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Purinyl-cobamide is a native prosthetic group of reductive dehalogenases

Nature Chemical Biology volume 14, pages 8–14 (2018)Cite this article

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Abstract

Cobamides such as vitamin B12 are structurally conserved, cobalt-containing tetrapyrrole biomolecules that have essential biochemical functions in all domains of life. In organohalide respiration, a vital biological process for the global cycling of natural and anthropogenic organohalogens, cobamides are the requisite prosthetic groups for carbon–halogen bond-cleaving reductive dehalogenases. This study reports the biosynthesis of a new cobamide with unsubstituted purine as the lower base and assigns unsubstituted purine a biological function by demonstrating that Coα-purinyl-cobamide (purinyl-Cba) is the native prosthetic group in catalytically active tetrachloroethene reductive dehalogenases of Desulfitobacterium hafniense. Cobamides featuring different lower bases are not functionally equivalent, and purinyl-Cba elicits different physiological responses in corrinoid-auxotrophic, organohalide-respiring bacteria. Given that cobamide-dependent enzymes catalyze key steps in essential metabolic pathways, the discovery of a novel cobamide structure and the realization that lower bases can effectively modulate enzyme activities generate opportunities to manipulate functionalities of microbiomes.

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Figure 1: Cobamide general structure and the 16 known lower bases found in naturally occurring cobamides.
Figure 2: Spectrophotometric and structural features of Desulfitobacterium native corrinoids.
Figure 3: Identification of purinyl-Cba as the native prosthetic group in Dsf PCE RDase following nondenaturing, gel-electrophoretic separation of Dsf crude protein extracts using BN–PAGE.
Figure 4: Phylogenetic analysis of CobT homologous proteins and substrate specificity of Dsf CobT.
Figure 5: Effects of purinyl-Cba substitution for vitamin B12 on the activity of corrinoid-auxotrophic, organohalide-respiring bacteria.

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Acknowledgements

We thank J. Maillard, École Polytechnique Fédérale de Lausanne, France, for providing Dehalobacter restrictus strain PER-K23. We also thank R. Flick from the BioZone Mass Spectrometry facility, Toronto, for LC/MS assistance. This research was supported by a grant from the Superfund Research Program under the National Institute of Environmental Health Sciences (R01ES024294) to F.E.L., with additional support provided by the Strategic Environmental Research and Development Program (SERDP project ER-2312) to F.E.L. and by the Natural Science and Engineering Research Council of Canada (NSERC) Industrial Biocatalysis Network to E.A.E. Y. Yin acknowledges the financial support from the China-UT One-Hundred Scholars Program by the China Scholarship Council and the University of Tennessee.

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Authors and Affiliations

  1. Department of Microbiology, University of Tennessee, Knoxville, Tennessee, USA

    Jun Yan, Meng Bi, Yongchao Yin & Frank E Löffler

  2. Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, Liaoning, P.R. China

    Jun Yan

  3. Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee, USA

    Jun Yan, Nannan Jiang, Yi Yang, Burcu Şimşir & Frank E Löffler

  4. Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA

    Jun Yan, Nannan Jiang & Frank E Löffler

  5. Joint Institute for Biological Sciences (JIBS), Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA

    Jun Yan, Nannan Jiang & Frank E Löffler

  6. Department of Chemistry, University of Tennessee, Knoxville, Tennessee, USA

    Allen K Bourdon, Abigail T Farmer & Shawn R Campagna

  7. Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada

    Po-Hsiang Wang, Olivia Molenda, Andrew T Quaile & Elizabeth A Edwards

  8. Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee, Knoxville, Tennessee, USA

    Nannan Jiang & Frank E Löffler

  9. Department of Civil and Environmental Engineering, University of Tennessee, Knoxville, Tennessee, USA

    Yi Yang, Burcu Şimşir & Frank E Löffler

  10. Department of Biosystems Engineering and Soil Science, University of Tennessee, Knoxville, Tennessee, USA

    Frank E Löffler

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Contributions

F.E.L., J.Y., and S.R.C. conceptualized the research and designed experiments. J.Y., M.B., B.S., Y. Yang, and Y. Yin performed cultivation work, corrinoid extraction and purification, and phylogenetic analyses. A.K.B. and A.T.F. performed LC–MS and structural analyses. P.W., O.M. and A.T.Q. performed BN–PAGE, enzyme assays, and proteomic analysis. N.J. generated cobT expression clones. All authors contributed to data analysis and interpretation, and J.Y., S.R.C., E.A.E., and F.E.L. wrote the manuscript.

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Correspondence to Jun Yan or Frank E Löffler.

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Yan, J., Bi, M., Bourdon, A. et al. Purinyl-cobamide is a native prosthetic group of reductive dehalogenases. Nat Chem Biol 14, 8–14 (2018). https://doi.org/10.1038/nchembio.2512

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