Post-translational thioamidation of methyl-coenzyme M reductase, a key enzyme in methanogenic and methanotrophic Archaea

AbstractThe enzyme methyl-coenzyme M reductase (MCR), found in strictly anaerobic methanogenic and methanotrophic archaea, catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The α subunit of this enzyme (McrA) contains several unusual post-translational modifications, including an exceptionally rare thioamidation of glycine. Based on the presumed function of homologous genes involved in the biosynthesis of thioamide-containing natural products, we hypothesized that the archaealtfuAandycaOgenes would be responsible for post-translational installation of thioglycine into McrA. Mass spectrometric characterization of McrA in a ΔycaO-tfuAmutant of the methanogenic archaeonMethanosarcina acetivoransrevealed the presence of glycine, rather than thioglycine, supporting this hypothesis. Physiological characterization of this mutant suggested a new role for the thioglycine modification in enhancing protein stability, as opposed to playing a direct catalytic role. The universal conservation of this modification suggests that MCR arose in a thermophilic ancestor.

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Post-translational thioamidation of methyl-coenzyme M reductase, a key enzyme in methanogenic and methanotrophic Archaea

eLife ◽

10.7554/elife.29218 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 41

Author(s):

Dipti D Nayak ◽

Nilkamal Mahanta ◽

Douglas A Mitchell ◽

William W Metcalf

Keyword(s):

Elevated Temperatures ◽

Protein Secondary Structure ◽

Phenotypic Characterization ◽

Strictly Anaerobic ◽

Methanosarcina Acetivorans ◽

Coenzyme M ◽

Α Subunit ◽

Key Enzyme ◽

Methyl Coenzyme M Reductase

Methyl-coenzyme M reductase (MCR), found in strictly anaerobic methanogenic and methanotrophic archaea, catalyzes the reversible production and consumption of the potent greenhouse gas methane. The α subunit of MCR (McrA) contains several unusual post-translational modifications, including a rare thioamidation of glycine. Based on the presumed function of homologous genes involved in the biosynthesis of thioviridamide, a thioamide-containing natural product, we hypothesized that the archaeal tfuA and ycaO genes would be responsible for post-translational installation of thioglycine into McrA. Mass spectrometric characterization of McrA from the methanogenic archaeon Methanosarcina acetivorans lacking tfuA and/or ycaO revealed the presence of glycine, rather than thioglycine, supporting this hypothesis. Phenotypic characterization of the ∆ycaO-tfuA mutant revealed a severe growth rate defect on substrates with low free energy yields and at elevated temperatures (39°C - 45°C). Our analyses support a role for thioglycine in stabilizing the protein secondary structure near the active site.

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Genetic techniques for studies of methyl-coenzyme M reductase from Methanosarcina acetivorans C2A

Enzymes of Energy Technology - Methods in Enzymology ◽

10.1016/bs.mie.2018.10.012 ◽

2018 ◽

pp. 325-347 ◽

Cited By ~ 3

Author(s):

Dipti D. Nayak ◽

William W. Metcalf

Keyword(s):

Methanosarcina Acetivorans ◽

Coenzyme M ◽

Genetic Techniques ◽

Methyl Coenzyme M Reductase

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Expression, Purification, and Characterization of (R)-Sulfolactate Dehydrogenase (ComC) from the Rumen MethanogenMethanobrevibacter milleraeSM9

Archaea ◽

10.1155/2017/5793620 ◽

2017 ◽

Vol 2017 ◽

pp. 1-6

Author(s):

Yanli Zhang ◽

Linley R. Schofield ◽

Carrie Sang ◽

Debjit Dey ◽

Ron S. Ronimus

Keyword(s):

