mcrA (MA_4546) encodes the alpha subunit of methyl-coenzyme M reductase (MCR) in the metabolically versatile marine methanogen Methanosarcina acetivorans C2A, which forms methane from acetate (acetoclastic route), from methanol and methylamines (methylotrophic route) and, in stationary phase, from carbon monoxide, but cannot use H2 plus CO2. MCR is a soluble cytoplasmic alpha2beta2gamma2 heterohexamer that catalyzes the terminal, methane-forming step shared by all of these routes, the reduction of methyl-coenzyme M by coenzyme B to methane and the CoM-S-S-CoB heterodisulfide (EC 2.8.4.1). Each holoenzyme has two active sites formed at the alpha, alpha', beta, gamma interface, each holding one non-covalently bound nickel porphinoid coenzyme F430; the alpha subunit supplies the axial glutamine ligand (Gln161) to the F430 nickel and most coenzyme M and coenzyme B contacts. The alpha subunit carries conserved active-site post-translational modifications (thioglycine, methylhistidine, methylcysteine, didehydroaspartate) and a C5-methylarginine (Arg285) installed by the radical SAM methyltransferase MA_4551, which is dispensable for growth but contributes to MCR stability. Cryo-EM structures of assembly intermediates show that the McrD protein binds the alpha subunit of apo-MCR and promotes F430 loading.
Definition: Binding to coenzyme F430, a nickel-containing tetrapyrrole (hydrocorphinoid) cofactor of methyl-coenzyme M reductase and related alkyl-coenzyme M reductases.
Justification: Every MCR alpha subunit binds F430 through an axial glutamine ligand to its nickel. GO has heme, chlorophyll and cobalamin binding under tetrapyrrole binding but no F430 term, so keyword mappings fall back to metal ion binding.
Parent term: tetrapyrrole binding
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping (Cytoplasm, by similarity to the Methanothermobacter marburgensis MCR I alpha subunit P11558). MCR is a soluble, very abundant cytoplasmic enzyme; immunolabeling studies (including in Methanosarcina) suggest proximity to the membrane, but no MCR subunit has a membrane anchor. Reason: Cytoplasm is the correct location for this soluble enzyme complex. It is the same claim as the ISS row and the two reinforce each other. The earlier draft supported this row with a non-verbatim placeholder quote from the falcon deep-research file; that is replaced with verbatim support from the UniProt record and the Thauer 2019 review. Possible peripheral membrane association is unresolved and does not warrant a membrane term. Supporting Evidence: file:METAC/mcrA/mcrA-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000250|UniProtKB:P11558}. PMID:30951290 in the soluble cell fraction, whole-cell immune-labeling experiments PMID:30951290 However, none of the genes associated with MCR synthesis |
| GO:0015948 methanogenesis | IEA GO_REF:0000120 | ACCEPT | Summary: Historical combined automated annotation (InterPro plus the Methanogenesis keyword). In the current GOA snapshot this assertion is carried by the InterPro2GO row (GO_REF:0000002), so this record is retired as a source change, not as a biological judgment. Reason: Same claim as the current GO_REF:0000002 row and reviewed identically. MCR catalyzes the methane-forming step of every methanogenic route used by M. acetivorans (acetate, methanol, methylamines, CO), so the generic term is the appropriate level for this organism (see the GO_REF:0000002 row). Supporting Evidence: PMID:16346552 Sodium acetate, methanol, methylamine, dimethylamine, and trimethylamine were substrates for growth and methanogenesis; H(2)-CO(2) (80:20) and sodium formate were not. |
| GO:0016740 transferase activity | IEA GO_REF:0000043 | TOO GENERAL | Summary: Historical UniProt keyword mapping (KW-0808 Transferase). The term is true, since EC 2.8.4.1 coenzyme-B sulfoethylthiotransferase activity is classified as a transferase, but it is uninformative. The row is no longer in the current GOA snapshot and is retired as a source change. Reason: GO:0050524 coenzyme-B sulfoethylthiotransferase activity, accepted elsewhere in this review, is an is_a descendant of GO:0016740 (via GO:0016782 and GO:0050497, checked in QuickGO), so transferase activity is entailed and adds nothing. The earlier draft marked this row REMOVE, which is wrong for a true-but-broad term. Supporting Evidence: file:METAC/mcrA/mcrA-uniprot.txt Reaction=coenzyme B + methyl-coenzyme M = methane + coenzyme M-coenzyme B heterodisulfide |
