794 (2013).
Pollak, A. et al. Enzyme immobilization by condensation copolymerization into crosslinked polyacrylamide gels. J. Am. Chem. Soc. 102, 6324–6336 (1980).
Chen, Y. Development and application of co-culture for ethanol production by co-fermentation of glucose and xylose: a systematic review. J. Ind. Microbiol. Biotechnol. 38, 581–597 (2011).
Gonzalez-Saiz, J. M. & Pizarro, C. Polyacrylamide gels as support for enzyme immobilization by entrapment. Effect of polyelectrolyte carrier, pH and temperature on enzyme action and kinetics parameters. Eur. Polym. J. 37, 435–444 (2001).
Kumar, S., Yadav, R. V. & Negi, S. A Comparative study of immobilized lipase produced from Penicillium chrysogenum SNP5 on two different anionic carriers for its pH and thermostability. Indian J. Biotechnol. 13, 301–305 (2014).
Blanchette, C. D. et al. Printable enzyme-embedded materials for methane to methanol conversion. Nat. Commun. 7, 11900 (2016).
Culpepper, M. A. et al. Identification of the valence and coordination environment of the particulate methane monooxygenase copper centers by advanced EPR characterization. J. Am. Chem. Soc. 136, 11767–11775 (2014).
Page, C. C., Moser, C. C., Chen, X. & Dutton, P. L. Natural engineering principles of electron tunneling in biological oxidation reduction. Nature 402, 47–52 (1999).
Hempstead, P. D. et al. Comparison of the three-dimensional structures of recombinant human H and horse L ferritins at high resolution. J. Mol. Biol. 268, 424–448 (1997).
Ro, S. Y. et al. From micelles to bicelles: effect of the membrane on particulate methane monooxygenase activity. J. Biol. Chem. 293, 10457–10465 (2018).
Mayne, C. G., Saam, J., Schulten, K. & Tajkhorshid, E. Rapid parameterization of small molecules using the Force Field Toolkit. J. Comput. Chem. 34, 2757–2770 (2013).
Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996).
Kale, L. et al. NAMD2: greater scalability for parallel molecular dynamics. J. Comput. Phys. 151, 283–312 (1999).
Feller, S. E., Zhang, Y., Pastor, R. W. & Brooks, B. R. Constant-pressure molecular-dynamics simulation – the Langevin piston method. J. Chem. Phys. 103, 4613–4621 (1995).
Darden, T., York, D. & Pederson, L. Particle mesh Ewald: an N·log(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089–10092 (1993).
Andersen, H. C. Rattle: a “velocity” version of the shake algorithm for molecular dynamics calculations. J. Comput. Phys. 52, 24–34 (1983).
Ravel, B. & Newville, M. ATHENA, ARTEMIS, HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT. J. Synchrotron Radiat. 12, 537–541 (2005).
Stoll, S. & Schweiger, A. EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J. Magn. Reson. 178, 42–55 (2006).
Liu, X., Yu, M., Kim, H., Mameli, M. & Stellacci, F. Determination of monolayer-protected gold nanoparticle ligand–shell morphology using NMR. Nat. commun. 3, 1182 (2012).
Acknowledgements
This study was supported by the 2015 NLRL (National Leading Research Lab.) Project (grant no. NRF (National Research Foundation Korea)-2015R1A2A1A05001861), Bio & Medical Technology Development Program (grant no. NRF‐2017M3A9F5032628), and also partly by NRF-Korea (NRF-2016R1A6A3A11933393).
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J.L. conceived the design and synthesis of pMMO-mimics, conducted the data analysis and wrote the paper. H.J.K. performed the synthesis and activity analyses of the designed pMMO-mimics and collected the data. J.H. performed the MD simulation based on the design of pMMO-mimics, collected the data and co-wrote the paper. Y.W.K. performed the data collection and analyses of XANES, EXAFS and EPR spectroscopy. D.P., Y.Y., Y.E.J., B.-R.L. and E.J. performed the experiments of pMMO-mimics synthesis. Y.H. and W.L. performed SEC chromatographic and CD spectroscopic analyses. E.J.L. analysed the experimental data. We also appreciate the contribution of P. Shrestha at Yonsei University in data collection.
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Supplementary Discussion, Supplementary Methods, Supplementary Figures 1–21, Supplementary Tables 1 and 2, Supplementary Note 1 and Supplementary References
Supplementary Data 1
Cartesian coordinates of Figure 1b
Supplementary Movie 1
MD simulations of pmoB with duroquinol and hydroquinone
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Kim, H.J., Huh, J., Kwon, Y.W. et al. Biological conversion of methane to methanol through genetic reassembly of native catalytic domains. Nat Catal 2, 342–353 (2019). https://doi.org/10.1038/s41929-019-0255-1
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DOI: https://doi.org/10.1038/s41929-019-0255-1
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