Biosynthetic Pathway ◽

Critical Role ◽

Reduction Reaction ◽

Coenzyme M ◽

Purification And Characterization ◽

The Third ◽

Optimal Activity ◽

Methyl Coenzyme M Reductase

(R)-Sulfolactate dehydrogenase (EC 1.1.1.337), termed ComC, is a member of an NADH/NADPH-dependent oxidoreductase family of enzymes that catalyze the interconversion of 2-hydroxyacids into their corresponding 2-oxoacids. The ComC reaction is reversible and in the biosynthetic direction causes the conversion of (R)-sulfolactate to sulfopyruvate in the production of coenzyme M (2-mercaptoethanesulfonic acid). Coenzyme M is an essential cofactor required for the production of methane by the methyl-coenzyme M reductase complex. ComC catalyzes the third step in the first established biosynthetic pathway of coenzyme M and is also involved in methanopterin biosynthesis. In this study, ComC fromMethanobrevibacter milleraeSM9 was cloned and expressed inEscherichia coliand biochemically characterized. Sulfopyruvate was the preferred substrate using the reduction reaction, with 31% activity seen for oxaloacetate and 0.2% seen forα-ketoglutarate. Optimal activity was observed at pH 6.5. The apparentKMfor coenzyme (NADH) was 55.1 μM, and for sulfopyruvate, it was 196 μM (for sulfopyruvate theVmaxwas 93.9 μmol min−1 mg−1andkcatwas 62.8 s−1). The critical role of ComC in two separate cofactor pathways makes this enzyme a potential means of developing methanogen-specific inhibitors for controlling ruminant methane emissions which are increasingly being recognized as contributing to climate change.

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Spectroscopic and computational characterization of the nickel-containing F430 cofactor of methyl-coenzyme M reductase

JBIC Journal of Biological Inorganic Chemistry ◽

10.1007/s00775-003-0499-7 ◽

2003 ◽

Vol 9 (1) ◽

pp. 77-89 ◽

Cited By ~ 14

Author(s):

Jennifer L. Craft ◽

Yih-Chern Horng ◽

Stephen W. Ragsdale ◽

Thomas C. Brunold

Keyword(s):

Coenzyme M ◽

Methyl Coenzyme M Reductase

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Isolation and physiological characterization of Syntrophus buswellii strain GA from a syntrophic benzoate-degrading, strictly anaerobic coculture

Applied Microbiology and Biotechnology ◽

10.1007/bf00164509 ◽

1995 ◽

Vol 44 (1-2) ◽

pp. 241-248 ◽

Cited By ~ 15

Author(s):

G. Auburger ◽

J. Winter

Keyword(s):

Strictly Anaerobic ◽

Physiological Characterization

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Mmp10 is required for post-translational methylation of arginine at the active site of methyl-coenzyme M reductase

10.1101/211441 ◽

2017 ◽

Cited By ~ 3

Author(s):

Zhe Lyu ◽

Chau-wen Chou ◽

Hao Shi ◽

Ricky Patel ◽

Evert C. Duin ◽

...

Keyword(s):

Active Site ◽

Methane Formation ◽

Wild Type ◽

Coenzyme M ◽

Methane Cycle ◽

Gene Encoding ◽

Post Translational Modifications ◽

Whole Cells ◽

Type Gene ◽

Methyl Coenzyme M Reductase

AbstractCatalyzing the key step for anaerobic methane production and oxidation, methyl-coenzyme M reductase or Mcr plays a key role in the global methane cycle. The McrA subunit possesses up to five post-translational modifications (PTM) at its active site. Bioinformatic analyses had previously suggested that methanogenesis marker protein 10 (Mmp10) could play an important role in methanogenesis. To examine its role, MMP1554, the gene encoding Mmp10 inMethanococcus maripaludis, was deleted with a new genetic tool, resulting in the specific loss of the 5-(S)-methylarginine PTM of residue 275 in the McrA subunit and a 40~60 % reduction in the maximal rates of methane formation by whole cells. Methylation was restored by complementations with the wild-type gene. However, the rates of methane formation of the complemented strains were not always restored to the wild type level. This study demonstrates the importance of Mmp10 and the methyl-Arg PTM on Mcr activity.

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