Letter pubs.acs.org/JAFC
Inhibition of Methyl-CoM Reductase from Methanobrevibacter ruminantium by 2‑Bromoethanesulfonate ABSTRACT: Cattle husbandry is a major contributor to atmospheric methane, which is considered as an important greenhouse gas. Moreover, the generation of methane in the intestine of domestic ruminants by methanogenic bacteria is a drag on feed efficacy. Studies on methanogenesis have typically implied model organisms that are, however, not relevant in the ruminant gut. This paper shows that methyl-CoM reductase catalyzing the final step of methanogenesis in Methanobrevibacter ruminantium, a major participant in methane production by cattle, is inhibited by 2-bromoethanesulfonate, a compound often used as a model in animal agriculture, with an apparent IC50 of 0.4 ± 0.04 μM. KEYWORDS: Methanobrevibacter ruminantium, inhibition of methanogenesis, methyl-CoM reductase, greenhouse gas
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INTRODUCTION Methane is a greenhouse gas having an impact that has been reported to be 28-fold higher as compared to that of carbon dioxide over a period of 100 years and is continuously introduced into the atmosphere by abiotic as well as biotic processes.1 Biogenic methane is contributed by anaerobic biota such as swampland and from the gastrointestinal tract of mammals, most notably by ruminant livestock.2−4 Whereas swampland is generally receding due to a wide variety of human activities, cattle husbandry is developing. Hence, methane generated by bovine livestock is a progressively increasing contribution to the methane content of the atmosphere. The global production of biogenic methane has been estimated to be in the range of 1012 kg per year. About one-third of that massive amount is presumed to enter the atmosphere.5,6 Besides being a severe environmental pollutant due to its global warming potential, methane is also an energy-rich compound. The oxidation level of the methane carbon atom changes from −4 to +4 upon oxidation to CO2, releasing heat of combustion of 55.5 kJ/g. For comparison, with 29.7 kJ/g the heat of combustion of ethanol is only about half. It is estimated that through methane eructation 6−12% of the gross energy in the feed taken up by the cattle is lost.7,8 The biochemistry of microbial methane generation has been studied in considerable detail. Predominantly, these studies were conducted with Methanothermobacter thermoautotrophicus and Methanosarcina barkeri, the workhorses of methanobacterial research. However, neither of these prototype methanobacterial genera plays a significant role in the microbiota of the cattle intestine, where the main contributors for methanogenesis are believed to be Methanobrevibacter ruminantium, Methanobrevibacter gottschalkii, and Methanosphaera stadtmanae.9 By comparison with Methanothermobacter thermoautotrophicus and Methanosarcina barkeri, the methane producers from the bovine rumen have been studied to a significantly lesser extent. Importantly, however, the complete genome of Methanobrevibacter ruminantium, a major methane producer in bovine rumen, has been reported recently and constitutes an important stepping stone for further studies on rumen methanogens.10 There is a general presumption that the down-regulation of methanogenesis in the cattle rumen11 by an appropriate pharmacologic could serve a dual purpose, that is, (i) reducing © 2014 American Chemical Society
the contribution of biogenic methane to the atmosphere and (ii) improving the food utilization by livestock, which could in turn translate into increased production parameters at the level of meat and milk yields. This last point, however, will need to be further investigated because methane reduction could lead to higher energy efficiency only if the hydrogen not disposed of in the form of methane is rechanneled toward useful pathways (through acetogenesis, for example). On the basis of the Methanobrevibacter ruminantium genome,10 a set of about 30 gene products has been identified as potential targets for pharmacological intervention; notably, proteins directly involved in methane formation are considered as important potential targets. Studies performed at the level of pure microbial cultures, rumen fluid, and animal experiments have identified a variety of compounds that can reduce methane production. Several structural analogues of coenzyme M, a key player in methanogenesis, have been shown to inhibit methane production in pure methanobacterial cultures, in rumen fluid, and in animals.12 Studies in the 1970s showed that the CoM analogue 2-bromoethanesulfonate restricts methanogenesis and growth of methanogens via the inhibition of methyl-CoM reductase. In numerous subsequent studies, the compound has been used to suppress methanogenesis and/or methanogen proliferation in a variety of experimental setups.13−17 The unfavorable toxicological profile of 2-bromoethanesulfonate would immediately prevent it from being authorized as a feed additive. However, the magnitude of its effect and consistency of result when used in live animals make it one of the most well used compounds when it comes to research on methane mitigation in the agricultural sector.12,18 The compound has been shown to act as an inhibitor of methyl-CoM reductase of Methanothermobacter thermoautotrophicus, the final enzyme in the methanogenesis cascade,6,15 and has also been shown to inhibit methane production by cultured Methanobrevibacter ruminantium,12 but in vitro experiments with Methanobrevibacter ruminantium protein have not been reported hitherto to the best of our knowledge. We compared the impact of the low molecular weight inhibitor at the level of intact MethanoReceived: Revised: Accepted: Published: 12487
October 24, 2014 December 2, 2014 December 6, 2014 December 6, 2014 dx.doi.org/10.1021/jf505056g | J. Agric. Food Chem. 2014, 62, 12487−12490
Journal of Agricultural and Food Chemistry
Letter
brevibacter ruminantium cells and of partially purified methylCoM reductase with the aim to obtain additional information on the inhibitor’s molecular target.
