Fig 1.
Crystal structure of MCR from M. acetivorans.
(A) The α2β2γ2 configuration of the inactive MCR complex affinity-purified from M. acetivorans under aerobic conditions. The location of posttranslational modifications, the F430 cofactor, as well as CoM-SH and CoB-SH are highlighted. No electron density corresponding to the affinity tag was observed. (B) A close-up of the active site within the McrA subunit in M. acetivorans. The red shading of the MeHis label indicates that this residue is from the other α-subunit, illustrating the fact that residues from both α-subunits are present in each active site. CoB, methyl-coenzyme B; CoM, methyl-coenzyme M; Dya, Didehydroasparate F430, factor 430; MCR, methyl-coenzyme M reductase; McrA, alpha subunit of MCR; MeArg, 5-(S)-methylarginine; MeCys, S-methylcysteine; MeHis, N1-methylhistidine; ThioGly, thioglycine.
Fig 2.
(A) Chromosomal organization of genes near the mcr operon in M. acetivorans. Two SAM-dependent methyltransferases are encoded on either side of the mcr operon. MA4551 encodes a radical SAM methyltransferase that was recently shown to be involved in the conversion of Arg285 in McrA to 5-(S)-methylarginine [27]. We have named this gene mamA based on its proposed role (see text for details). (B) A maximum-likelihood phylogenetic tree of the amino acid sequence of mcmA homologs in archaea. The node labels indicate support values calculated using the Shiomdaira–Hasegawa test using 1,000 resamples. Support values less than 0.6 have not been shown. The outgroup is from bacterial McmA homologs (in black). ANME, anaerobic methane-oxidizing archaea; mamA, methylarginine modification; mcmA, methylcysteine modification; mcr, methyl-coenzyme M reductase; McrA, alpha subunit of MCR; SAM, S-adenosylmethionine.
Fig 3.
MALDI-TOF-MS analysis of McrA.
(A) A list of all the posttranslational modifications found in McrA derived from the various mutants as indicated. (B) Spectrum of the indicated peptides, which were obtained after AspN and GluC digestion of MCR from wild type (WWM60) and mutants lacking ycaO-tfuA, mcmA, and mamA in all possible combinations. The M280-S301 peptide contains the Arg285 that is modified to 5-(S)-methylarginine by MamA. (C) Spectrum of the indicated MCR tryptic peptide obtained from wild type (WWM60) and mutants lacking ycaO-tfuA, mcmA, and mamA in all possible combinations. The L461-R491 peptide contains the Gly465 and Cys472 that are modified to thioglycine and S-methylcysteine by ycaO-tfuA and mcmA, respectively, as well as the didehydroaspartate at Asp470. Modified residues are red in the peptide sequence. Individual spectra are labeled with numbers in parentheses as indicated in panel A. MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; mamA, methylarginine modification; mcmA, methylcysteine modification; MCR, methyl-coenzyme M reductase; McrA, alpha subunit of MCR.
Fig 4.
Phenotypic analyses on methanogenic substrates.
Heat maps depicting (A) growth rate or (B) growth yield (measured as the maximum optical density at 600 nm) of mutants lacking ycaO-tfuA, mcmA, and mamA in all possible combinations relative to wild type in bicarbonate-buffered HS medium supplemented with 125 mM MeOH, 50 mM TMA, 20 mM DMS, or 40 mM Ac as the methanogenic substrates. All growth assays were performed at 36°C. Heat maps depicting (C) growth rate or (D) growth yield (measured as the maximum optical density at 600 nm) of mutants in bicarbonate-buffered HS medium supplemented with 50 mM TMA at three different temperatures as indicated. Statistically significant differences in growth parameters (p < 0.05 or p < 0.01) relative to the wild type as determined by a two-sided t test are indicated with * and **, respectively. The black box for the ΔmcmAΔycaO-tfuA mutant in DMS-supplemented HS medium indicates that no measurable growth was detected after 6 mo of incubation. The primary data used to construct the heat maps are presented in S2–S13 Tables. Ac, sodium acetate; DMS, dimethyl sulfide; HS, high-salt; mamA, methylarginine modification; mcmA, methylcysteine modification; MeOH, methanol; TMA, trimethylamine.
Fig 5.
The thermal stability of the tandem-affinity-tagged MCR complex purified from (A) WT and single mutants lacking ycaO-tfuA, mcmA, or mamA; (B) WT and double mutants lacking two of ycaO-tfuA, mcmA, and mamA in all possible combinations; and (C) WT and the triple mutant lacking ycaO-tfuA, mcmA, and mamA was measured using the SYPRO Orange dye–based Thermofluor assay. The inflection point of the first differential curve for the fluorescence intensity relative to the temperature (−dI/dT) for each of three technical replicates was used to calculate the mean Tm ± standard deviation (in°C) of the MCR complex. The no-dye control (in gray) lacks the SYPRO Orange dye, and the no-protein control (in black) was performed with elution buffer instead of purified protein. The assays reported in each panel were conducted on the same day with freshly purified protein using the WT as a control. Slight variations from day to day in the control are due to variations in the individual protein preparations. The primary data used to construct this figure are provided in S14 Table. mamA, methylarginine modification; mcmA, methylcysteine modification; MCR, methyl-coenzyme M reductase; Tm, melting temperature; WT, wild-type.
Fig 6.
Absence of modified amino acids does not change the structure of MCR.
(A–C) The local structure near Gly465 (modified to ThioGly), Cys472 (modified to MeCys), and Arg285 (modified to MeArg) in wild type. (D–F) The local structure near the unmodified Gly465 residue, unmodified Cys472 residue, and unmodified Arg285 residue in MCR derived from the ΔycaO-tfuA, ΔmcmA, ΔmamA single mutants, respectively. The structure of MCR purified from the double and triple mutants was indistinguishable from that shown here, with RMSD between all pairs ranging between 0.1–0.3 Å. mamA, methylarginine modification; mcmA, methylcysteine modification; MCR, methyl-coenzyme M reductase; MeArg, 5-(S)-methylarginine; MeCys, S-methylcysteine; RMSD, root mean square deviation; ThioGly, thioglycine.