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Elucidation of the biosynthesis of the methane catalyst coenzyme F430

Nature volume 543, pages 78–82 (2017)Cite this article

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A Corrigendum to this article was published on 04 May 2017

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Abstract

Methane biogenesis in methanogens is mediated by methyl-coenzyme M reductase, an enzyme that is also responsible for the utilization of methane through anaerobic methane oxidation. The enzyme uses an ancillary factor called coenzyme F430, a nickel-containing modified tetrapyrrole that promotes catalysis through a methyl radical/Ni(ii)-thiolate intermediate. However, it is unclear how coenzyme F430 is synthesized from the common primogenitor uroporphyrinogen iii, incorporating 11 steric centres into the macrocycle, although the pathway must involve chelation, amidation, macrocyclic ring reduction, lactamization and carbocyclic ring formation. Here we identify the proteins that catalyse the biosynthesis of coenzyme F430 from sirohydrochlorin, termed CfbA–CfbE, and demonstrate their activity. The research completes our understanding of how the repertoire of tetrapyrrole-based pigments are constructed, permitting the development of recombinant systems to use these metalloprosthetic groups more widely.

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Figure 1: Coenzyme F430 and biosynthesis gene clusters in methanogens.
Figure 2: EPR characterization of CfbC and CfbD.
Figure 3: Enzymatic activity of CfbC and CfbD.
Figure 4: Enzymatic activity of CfbB.
Figure 5: Biosynthesis of coenzyme F430 from sirohydrochlorin.

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  • 01 March 2017

    In Fig. 1a, a missing bond was added to ring F.

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Acknowledgements

We thank M. Höninger, T. Schnitzer and J. Streif for conducting initial experiments with CfbA and CfbC/CfbD. We thank R. Thauer and S. Shima for the gift of the F430 standard. This work was supported by grants from the Boehringer Ingelheim Foundation (Exploration Grant) and the Deutsche Forschungsgemeinschaft (LA2412/6-1) to G.L. and from the Biotechnology and Biological Sciences Research Council (BBSRC; 68/B19356 and BB/I012079) to M.J.W.

Author information

Authors and Affiliations

  1. School of Biosciences, University of Kent, Giles Lane, Canterbury, Kent CT2 7NJ, UK

    Simon J. Moore, Evelyne Deery, Andrew D. Lawrence, Mark J. Howard & Martin J. Warren

  2. Institute of Biochemistry, Leipzig University, Brüderstrasse 34, 04103 Leipzig, Germany,

    Sven T. Sowa, Christopher Schuchardt, José Vazquez Ramos & Gunhild Layer

  3. Institute of Analytical Chemistry, Leipzig University, Linnéstrasse 3, 04103 Leipzig, Germany,

    Susan Billig & Claudia Birkemeyer

  4. Department of Chemistry and Department of Biosciences, Durham University, Durham, DH1 3LE, UK

    Peter T. Chivers

  5. Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK

    Stephen E. J. Rigby

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Contributions

S.J.M., S.T.S., C.S., E.D., A.D.L., J.V.R., S.B. and C.B. all undertook aspects of the experimental work, cloning, protein purification and enzyme assays, and helped with the interpretation of the data. P.T.C. provided the nixA clone and helped design the nickel uptake system. M.J.H., S.J.M. and A.D.L. designed and interpreted the NMR experiments and S.E.J.R., together with S.J.M. and A.D.L., provided the EPR data. S.J.M., M.J.W. and G.L. designed the experiments and wrote the paper.

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Correspondence to Gunhild Layer or Martin J. Warren.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Nickel chelatase activity of CfbA.

a, b, In vitro activity assay of CfbA. Purified CfbA was incubated with sirohydrochlorin and NiSO4 at 37 °C (a). The insertion of nickel was monitored by UV/Vis absorption spectroscopy every 15 min. When CfbA was omitted from the assay mixture (b), no nickel insertion was observed. c, In vivo activity of CfbA. Cell pellets of E. coli cells transformed with either pETcoco-2-cobA-sirC-cfbA or pETcoco-2-cobA-sirC-cfbA-nixA grown in the presence of nickel.