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Fractions were analyzed for in vitro methane production using methyl-CoM and coenzyme B as substrates (Figures 2 and 3).
MATERIALS AND METHODS
Please see the Supporting Information.
RESULTS AND DISCUSSION The impact of 2-bromoethanesulfonate on methane production was determined using cultures of Methanobrevibacter ruminantium growing in 50 mL serum vessels using a complex medium as described in the Supporting Information and an atmosphere containing 80 mol % of H2 and 20 mol % of CO2 at an initial pressure of 210 kPa. Individual culture vessels were doped with the inhibitor at concentrations extending from 3 nM to 30 μM. The methane concentration in the overhead space was determined by gas chromatography after incubation for 3 days. The semilogarithmic plot in Figure 1A shows a sigmoidal, essentially symmetrical curve. Evaluation using the software package Dynafit (Biokin) affords an IC50 value of 0.8 ± 0.2 μM.19
Figure 2. Methanogenesis in Methanobrevibacter ruminantium; methylCoM reductase reaction shown red. MF, methanofuran; THM, tetrahydromethanopterin.
Figure 3. (A) Coenzyme M (CoM); (B) 2-bromoethanesulfonate; (C) coenzyme B (CoB).
Under these specific conditions, one molecule of methyl-CoM and one molecule of coenzyme B gave rise to one molecule of methane plus one disulfide (CoM−CoB complex). In vivo, this CoM−CoB complex is subject to reductive cleavage conducive to the regeneration of coenzymes B and M. Because our in vitro assay did not include any enzyme for coenzyme recycling, the mixed disulfide was the final product. Using this assay, active methyl-CoM reductase was shown to be eluted from the column with a retention volume of 30−40 mL. All three subunit types of methyl-CoM reductase were confirmed in the partially purified (about 90%) enzyme preparation as revealed by mass spectrometry. In line with observations reported for Methanothermobacter thermoautotrophicus, the enzyme was subject to rapid activity loss under in vitro conditions.20 Hence, inhibition assays were carried out immediately after the purification procedure. Figure 1B demonstrates the impact of 2-bromoethanesulfonate on the in vitro formation of methane by the partially purified enzyme. The semilogarithmic plot shows a sigmoid curve with c2 symmetry. Evaluation with the software package Dynafit assigned an IC50 of 0.4 ± 0.04 μM. Thus, methyl-CoM reductase from Methanobrevibacter ruminantium appears to be more sensitive to 2-bromoethanesulfonate inhibition than the enzyme from Methanothermobacter thermoautotrophicus, for which an IC50 of 4 μM has been reported.21 Notably, the IC50 values obtained with Methanobrevibacter ruminantium cultures and with the partially purified Methanobrevibacter ruminantium enzyme are numerically similar. This is well in line
Figure 1. Inhibition of methyl-CoM reductase by 2-bromoethanesulfonate: (A) in vivo methane formation at different inhibitor concentrations; (B) in vitro methane formation at different inhibitor concentrations; (blue diamonds) mean of three independent measurements (error bars indicate standard deviation); (red curve) line of best fit.
