Abstract
Methyl coenzyme M reductase (MCR) catalyzes the last step in the biological production of methane by methanogenic archaea, as well as the first step in the anaerobic oxidation of methane to methanol by methanotrophic archaea. MCR contains a number of unique post-translational modifications in its α subunit, including thioglycine, 1-N-methylhistidine, S-methylcysteine, 5-C-(S)-methylarginine, and 2-C-(S)-methylglutamine. Recently, genes responsible for the thioglycine and methylarginine modifications have been identified in bioinformatics studies and in vivo complementation of select mutants; however, none of these reactions has been verified in vitro Herein, we purified and biochemically characterized the radical S-adenosylmethionine (SAM) protein MaMmp10, the product of the methanogenesis marker protein 10 gene in the methane-producing archaea Methanosarcina acetivorans Using an array of approaches, including kinetic assays, LC-MS-based quantification, and MALDI TOF-TOF MS analyses, we found that MaMmp10 catalyzes the methylation of the equivalent of Arg285 in a peptide substrate surrogate, but only in the presence of cobalamin. We noted that the methyl group derives from SAM, with cobalamin acting as an intermediate carrier, and that MaMmp10 contains a C-terminal cobalamin-binding domain. Given that Mmp10 has not been annotated as a cobalamin-binding protein, these findings suggest that cobalamin-dependent radical SAM proteins are more prevalent than previously thought.
Highlights
Methyl coenzyme M reductase (MCR) catalyzes the last step in the biological production of methane by methanogenic archaea, as well as the first step in the anaerobic oxidation of methane to methanol by methanotrophic archaea
Given that methanogenesis marker protein 10 (Mmp10) has not been annotated as a cobalamin-binding protein, these findings suggest that cobalamin-dependent radical SAM proteins are more prevalent than previously thought
The gene was cloned into pET-26b such that the encoded protein would contain a hexahistidine tag separated from its native C-terminal amino acid by a linker of two amino acids
Summary
Methyl coenzyme M reductase (MCR) catalyzes the last step in the biological production of methane by methanogenic archaea, as well as the first step in the anaerobic oxidation of methane to methanol by methanotrophic archaea. The X-ray crystal structure of MCR from Methanothermobacter thermoautotrophicum, solved to 1.45 Å resolution, and those from other organisms, solved to even higher resolutions [9, 10], revealed the presence of five modified amino acids in its ␣ subunit, McrA, near the active site: 1-N-methylhistidine (MeHis), S-methylcysteine (MeCys), 5-C-(S)-methylarginine (MeArg), 2-C-(S)-methylglutamine (MeGln), and thioglycine [9]. Since those initial discoveries, didehydroaspartate, 6-hydroxytryptophan, and 7-hydroxytryptophan have been observed in MCRs of other methanogenic or methanotrophic archaea [11, 12]. In the presence of the required low-potential reductant, Ti(III) citrate, MaMmp exhibits a kcat of 1.87 minϪ1, which is comparable with that of other class B methylases that catalyze reactions via a 5Ј-dA1⁄7 intermediate [24]
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