Extended Data Figure 2 Amidotransferase activity of CfbE.

a, In vivo activity of CfbE. E. coli cells transformed with pETcoco-2-cobA-sirC-cfbA-nixA and pET14b-cfbE and grown in the presence of nickel produce a dark violet pigment that co-purifies with CfbE during IMAC. b, c, 15N labelling of nickel-sirohydrochlorin a,c-diamide. b, Reverse-phase HPLC chromatogram of nickel-sirohydrochlorin substrate, m/z = 919 (i); unlabelled nickel-sirohydrochlorin a,c-diamide, m/z = 917 (ii); and 15N labelled nickel-sirohydrochlorin a,c-diamide, m/z = 919 (iii). c, 1H–15N HSQC of an ATP limited titration with nickel-sirohydrochlorin, CfbE and 15NH3. The a and c amide groups increase proportionally in intensity as the level of ATP increases.

Extended Data Figure 3 NMR characterization of Ni2+-sirohydrochlorin a,c-diamide.

a, b, 1H–13C HSQC (a) and 1H–15N HSQC (b) of 4 mM Ni2+-sirohydrochlorin a,c-diamide in D2O.

Extended Data Figure 4 Steady-state kinetics of the M. barkeri CfbE amidotransferase with glutamine or ATP as a variable.

a, 1 mM glutamine with ATP varied between 0.05 and 1.5 mM ATP. b, 0.5 mM ATP with glutamine varied between 0.05 and 10 mM. Fixed conditions: buffer B, 20 °C, 2.5 μM M. barkeri CfbE, 25 μM nickel-sirohydrochlorin, 5 mM MgCl2. The mean and error bars were calculated from 3 technical repeats.

Extended Data Figure 5 Characterization of the CfbC/CfbD assay reaction products by mass spectrometry after HPLC separation.

a, Mass spectrum with the isotopic pattern of the reaction product after 1.5 h of incubation measured in positive ion mode. b, Mass spectrum with the isotopic pattern of the reaction product after 22 h of incubation measured in positive ion mode.

Extended Data Figure 6 NMR characterization of seco-F430.

a, b, 1H–13C HSQC (a) and 1H–15N HSQC (b) of 4 mM seco-F430 in D2O.

Extended Data Figure 7 Characterization of the CfbB assay reaction products.

a, UV/Vis absorption spectrum of an F430 standard in 0.01% formic acid/acetonitrile. b, CfbB assay with Ni2+-hexahydrosirohydrochlorin a,c-diamide as the substrate. Mass spectrum with the isotopic pattern of the reaction product after 2 h of incubation measured in positive ion mode after HPLC separation. c, CfbB assay with seco-F430 as the substrate. Mass spectrum with the isotopic pattern of the reaction product after 22 h of incubation measured in positive ion mode after HPLC separation.

Extended Data Figure 8 NMR characterization of F430 synthesized by CfbB.

1H–13C HSQC and 1H–15N HSQC of F430 in TFE-d3.

Extended Data Figure 9 Proposed mechanism for the reaction catalysed by CfbB.

Initially, CfbB promotes the ATP-dependent phosphorylation of the propionic acid side chain on ring D of seco-F430. This activated side chain is then able to undergo cyclisation to form ring F and thereby generate coenzyme F430.

Extended Data Table 1 Plasmids and primers used in this study
Full size table

Supplementary information

Supplementary Table 1

NMR chemical shift assignments for Ni2+-sirohydrochlorin a,c-diamide 50 mM KPi pH 8.0 (D2O). (XLSX 12 kb)

Supplementary Table 2

NMR chemical shift assignments for seco-F430 in D2O (XLSX 12 kb)

Supplementary Table 3

NMR chemical shift assignments for F430 in TFE-d3 (XLSX 9 kb)

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Moore, S., Sowa, S., Schuchardt, C. et al. Elucidation of the biosynthesis of the methane catalyst coenzyme F430. Nature 543, 78–82 (2017). https://doi.org/10.1038/nature21427

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