To measure the in vitro impact of 2-bromoethanesulfonate on methyl-CoM reductase, we cultured Methanobrevibacter ruminantium to late log phase under an atmosphere containing 80 mol % of H2 and 20 mol % of CO2 in 1 L Schott flasks. The cells were harvested under anaerobic conditions and were disrupted by sonication. SDS-PAGE analysis of the crude extract revealed prominent bands at 59.8, 47.1, and 28.7 kDa most likely visualizing the α, β, and γ subunit types, respectively, of methyl-CoM reductase (not shown). The specific activity of the peak fraction was 6 nmol/mg·min. The crude cell extract was placed on a column of Sepharose Q that was kept under anaerobic conditions and developed with a gradient of 0.4−1.0 M NaCl in 100 mM MOPS, pH 7.2. 12488
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(2) Hook, S. E.; Wright, A.-D. G.; McBride, B. W. Methanogens: methane producers of the rumen and mitigation strategies. Archaea 2010, 945785, 11 pages. (3) Janssen, P. H.; Kirs, M. Structure of the archaeal community of the rumen. Appl. Environ. Microbiol. 2008, 74, 3619−3625. (4) Johnson, K. A.; Johnson, D. E. Methane emissions from cattle. J. Anim. Sci. 1995, 73, 2483−2492. (5) Scheehle, E. A.; Kruger, D. Global anthropogenic methane and nitrous oxide emissions. Energy J. 2006, 3 (Special Issue), 33−44. (6) Ermler, U. On the mechanism of methyl-coenzyme M reductase. Dalton Trans. 2005, 21, 3451−3458. (7) Vermorel, M. Emissions annuelles de méthane d’origine digestive par les bovins en France. INRA Prod. Anim. 1995, 8, 265−272. (8) Hristov, A. N.; Oh, J.; Lee, C.; Meinen, R.; Montes, F.; Ott, T.; Firkins, J.; Rotz, A.; Dell, C.; Adesogan, A.; Yang, W. Z.; Tricarico, J.; Kebreab, E.; Waghorn, G.; Dijkstra, J.; Oosting, S. Mitigation of greenhouse gas emissions in livestock production − a review of technical options for non-CO2 emissions. In FAO Animal Production and Health Paper 177; Gerber, P., Henderson, B., Makkar, H., Eds.; Food and Agriculture Organization of the United Nations: Rome, Italy, 2013; pp 1−226. (9) Whitford, M. F.; Teather, R. M.; Forster, R. J. Phylogenetic analysis of methanogens from the bovine rumen. BMC Microbiol. 2001, 1. (10) Leahy, S. C.; Kelly, W. J.; Altermann, E.; Ronimus, R. S.; Yeoman, C. J.; Pacheco, D. M.; Dong, L.; Zhanhao, K.; McTavish, S.; Sang, C.; Lambie, S. C.; Janssen, P. H.; Dey, D.; Attwood, G. T. The genome sequence of the rumen methanogen Methanobrevibacter ruminantium reveals new possibilities for controlling ruminant methane emissions. PLoS One 2010, 5, e8926. (11) McAllister, T. A.; Newbold, C. J. Redirecting rumen fermentation to reduce methanogenesis. Aust. J. Exp. Agric. 2008, 48, 7−13. (12) Ungerfeld, E. M.; Rust, S. R.; Boone, D. R.; Liu, Y. Effects of several inhibitors on pure cultures of ruminal methanogens. J. Appl. Microbiol. 2004, 97, 520−526. (13) Gunsalus, R. P.; Wolfe, R. S. Methyl coenzyme M reductase from Methanobacterium thermoautotrophicum. Resolution and properties of the components. J. Biol. Chem. 1980, 255, 1891−1895. (14) Rospert, S.; Böcher, R.; Albracht, S. P.; Thauer, R. K. Methylcoenzyme M reductase preparations with high specific activity from H2-preincubated cells of Methanobacterium thermoautotrophicum. FEBS Lett. 1991, 291, 371−375. (15) Rospert, S.; Voges, M.; Berkessel, A.; Albracht, S. P.; Thauer, R. K. Substrate-analogue-induced changes in the nickel-EPR spectrum of active methyl-coenzyme-M reductase from Methanobacterium thermoautotrophicum. Eur. J. Biochem. 1992, 210, 101−107. (16) Gunsalus, R. P.; Romesser, J. A.; Wolfe, R. S. Preparation of coenzyme M analogues and their activity in the methyl coenzyme M reductase system of Methanobacterium thermoautotrophicum. Biochemistry 1978, 17, 2374−2377. (17) Smith, M. R.; Mah, R. A. Growth and methanogenesis by Methanosarcina strain 227 on acetate and methanol. Appl. Environ. Microbiol. 