Methanogenesis marker protein 10 (Mmp10) from Methanosarcina acetivorans is a radical S-adenosylmethionine methylase that unexpectedly requires cobalamin

Abstract

Edited by Ruma Banerjee 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 ␣ 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 methaneproducing 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 Arg 285 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. Methyl coenzyme M reductase (MCR) 2 catalyzes the final and rate-limiting step in methanogenesis, which is the conver

Functional interactions between posttranslationally modified amino acids of methyl-coenzyme M reductase in Methanosarcina acetivorans

“…The core modified amino acids are widely conserved and include N 1 -methylhistidine (or 3-methylhistidine), 5-(S)-methylarginine, and thioglycine ( Fig 1B). The widespread conservation of these core modifications is exemplified by the recently discovered enzymes responsible for installation of the thioglycine and 5-(S)-methylarginine modifications, whose encoding genes were originally designated as methanogenesis marker genes because of their universal occurrence in the genomes of sequenced methanogens [26][27][28][29][30]. Given that 5-(S)methylarginine and thioglycine are extremely rare in nature, yet universally conserved in MCR, it was speculated that these residues must be essential for catalysis [24].…”

Surprise! A hidden B12 cofactor catalyzes a radical methylation

“…Although these data are consistent with a class B RS methylase mechanism (Fig. 1), the low sequence homology with known class B methylases led the authors to conclude that Mmp10 represents the first example of a new subclass of class B RS methylases (8).…”

“…As now reported by Radle et al (8), Mmp10 is an RS enzyme that incorporates a C-terminal domain annotated as DUF512 (domain of unknown function). Radle et al (8) demonstrate that Mmp10 catalyzes the SAM-and B 12 -dependendent methylation of arginine in a peptide substrate and suggest that DUF512 may be a novel B 12 -binding domain.…”

“…Radle et al (8) observed that Mmp10, heterologously expressed in Escherichia coli and purified under anaerobic conditions, exhibits a broad UV-visible absorbance expected for the catalytic [4Fe-4S] 2ϩ cluster found in RS enzymes, but they found that the enzyme has very low activity. Suspecting that cobalamin could play an important role in the methylation reaction, the authors noted the presence of a trace mixture of tightly-bound cobalamins whose presence was hidden by the iron-sulfur cluster, and they show that reconstitution of the Mmp10 with either methylcobalamin or hydroxocobalamin dramatically increases activity.…”

Functional interactions between post-translationally modified amino acids of methyl-coenzyme M reductase inMethanosarcina acetivorans

“…To test this, we generated deletion mutants lacking the genes responsible for installing S -methylcysteine, thioglycine and 5-(S)-methylarginine in all possible combinations (Supplementary Figure S3A). These include MA4551, which we have renamed mamA ( m ethyl a rginine m odification) to better reflect its function (Supplementary Figure S4 and (27, 28)) and ycaO-tfuA (MA0165/MA0164), which coverts glycine to thioglycine (26, 29). The phenotypic analyses described below were carried out in mutants that encode wild-type MCR, while biochemical and structural studies were conducted with MCR purified from a second set of mutants that encode a modified mcrG allele (encoding the γ subunit of MCR) with an N-terminal a tandem-affinity purification tag comprising of a twin-Strep tag and a 3×FLAG tag (hereafter referred to as TAP tag) (Supplementary Figure S3B).…”