MtcB, a member of the MttB superfamily from the human gut acetogen Eubacterium limosum, is a cobalamin-dependent carnitine demethylase
2020; Elsevier BV; Volume: 295; Issue: 34 Linguagem: Inglês
10.1074/jbc.ra120.012934
ISSN1083-351X
AutoresDuncan J. Kountz, Edward J. Behrman, Liwen Zhang, Joseph A. Krzycki,
Tópico(s)Gut microbiota and health
ResumoThe trimethylamine methyltransferase MttB is the first described member of a superfamily comprising thousands of microbial proteins. Most members of the MttB superfamily are encoded by genes that lack the codon for pyrrolysine characteristic of trimethylamine methyltransferases, raising questions about the activities of these proteins. The superfamily member MtcB is found in the human intestinal isolate Eubacterium limosum ATCC 8486, an acetogen that can grow by demethylation of l-carnitine. Here, we demonstrate that MtcB catalyzes l-carnitine demethylation. When growing on l-carnitine, E. limosum excreted the unusual biological product norcarnitine as well as acetate, butyrate, and caproate. Cellular extracts of E. limosum grown on l-carnitine, but not lactate, methylated cob-(I)alamin or tetrahydrofolate using l-carnitine as methyl donor. MtcB, along with the corrinoid protein MtqC and the methylcorrinoid:tetrahydrofolate methyltransferase MtqA, were much more abundant in E. limosum cells grown on l-carnitine than on lactate. Recombinant MtcB methylates either cob(I)alamin or Co(I)-MtqC in the presence of l-carnitine and, to a much lesser extent, γ-butyrobetaine. Other quaternary amines were not substrates. Recombinant MtcB, MtqC, and MtqA methylated tetrahydrofolate via l-carnitine, forming a key intermediate in the acetogenic Wood–Ljungdahl pathway. To our knowledge, MtcB methylation of cobalamin or Co(I)-MtqC represents the first described mechanism of biological l-carnitine demethylation. The conversion of l-carnitine and its derivative γ-butyrobetaine to trimethylamine by the gut microbiome has been linked to cardiovascular disease. The activities of MtcB and related proteins in E. limosum might demethylate proatherogenic quaternary amines and contribute to the perceived health benefits of this human gut symbiont. The trimethylamine methyltransferase MttB is the first described member of a superfamily comprising thousands of microbial proteins. Most members of the MttB superfamily are encoded by genes that lack the codon for pyrrolysine characteristic of trimethylamine methyltransferases, raising questions about the activities of these proteins. The superfamily member MtcB is found in the human intestinal isolate Eubacterium limosum ATCC 8486, an acetogen that can grow by demethylation of l-carnitine. Here, we demonstrate that MtcB catalyzes l-carnitine demethylation. When growing on l-carnitine, E. limosum excreted the unusual biological product norcarnitine as well as acetate, butyrate, and caproate. Cellular extracts of E. limosum grown on l-carnitine, but not lactate, methylated cob-(I)alamin or tetrahydrofolate using l-carnitine as methyl donor. MtcB, along with the corrinoid protein MtqC and the methylcorrinoid:tetrahydrofolate methyltransferase MtqA, were much more abundant in E. limosum cells grown on l-carnitine than on lactate. Recombinant MtcB methylates either cob(I)alamin or Co(I)-MtqC in the presence of l-carnitine and, to a much lesser extent, γ-butyrobetaine. Other quaternary amines were not substrates. Recombinant MtcB, MtqC, and MtqA methylated tetrahydrofolate via l-carnitine, forming a key intermediate in the acetogenic Wood–Ljungdahl pathway. To our knowledge, MtcB methylation of cobalamin or Co(I)-MtqC represents the first described mechanism of biological l-carnitine demethylation. The conversion of l-carnitine and its derivative γ-butyrobetaine to trimethylamine by the gut microbiome has been linked to cardiovascular disease. The activities of MtcB and related proteins in E. limosum might demethylate proatherogenic quaternary amines and contribute to the perceived health benefits of this human gut symbiont. At present, ∼10,000 representatives of the MttB protein superfamily can be found in nearly 2000 different archaeal and bacterial genomes maintained at the National Center for Biotechnology. The first-described member of this large and well-distributed superfamily is the trimethylamine methyltransferase MttB, which catalyzes the corrinoid-dependent demethylation of trimethylamine (TMA) (1Ferguson Jr., D.J. Krzycki J.A. Reconstitution of trimethylamine-dependent coenzyme M methylation with the trimethylamine corrinoid protein and the isozymes of methyltransferase II from Methanosarcina barkeri.J. Bacteriol. 