Mode of action uncovered for the specific reduction of methane emissions from ruminants by the small molecule 3-nitrooxypropanol

Edited by Lonnie O. Ingram, University of Florida, Gainesville, FL, and approved March 30, 2016 (received for review January 8, 2016)
May 2, 2016
113 (22) 6172-6177

Significance

Methane emission from the ruminant livestock sector—a by-product from enteric fermentation of plant biomass in the ruminant digestive system—is produced by methanogenic archaea and represents not only a significant amount of anthropogenic greenhouse gases contributing to climate change but also an energy loss and a reduction in feed efficacy. The present study elucidates the development and the unique mode of action of the highly specific inhibitor 3-nitrooxypropanol (3-NOP), which is targeting the nickel enzyme methyl-coenzyme M reductase in rumen archaea that catalyzes the methane-forming reaction. At the very low effective concentrations recently applied in vivo (dairy and beef cattle), 3-NOP appears to inhibit only methanogens and thus to be attractive for development as a feed supplement.

Abstract

Ruminants, such as cows, sheep, and goats, predominantly ferment in their rumen plant material to acetate, propionate, butyrate, CO2, and methane. Whereas the short fatty acids are absorbed and metabolized by the animals, the greenhouse gas methane escapes via eructation and breathing of the animals into the atmosphere. Along with the methane, up to 12% of the gross energy content of the feedstock is lost. Therefore, our recent report has raised interest in 3-nitrooxypropanol (3-NOP), which when added to the feed of ruminants in milligram amounts persistently reduces enteric methane emissions from livestock without apparent negative side effects [Hristov AN, et al. (2015) Proc Natl Acad Sci USA 112(34):10663–10668]. We now show with the aid of in silico, in vitro, and in vivo experiments that 3-NOP specifically targets methyl-coenzyme M reductase (MCR). The nickel enzyme, which is only active when its Ni ion is in the +1 oxidation state, catalyzes the methane-forming step in the rumen fermentation. Molecular docking suggested that 3-NOP preferably binds into the active site of MCR in a pose that places its reducible nitrate group in electron transfer distance to Ni(I). With purified MCR, we found that 3-NOP indeed inactivates MCR at micromolar concentrations by oxidation of its active site Ni(I). Concomitantly, the nitrate ester is reduced to nitrite, which also inactivates MCR at micromolar concentrations by oxidation of Ni(I). Using pure cultures, 3-NOP is demonstrated to inhibit growth of methanogenic archaea at concentrations that do not affect the growth of nonmethanogenic bacteria in the rumen.

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Data Availability

Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 5G0R).

Acknowledgments

We thank Peter Livant (Auburn University), Elisabeth Jimenez (Consejo Superior de Investigaciones Cientificas), John Wallace (University of Aberdeen), and Jamie Newbold (Aberystwyth University) for the use of the gas chromatograph, for the in vitro culture work, and for providing stock pure cultures, respectively; Ulrich Ermler and the staff of the PXII beam line at Swiss Light Source (Villigen, Switzerland) for helping with the X-ray data collection; and David Rinaldo (Schrödinger, LLC) for molecular modeling support.

Supporting Information

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Information & Authors

Information

Published in

The cover image for PNAS Vol.113; No.22
Proceedings of the National Academy of Sciences
Vol. 113 | No. 22
May 31, 2016
PubMed: 27140643

Classifications

Data Availability

Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 5G0R).

Submission history

Published online: May 2, 2016
Published in issue: May 31, 2016

Keywords

  1. methanogenesis
  2. methyl-coenzyme M reductase
  3. enteric methane
  4. greenhouse gas
  5. climate change

Acknowledgments

We thank Peter Livant (Auburn University), Elisabeth Jimenez (Consejo Superior de Investigaciones Cientificas), John Wallace (University of Aberdeen), and Jamie Newbold (Aberystwyth University) for the use of the gas chromatograph, for the in vitro culture work, and for providing stock pure cultures, respectively; Ulrich Ermler and the staff of the PXII beam line at Swiss Light Source (Villigen, Switzerland) for helping with the X-ray data collection; and David Rinaldo (Schrödinger, LLC) for molecular modeling support.

Notes

This article is a PNAS Direct Submission.
See Commentary on page 6100.

Authors

Affiliations

Evert C. Duin
Department of Chemistry and Biochemistry, Auburn University, Auburn, AL 36849;
Tristan Wagner
Max Planck Institute for Terrestrial Microbiology, D-35043 Marburg, Germany;
Seigo Shima
Max Planck Institute for Terrestrial Microbiology, D-35043 Marburg, Germany;
Divya Prakash
Department of Chemistry and Biochemistry, Auburn University, Auburn, AL 36849;
Present address: Althouse Laboratory, Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802.
Bryan Cronin
Department of Chemistry and Biochemistry, Auburn University, Auburn, AL 36849;
David R. Yáñez-Ruiz
Estación Experimental del Zaidín, Consejo Superior de Investigaciones Cientificas, 18008 Granada, Spain;
Stephane Duval
Research Centre for Animal Nutrition and Health, DSM Nutritional Products France, 68305 Saint Louis, France;
Robert Rümbeli
Research and Development, DSM Nutritional Products, 4002 Basel, Switzerland
René T. Stemmler
Research and Development, DSM Nutritional Products, 4002 Basel, Switzerland
Rudolf Kurt Thauer2 [email protected]
Max Planck Institute for Terrestrial Microbiology, D-35043 Marburg, Germany;
Maik Kindermann2 [email protected]
Research and Development, DSM Nutritional Products, 4002 Basel, Switzerland

Notes

2
To whom correspondence may be addressed. Email: [email protected] or [email protected].
Author contributions: E.C.D., D.R.Y.-R., S.D., R.T.S., R.K.T., and M.K. designed research; E.C.D., T.W., S.S., D.P., B.C., D.R.Y.-R., S.D., R.R., and R.T.S. performed research; E.C.D., S.S., R.T.S., R.K.T., and M.K. analyzed data; and R.K.T. and M.K. wrote the paper.

Competing Interests

Conflict of interest statement: The authors working at Auburn University and at the Max Planck Institute in Marburg (E.C.D., T.W., S.S., D.P., B.C., and R.K.T.) declare no competing financial interests. The authors affiliated with Estation Experimental del Zaidin (D.R.Y.-R.) or DSM Nutritional Products (S.D., R.R., R.T.S., and M.K.) have filed patent applications for nitrate esters as inhibitors of rumen methanogenesis.

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    Mode of action uncovered for the specific reduction of methane emissions from ruminants by the small molecule 3-nitrooxypropanol
    Proceedings of the National Academy of Sciences
    • Vol. 113
    • No. 22
    • pp. 6083-E3186

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