| GO:0046872 metal ion binding | IEA GO_REF:0000043 | MODIFY | Summary: Historical UniProt keyword mapping (KW-0479 Metal-binding). The only metal is the nickel of coenzyme F430, which the alpha subunit binds through the axial Gln161 ligand. The row is no longer in the current GOA snapshot and is retired as a source change. Reason: The protein does not bind a free metal ion; it binds the nickel tetrapyrrole cofactor F430, and the residue contact to the nickel is part of binding that cofactor. By analogy with heme proteins, which GO annotates with heme binding rather than iron ion binding, the informative term is cofactor binding. GO has no coenzyme F430 binding term, so the closest existing term is GO:0046906 tetrapyrrole binding (the parent of heme, chlorophyll and cobalamin binding; ChEBI classifies coenzyme F430 as a nickel tetrapyrrole). The earlier draft proposed nickel cation binding (GO:0016151), which mis-describes binding of a metal held in a tetrapyrrole cofactor. A coenzyme F430 binding term is proposed under proposed_new_terms. Proposed replacements: tetrapyrrole binding Supporting Evidence: PMID:37307484 containing the F430 axial ligand GlnΞ±161 file:METAC/mcrA/mcrA-uniprot.txt Coenzyme F430 is a yellow nickel porphinoid |
| GO:0050524 coenzyme-B sulfoethylthiotransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Historical combined automated annotation (InterPro, Rhea and EC) for the MCR alpha-subunit activity. The current GOA row for the same term, evidence and reference adds an ARBA source and is reviewed separately; this record is retired as a source change. Reason: Same claim as the current IEA and ISS rows, reviewed identically. The earlier draft cited PMID:29743535 (a radical SAM methyltransferase paper) as support for the activity; that paper does not address the catalytic activity and has been replaced as support (see its reference_review). Supporting Evidence: PMID:37307484 Each active site contains F430, a unique Ni porphinoid that catalyzes the reduction of methyl-coenzyme M (CH3-CoM) by coenzyme B (CoB), yielding methane and a heterodisulfide |
| GO:0005737 cytoplasm | ISS GO_REF:0000024 | ACCEPT | Summary: Curator-judged sequence-similarity transfer from the M. marburgensis MCR I alpha subunit (P11558), which has an experimental cytoplasm annotation. Reason: MCR is a soluble cytoplasmic enzyme throughout methanogens, and the alpha subunit family is highly conserved. Same claim as the IEA row; the two reinforce each other. Supporting Evidence: file:METAC/mcrA/mcrA-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000250|UniProtKB:P11558}. PMID:30951290 in the soluble cell fraction, whole-cell immune-labeling experiments |
| GO:0015948 methanogenesis | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the MCR alpha-subunit family signatures. MCR catalyzes the terminal methane-forming step, so the alpha subunit directly participates in methanogenesis. Reason: Unlike the obligately hydrogenotrophic Methanothermobacter thermautotrophicus (where the ortholog review narrows this term to GO:0019386 methanogenesis, from carbon dioxide), M. acetivorans cannot grow on H2 plus CO2 and forms methane from acetate, methanol, mono-, di- and trimethylamine, and (in stationary phase) CO. Its single MCR therefore catalyzes the final step of several routes. GO has specific children only for the acetate (GO:0019385) and methanol (GO:0019387) routes, not for methylamine or CO-derived methanogenesis, so replacing the generic term with those children would drop routes. The generic term is as precise as warranted for this organism. Supporting Evidence: PMID:16346552 Sodium acetate, methanol, methylamine, dimethylamine, and trimethylamine were substrates for growth and methanogenesis; H(2)-CO(2) (80:20) and sodium formate were not. PMID:37307484 strains were routinely maintained in 10 mL of high salt (HS) media with either acetate (40 mM), methanol (125 mM), or trimethylamine (50 mM) as the sole carbon and energy source PMID:15550538 Methane formation surpassed acetate/formate formation only when the cultures entered stationary growth phase |
| GO:0050524 coenzyme-B sulfoethylthiotransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated annotation (ARBA, InterPro, Rhea/EC) for the MCR alpha-subunit activity, EC 2.8.4.1. Reason: The MCR alpha-subunit family (IPR016212) is uniformly the F430-bearing catalytic subunit of methyl-coenzyme M reductase, and the active-site architecture is conserved. The M. acetivorans cryo-EM structure resolves F430, coenzyme M and coenzyme B in the active site with the alpha subunit supplying the F430 axial ligand. The activity is that of the alpha2beta2gamma2 holoenzyme, captured by the NEW complex annotation; enables is retained for the catalytic subunit, as in the Methanothermobacter ortholog review. Supporting Evidence: PMID:37307484 Each active site contains F430, a unique Ni porphinoid that catalyzes the reduction of methyl-coenzyme M (CH3-CoM) by coenzyme B (CoB), yielding methane and a heterodisulfide PMID:37307484 Strong density is present throughout the entire active-site cavity, allowing for unambiguous identification of CoM, CoB, and F430 as well as the loop containing GlnΞ±161. |