1997; 179 (9006042): 846-85210.1128/jb.179.3.846-852.1997Crossref PubMed Google Scholar, 2Krzycki J.A. Function of genetically encoded pyrrolysine in corrinoid-dependent methylamine methyltransferases.Curr. Opin. Chem. Biol. 2004; 8 (15450490): 484-49110.1016/j.cbpa.2004.08.012Crossref PubMed Scopus (73) Google Scholar). MttB is one of the few proteins known to possess the rare genetically encoded amino acid pyrrolysine (1Ferguson Jr., D.J. Krzycki J.A. Reconstitution of trimethylamine-dependent coenzyme M methylation with the trimethylamine corrinoid protein and the isozymes of methyltransferase II from Methanosarcina barkeri.J. Bacteriol. 1997; 179 (9006042): 846-85210.1128/jb.179.3.846-852.1997Crossref PubMed Google Scholar, 3Paul L. Ferguson D.J. Krzycki J.A. The trimethylamine methyltransferase gene and multiple dimethylamine methyltransferase genes of Methanosarcina barkeri contain in-frame and read-through amber codons.J. Bacteriol. 2000; 182 (10762254): 2520-252910.1128/jb.182.9.2520-2529.2000Crossref PubMed Scopus (78) Google Scholar, 4Soares J.A. Zhang L. Pitsch R.L. Kleinholz N.M. Jones R.B. Wolff J.J. Amster J. Green-Church K.B. Krzycki J.A. The residue mass of l-pyrrolysine in three distinct methylamine methyltransferases.J. Biol. Chem. 2005; 280 (16096277): 36962-3696910.1074/jbc.M506402200Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). However, the genes encoding the vast majority of the superfamily lack the amber codon necessary for co-translational insertion of the pyrrolysine residue that is characteristic of verified TMA methyltransferases (5Ticak T. Kountz D.J. Girosky K.E. Krzycki J.A. Ferguson Jr., D.J. A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase.Proc. Natl. Acad. Sci. U. S. A. 2014; 111 (25313086): E4668-E467610.1073/pnas.1409642111Crossref PubMed Scopus (38) Google Scholar), leaving the function of their gene products an open question. This conundrum was in part resolved by the discovery of MtgB, a nonpyrrolysine MttB homolog from nitrite-respiring Desulfitobacterium hafniense Y51 (5Ticak T. Kountz D.J. Girosky K.E. Krzycki J.A. Ferguson Jr., D.J. A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase.Proc. Natl. Acad. Sci. U. S. A. 2014; 111 (25313086): E4668-E467610.1073/pnas.1409642111Crossref PubMed Scopus (38) Google Scholar). MtgB initiates the corrinoid-dependent demethylation of glycine betaine to dimethylglycine as part of a multicomponent glycine betaine:THF methyltransferase system that also requires MtgC, a corrinoid-binding protein, and MtgA, a methylcorrinoid:THF methyltransferase (see Fig. 1 for a schematic of reactions involved in quaternary amine and TMA metabolism). Highly similar homologs of MtgB, MtgC, and MtgA were recently implicated in glycine betaine demethylation catalyzed by Acetobacterium woodii, an acetogen (6Lechtenfeld M. Heine J. Sameith J. Kremp F. Müller V. Glycine betaine metabolism in the acetogenic bacterium Acetobacterium woodii.Environ. Microbiol. 2018; 20 (30136352): 4512-452510.1111/1462-2920.14389Crossref PubMed Scopus (14) Google Scholar). The notable sequence divergence among MttB superfamily members led Ticak et al. (5Ticak T. Kountz D.J. Girosky K.E. Krzycki J.A. Ferguson Jr., D.J. A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase.Proc. Natl. Acad. Sci. U. S. A. 2014; 111 (25313086): E4668-E467610.1073/pnas.1409642111Crossref PubMed Scopus (38) Google Scholar) to hypothesize that different members of the family may have evolved specificity for other quaternary amines beyond glycine betaine. If so, the impact of the MttB superfamily could be significant in environments where organisms