1978, 36, 870−879. (18) Nevel, C. J.; Demeyer, D. Feed additives and other interventions for decreasing methane emissions. In Biotechnology and Animal Feeds and Animal Feeding; Wallace, R. J., Chesson, A., Eds.; Wiley-VCH Verlag: Weinheim, Germany, 1995; pp 329−349. (19) Kuzmic, P. Program Dynafit for the analysis of enzyme kinetic data: application to HIV proteinase. Anal. Biochem. 1996, 237, 260− 273. (20) Bonacker, L. G.; Baudner, S.; Mörschel, E.; Böcher, R.; Thauer, R. K. Properties of the two isoenzymes of methyl-coenzyme M reductase in Methanobacterium thermoautotrophicum. Eur. J. Biochem. 1993, 217, 587−595. (21) Holliger, C.; Kengen, S. W.; Schraa, G.; Stams, A. J.; Zehnder, A. J. Methyl-coenzyme M reductase of Methanobacterium thermoautotrophicum delta H catalyzes the reductive dechlorination of 1,2-
with the hypothesis that the terminal enzyme of methanogenesis is the actual target of the inhibitor, which is a structural analogue of coenzyme M. Methyl-CoM reductase has been identified as a potential target for inhibition of methanogenesis.10 The results of our study unequivocally support this hypothesis and confirm the relevance of using 2-bromoethanesulfonate as a model compound to test the effects of reduced methanogenesis in complex ecosystems such as the rumen or as a positive control to develop compounds working according to the same mode of action while benefiting from more favorable toxicological profiles. Whereas methyl-CoM reductase from Methanobrevibacter ruminantium is not well suited for high-throughput screening, due to the limited storing options, it is, however, possible to directly test potential new inhibitors in our in vivo system. Currently, we are downsizing the required culture volume with the scope to run growth studies in 1.5 mL GC vials.
Tobias Graw ̈ ert† Hans-Peter Hohmann§ Maik Kindermann§ Stephane Duval§ Adelbert Bacher† Markus Fischer*,†
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† Hamburg School of Food Science, Institute of Food Chemistry, Grindelallee 117, D-20146 Hamburg, Germany § DSM Nutritional Products, Wurmisweg 576, CH-4303 Kaiseraugst, Switzerland
ASSOCIATED CONTENT
S Supporting Information *
Materials and methods, Tables S1−S3, and additional references. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(M.F.) Phone: +49-40-428384342. Fax: +49-40-428384342. E-mail: markus.fi
[email protected]. Funding
We thank the Hans-Fischer-Gesellschaft for funding. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED CoB, coenzyme B; CoM, coenzyme M; GC- FID, gas chromatograph−flame ionization detector; IC50, half-maximal inhibitory concentration; MF, methanofuran; MOPS, 3-(Nmorpholino)propanesulfonic acid; THM, tetrahydromethanopterin
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REFERENCES
(1) Myhre, G.; Shindell, D.; Bréon, F.-M.; Collins, W.; Fuglestvedt, J.; Huang, J.; Koch, D.; Lamarque, J.-F.; Lee, D.; Mendoza, B.; Nakajima, T.; Robock, A.; Stephens, G.; Takemura, T.; Zhang, H. Anthropogenic and natural radiative forcing. In Climate Change 2013: The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change; Stocker, T. F., Qin, D., Plattner, G.-K., Tignor, M., Allen, S. K., Boschung, J., Nauels, A., Xia,Y., Bex, V., Midgley, P. M., Eds.; Cambridge University Press: Cambridge, UK, 2013; pp 659−740. 12489
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dichloroethane to ethylene and chloroethane. J. Bacteriol. 1992, 174, 4435−4434.
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