| GO:0050524 coenzyme-B sulfoethylthiotransferase activity | ISS GO_REF:0000024 | ACCEPT | Summary: Curator-judged sequence-similarity transfer from the M. marburgensis MCR I alpha subunit (P11558), whose activity is experimentally established (several EXP/IDA annotations on purified enzyme). Reason: The donor is the best characterized MCR alpha subunit, and MCR active sites are conserved across methanogen lineages. Same claim as the IEA rows. Supporting Evidence: PMID:37307484 Each active site contains F430, a unique Ni porphinoid that catalyzes the reduction of methyl-coenzyme M (CH3-CoM) by coenzyme B (CoB), yielding methane and a heterodisulfide |
| GO:0044674 methyl coenzyme M reductase complex | IDA PMID:37307484 McrD binds asymmetrically to methyl-coenzyme M reductase imp... | NEW | Summary: McrA is the alpha subunit of the alpha2beta2gamma2 MCR holoenzyme. Cryo-EM structures of MCR purified from M. acetivorans (PDB 8GF5/8GF6) show the assembled heterohexamer with active sites at the alpha, alpha', beta, gamma interface. Reason: No complex (cellular component) annotation exists in GOA. GO:0044674 (verified in QuickGO; alpha2beta2gamma2 hexamer) captures that the activity is executed by the holoenzyme, with the alpha subunit contributing the F430 ligand and most of each shared active site. Evidence type changed from IPI to IDA, since the structure of the purified complex is direct observation. Consistent with the Methanothermobacter ortholog review. Supporting Evidence: PMID:37307484 MCR is a C2-symmetric heterohexamer defined by Ξ±2Ξ²2Ξ³2 subunit stoichiometry PMID:37307484 two active sites formed at the interface of the Ξ±, Ξ±β², Ξ², and Ξ³ subunits file:METAC/mcrA/mcrA-falcon-research.md The enzyme assembles as a heterohexameric complex (Ξ±βΞ²βΞ³β) |
| GO:0046906 tetrapyrrole binding | IDA PMID:37307484 McrD binds asymmetrically to methyl-coenzyme M reductase imp... | NEW | Summary: The cryo-EM structure of M. acetivorans MCR resolves the nickel tetrapyrrole coenzyme F430 in the active site, with the alpha-subunit Gln161 as its axial ligand. MCR from a mcrD deletion strain has reduced F430 occupancy, and in the empty site Gln161 moves away from the cavity. Reason: Replaces the earlier draft's NEW GO:0016151 nickel cation binding, which described the nickel of a tetrapyrrole cofactor as a free cation. GO has no coenzyme F430 binding term; GO:0046906 tetrapyrrole binding (verified in QuickGO; parent of heme, chlorophyll and cobalamin binding) is the closest existing term, and a specific child is proposed under proposed_new_terms. F430 sits at the interface of several subunits, but the alpha subunit provides the axial ligand to its nickel, so the binding is a property of this subunit. Non-core relative to the catalytic activity it supports. Supporting Evidence: PMID:37307484 Strong density is present throughout the entire active-site cavity, allowing for unambiguous identification of CoM, CoB, and F430 as well as the loop containing GlnΞ±161. PMID:37307484 Notably, GlnΞ±161 has shifted ~7 Γ
away from the active-site cavity, consistent with a lack of F430. |
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Download this section (compressed HTML)Q: Should GO add a coenzyme F430 binding term (child of tetrapyrrole binding) for MCR alpha subunits, rather than relying on generic metal ion or nickel cation binding mappings?
Q: Should GO add a methanogenesis-from-methylamines child of GO:0015948, so that MCR in methylotrophic methanogens can be annotated with route-specific terms?
Q: What sequence or structural features, if any, determine whether an MCR operates mainly in methanogenesis or in anaerobic methane oxidation?
Experiment: Compare MCR purified from wild type and PTM-enzyme deletion strains grown on methanol, trimethylamine and acetate, measuring F430 occupancy, thermal stability and steady-state activity.
Hypothesis: McrA post-translational modifications tune MCR stability or catalysis under the different methanogenic growth substrates of M. acetivorans.
Type: Genetic and biochemical characterization
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