encoding nonpyrrolysine MttB family members are found. One such environment is the human intestine (5Ticak T. Kountz D.J. Girosky K.E. Krzycki J.A. Ferguson Jr., D.J. A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase.Proc. Natl. Acad. Sci. U. S. A. 2014; 111 (25313086): E4668-E467610.1073/pnas.1409642111Crossref PubMed Scopus (38) Google Scholar), where the metabolism of quaternary amines by members of the microbiome is now known to have a significant effect on human health (7Tang W.H.W. Bäckhed F. Landmesser U. Hazen S.L. Intestinal microbiota in cardiovascular health and disease: JACC state-of-the-art review.J. Am. Coll. Cardiol. 2019; 73 (31023434): 2089-210510.1016/j.jacc.2019.03.024Crossref PubMed Scopus (98) Google Scholar). In this work, we demonstrate for the first time that an MttB family member is an l-carnitine methyltransferase. l-Carnitine is widely used by eukaryotic cells for transport of fatty acids into the mitochrondria. As a result, l-carnitine is commonly found in many foodstuffs as a component of an omnivorous diet. Following ingestion, l-carnitine, as well as other quaternary amines such as choline and glycine betaine (8Wang Z. Klipfell E. Bennett B.J. Koeth R. Levison B.S. Dugar B. Feldstein A.E. Britt E.B. Fu X. Chung Y.M. Wu Y. Schauer P. Smith J.D. Allayee H. Tang W.H. et al.Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease.Nature. 2011; 472 (21475195): 57-6310.1038/nature09922Crossref PubMed Scopus (2740) Google Scholar, 9Koeth R.A. Wang Z. Levison B.S. Buffa J.A. Org E. Sheehy B.T. Britt E.B. Fu X. Wu Y. Li L. Smith J.D. DiDonato J.A. Chen J. Li H. Wu G.D. et al.Intestinal microbiota metabolism of l-carnitine, a nutrient in red meat, promotes atherosclerosis.Nat. Med. 2013; 19 (23563705): 576-58510.1038/nm.3145Crossref PubMed Scopus (2134) Google Scholar), enter the intestines, where they are either absorbed by the host or converted by members of the gut microbiota into TMA (10Al-Waiz M. Mikov M. Mitchell S.C. Smith R.L. 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Invest. 2019; 129 (30530985): 373-38710.1172/JCI94601Crossref PubMed Scopus (88) Google Scholar). Once in the bloodstream, TMA is transported to the liver and converted into trimethylamine N-oxide (TMAO), predominantly by flavin monooxygenase 3 (13Lang D.H. Yeung C.K. Peter R.M. Ibarra C. Gasser R. Itagaki K. Philpot R.M. Rettie A.E. Isoform specificity of trimethylamine N-oxygenation by human flavin-containing monooxygenase (FMO) and P450 enzymes: selective catalysis by FMO3.Biochem. Pharmacol. 1998; 56 (9776311): 1005-101210.1016/S0006-2952(98)00218-4Crossref PubMed Scopus (194) Google Scholar). High serum levels of TMAO have been shown to promote formation of atherosclerotic plaques in a mouse model (8Wang Z. Klipfell E. Bennett B.J. Koeth R. Levison B.S. Dugar B. Feldstein A.E. Britt E.B. Fu X. Chung Y.M. Wu Y. Schauer P. Smith J.D. Allayee H. 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Choline was shown to be directly converted to TMA by CutC, a glycyl radical enzyme that acts as a choline-TMA lyase (19Craciun S. Balskus E.P. Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme.Proc. Natl. Acad. Sci. U. S. A. 2012; 109 (23151509): 21307-2131210.1073/pnas.1215689109Crossref PubMed Scopus (333) Google Scholar). TMA may be produced from l-carnitine in a single step by the l-carnitine monoxygenase CntAB (20Zhu Y. Jameson E. Crosatti M. Schäfer H. Rajakumar K. Bugg T.D. Chen Y. Carnitine metabolism to trimethylamine by an unusual Rieske-type oxygenase from human microbiota.Proc. Natl. Acad. Sci. U. S. A. 2014; 111 (24591617): 4268-427310.1073/pnas.1316569111Crossref PubMed Scopus (149) Google Scholar). l-Carnitine can be converted to γ−butyrobetaine, which the oxygenase YeaWX can convert to TMA (11Koeth R.A. Levison B.S. Culley M.K. Buffa J.A. Wang Z. Gregory J.C. Org E. Wu Y. Li L. Smith J.D. Tang W.H. DiDonato J.A. Lusis A.J. Hazen S.L. γ-Butyrobetaine is a proatherogenic intermediate in gut microbial metabolism of l-carnitine to TMAO.Cell Metab. 2014; 20 (25440057): 799-81210.1016/j.cmet.2014.10.006Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). YeaWX also has some activity with l-carnitine and choline. Recent work has indicated that an anoxic uncharacterized pathway for TMA production from γ-butyrobetaine also exists in the gut microbiome (12Koeth R.A. Lam-Galvez B.R. Kirsop J. Wang Z. Levison B.S. Gu X. Copeland M.F. Bartlett D. Cody D.B. Dai H.J. Culley M.K. Li X.S. Fu X. Wu Y. Li L. et al.l-Carnitine in omnivorous diets induces an atherogenic gut microbial pathway in humans.J. Clin. Invest. 2019; 129 (30530985): 373-38710.1172/JCI94601Crossref PubMed Scopus (88) Google Scholar). Inhibitors of the major enzymes of quaternary amine degradation have been proposed as drugs to potentially decrease the net production of TMA in the gastrointestinal tract (21Kuka J. Liepinsh E. 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Different microbes thus might contribute to or interfere with the net synthesis of TMA (25Brown J.M. Hazen S.L. Metaorganismal nutrient metabolism as a basis of cardiovascular disease.Curr. Opin. Lipidol. 2014; 25 (24362355): 48-5310.1097/MOL.0000000000000036Crossref PubMed Scopus (53) Google Scholar). Only one group of microbes that might diminish TMA production has been previously identified (26Brugère J.F. Borrel G. Gaci N. Tottey W. O'Toole P.W. Malpuech-Brugere C. Archaebiotics: proposed therapeutic use of archaea to prevent trimethylaminuria and cardiovascular disease.Gut Microbes. 2014; 5 (24247281): 5-1010.4161/gmic.26749Crossref PubMed Scopus (118) Google Scholar, 27Ramezani A. Nolin T.D. Barrows I.R. Serrano M.G. Buck G.A. Regunathan-Shenk R. West 3rd, R.E. Latham P.S. Amdur R. Raj D.S. Gut colonization with methanogenic archaea lowers plasma trimethylamine N-oxide concentrations in apolipoprotein e−/− mice.Sci. Rep. 2018; 8 (30283097): 1475210.1038/s41598-018-33018-5Crossref PubMed Scopus (21) Google Scholar); the methanogenic archaea inhabiting the gut, whose genomes encode the pyrrolysyl-protein MttB, the TMA methyltransferase (1Ferguson Jr., D.J. Krzycki J.A. Reconstitution of trimethylamine-dependent coenzyme M methylation with the trimethylamine corrinoid protein and the isozymes of methyltransferase II from Methanosarcina barkeri.J. Bacteriol. 1997; 179 (9006042): 846-85210.1128/jb.179.3.846-852.1997Crossref PubMed Google Scholar, 2Krzycki J.A. Function of genetically encoded pyrrolysine in corrinoid-dependent methylamine methyltransferases.Curr. Opin. Chem. Biol. 2004; 8 (15450490): 484-49110.1016/j.cbpa.2004.08.012Crossref PubMed Scopus (73) Google Scholar). Another route that might conceivably limit TMA production would be competition for the quaternary amines that are precursors to TMA. However, such routes of quaternary amine degradation that would not eventually yield TMA under anaerobic conditions have been unknown, save for one, the demethylation of glycine betaine by the nonpyrrolysine (nonPyl) MttB family member, MtgB (5Ticak T. Kountz D.J. Girosky K.E. Krzycki J.A. Ferguson Jr., D.J. A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase.Proc. Natl. Acad. Sci. U. S. A. 2014; 111 (25313086): E4668-E467610.1073/pnas.1409642111Crossref PubMed Scopus (38) Google Scholar). Here we show that Eubacterium limosum ATCC 8486, an acetogenic and butyrogenic human gut isolate, consumes l-carnitine to produce norcarnitine. The latter is, to our knowledge, a novel biological product. The nonPyl MttB family member MtcB, along with a corrinoid protein and a corrinoid-dependent THF methyltransferase, were significantly more abundant in cells grown on l-carnitine than on lactate. These three proteins together catalyzed the methylation of THF with l-carnitine, thus providing a key intermediate toward the catabolic synthesis of acetate, butyrate, and carproate. MtcB initiates THF methylation by methylation of an abundant corrinoid protein specifically with l-carnitine. These results expand the known substrates of the MttB superfamily to include a proatherogenic dietary component and reveal a novel anoxic mechanism of l-carnitine degradation via demethylation. E. limosum strains have previously been reported to grow utilizing glycine betaine or choline (28Muller E. Fahlbusch K. Walther R. Gottschalk G. Formation of N N-dimethylglycine, acetic acid, and butyric acid from betaine by Eubacterium limosum.Appl. Environ. Microbiol. 1981; 42 (16345842): 439-44510.1128/AEM.42.3.439-445.1981Crossref PubMed Google Scholar). We found that E. limosum ATCC 8486 can also utilize l-carnitine as a growth substrate. Growth with l-carnitine was best in a medium supplemented with yeast extract and casamino acids with a doubling time of ∼6–7 h. Little or no growth was observed when E. limosum was inoculated into the same medium not supplemented with l-carnitine (Fig. 2A). E. limosum grew more slowly on l-carnitine when in a completely defined medium with a doubling time of ∼16–18 h, indicating that yeast extract and casamino acids were stimulatory but not necessary for growth with l-carnitine (Fig. S1). E. limosum strains were reported to demethylate glycine betaine and choline during growth (28Muller E. Fahlbusch K. Walther R. Gottschalk G. Formation of N N-dimethylglycine, acetic acid, and butyric acid from betaine by Eubacterium limosum.Appl. Environ. Microbiol. 1981; 42 (16345842): 439-44510.1128/AEM.42.3.439-445.1981Crossref PubMed Google Scholar). Therefore, we examined l-carnitine–grown cultures to determine whether l-carnitine was also demethylated during growth. Culture supernatants taken before and after growth were analyzed by TLC, followed by staining with bromocresol green (Fig. 2B). l-Carnitine was not detectable after growth. Instead, a compound was present that co-migrated to a position identical to that of a norcarnitine standard. To confirm this presumptive identification, the scraped spot was extracted with solvent and submitted to mass spectral analysis. The m/z value observed for the compound eluted from TLC plates was within 3 ppm of the theoretical value for norcarnitine (Table S1). MS/MS analysis of the parent ion revealed ions with m/z values predicted for fragmentation of norcarnitine. Additionally, the supernatants from l-carnitine cultures before and after growth were analyzed by MS following chromatography on an anion-exchange cartridge, which confirmed the presence of norcarnitine after growth (Fig. S2). E. limosum is capable of synthesizing acetyl-CoA from two one-carbon units (29Jeong J. Bertsch J. Hess V. Choi S. Choi I.G. Chang I.S. Müller V. Energy conservation model based on genomic and experimental analyses of a carbon monoxide-utilizing, butyrate-forming acetogen, Eubacterium limosum KIST612.Appl. Environ. Microbiol. 2015; 81 (25956767): 4782-479010.1128/AEM.00675-15Crossref PubMed Scopus (39) Google Scholar) and will also produce butyrate and (in some strains) caproate from acetyl-CoA (30Genthner B.R. Davis C.L. Bryant M.P. Features of rumen and sewage sludge strains of Eubacterium limosum, a methanol- and H2-CO2-utilizing species.Appl. Environ. Microbiol. 1981; 42 (6791591): 12-1910.1128/AEM.42.1.12-19.1981Crossref PubMed Google Scholar). Demethylation of l-carnitine did indeed support methylotrophic short-chain fatty acid production, as evidenced by the stoichiometry of l-carnitine degradation in defined medium in which l-carnitine and CO2 were the only carbon sources (aside from the defined vitamin mixture). In 10-ml cultures (n = 6), E. limosum demethylated 476 ± 51 µmol of l-carnitine to 534 ± 25 µmol of norcarnitine. In the process, 102 ± 1 µmol of CO2 were consumed to produce 77 ± 18 µmol of acetate, 78 ± 4 µmol of butyrate, and 10 ± 2 µmol of caproate. Any TMA produced was below the detection limit (∼50 μm TMA) by GC of stationary phase cultures. Total carbon recovery was 108 ± 11%. MS of the supernatant before and after growth provided no evidence of dehydration or further demethylation of l-carnitine beyond norcarnitine (Fig. S3). Given this and assuming a 1:1 stoichiometry between the l-carnitine consumed and norcarnitine produced, the carbon recovery of the methyl group of l-carnitine and CO2 in acid products was 91 ± 11%. Overall, our data support the following idealized equation for l-carnitine metabolism by E. limosum: 64 l-carnitine + 17 CO2 → 64 norcarnitine + 12 acetate + 12 butyrate + 1.5 caproate. 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