Ethylmalonyl-CoA mutase (Ecm) is a radical enzyme that catalyzes the interconversion of (2R)-ethylmalonyl-CoA to (2S)-methylsuccinyl-CoA using adenosylcobalamin (vitamin B12) as a cofactor. This enzyme is a key component of the ethylmalonyl-CoA pathway for acetyl-CoA assimilation, which is essential for Methylorubrum extorquens growth on one-carbon (methanol, methylamine) and two-carbon (ethanol, ethylamine) compounds as sole carbon sources. Unlike organisms that use the glyoxylate cycle for acetyl-CoA assimilation, M. extorquens lacks isocitrate lyase and instead employs this alternative pathway. Ecm acts as a metabolic control point, allowing efficient restoration of metabolic balance when challenged with sudden changes in growth substrate. The enzyme is assigned EC 5.4.99.63 based on characterization by Good et al. (2015). Disruption of ecm abolishes growth on C1 and C2 compounds but can be rescued by glyoxylate or glycolate addition, confirming its role in converting acetyl-CoA to glyoxylate.
Definition: Catalysis of the reaction: (2R)-ethylmalonyl-CoA = (2S)-methylsuccinyl-CoA. This reaction requires adenosylcobalamin as a cofactor.
Justification: Ethylmalonyl-CoA mutase (EC 5.4.99.63) is a distinct enzyme from methylmalonyl-CoA mutase (EC 5.4.99.2). The substrates are different: ethylmalonyl-CoA vs methylmalonyl-CoA. Currently, ecm is incorrectly annotated with GO:0004494 (methylmalonyl-CoA mutase activity) due to sequence similarity, but this annotation is biochemically incorrect. A specific term for ethylmalonyl-CoA mutase activity would enable accurate annotation of this enzyme and others in the ethylmalonyl-CoA pathway.
Parent term: intramolecular transferase activity
Mappings:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
|
GO:0003824
catalytic activity
|
IEA
GO_REF:0000002 |
MODIFY |
Summary: This annotation is technically correct but overly broad. Ecm has a well-defined catalytic activity as an ethylmalonyl-CoA mutase (EC 5.4.99.63), which is an intramolecular transferase that interconverts (2R)-ethylmalonyl-CoA to (2S)-methylsuccinyl-CoA. A more specific term should be used.
Reason: The term "catalytic activity" is too general for a well-characterized enzyme. While there is no specific GO term for "ethylmalonyl-CoA mutase activity", the most appropriate existing term is GO:0016866 (intramolecular transferase activity), which accurately describes the mechanistic class of this enzyme. This is preferable to the overly broad "catalytic activity" and avoids the incorrect implication of "methylmalonyl-CoA mutase activity" which involves different substrates.
Proposed replacements:
intramolecular transferase activity
|
|
GO:0004494
methylmalonyl-CoA mutase activity
|
IEA
GO_REF:0000002 |
MODIFY |
Summary: This annotation is INCORRECT. GO:0004494 (methylmalonyl-CoA mutase activity) is defined as catalyzing "(R)-methylmalonyl-CoA = succinyl-CoA". However, Ecm catalyzes a distinct reaction: "(2R)-ethylmalonyl-CoA = (2S)-methylsuccinyl-CoA" (EC 5.4.99.63). These are chemically different reactions with different substrates and products. The annotation appears to have been transferred based on sequence similarity to the methylmalonyl-CoA mutase family, but the substrate specificity of Ecm is different.
Reason: Ecm is an ethylmalonyl-CoA mutase, not a methylmalonyl-CoA mutase. The substrates are different: ethylmalonyl-CoA vs methylmalonyl-CoA. UniProt clearly assigns EC 5.4.99.63 (ethylmalonyl-CoA mutase) to this enzyme based on PMID:25448820. There is no specific GO term for ethylmalonyl-CoA mutase activity, so the best option is to use the parent term GO:0016866 (intramolecular transferase activity) which is mechanistically accurate. A new GO term for ethylmalonyl-CoA mutase activity should be proposed.
Proposed replacements:
intramolecular transferase activity
|
|
GO:0016853
isomerase activity
|
IEA
GO_REF:0000120 |
ACCEPT |
Summary: This annotation is correct. Ethylmalonyl-CoA mutase is an isomerase that catalyzes an intramolecular rearrangement, converting (2R)-ethylmalonyl-CoA to (2S)-methylsuccinyl-CoA. The "isomerase" classification aligns with the enzyme's EC number (5.4.99.63) where class 5 corresponds to isomerases.
Reason: The isomerase activity annotation correctly captures the enzyme class. Mutases are a subclass of isomerases that catalyze the intramolecular transfer of groups. The EC classification 5.4.99.63 places this enzyme under isomerases (class 5), intramolecular transferases (subclass 4), specifically transferring other groups (sub-subclass 99). This IEA annotation appropriately reflects the broad enzymatic class.
|
|
GO:0016866
intramolecular transferase activity
|
IEA
GO_REF:0000002 |
ACCEPT |
Summary: This annotation is correct and represents the most appropriate molecular function term for Ecm given current GO term availability. Ethylmalonyl-CoA mutase catalyzes an intramolecular rearrangement involving transfer of a group from one position to another within the same molecule, which is the defining feature of intramolecular transferases (EC 5.4).
Reason: The intramolecular transferase activity annotation accurately describes the mechanistic class of this enzyme. Given that there is no specific GO term for "ethylmalonyl-CoA mutase activity", GO:0016866 is the most informative and accurate term available. The enzyme belongs to EC 5.4.99.63, where 5.4 represents intramolecular transferases.
|
|
GO:0031419
cobalamin binding
|
IEA
GO_REF:0000120 |
ACCEPT |
Summary: This annotation is correct. Ecm requires adenosylcobalamin (coenzyme B12) as a cofactor. UniProt entry Q49115 documents the cofactor requirement with evidence from PMID:25448820. The protein contains a B12-binding domain (residues 530-659) with the characteristic axial His543 residue that coordinates the cobalt in the cobalamin cofactor.
Reason: The cobalamin binding annotation is well-supported by both computational domain analysis (B12-binding domain, PROSITE PS51332) and experimental evidence (PMID:25448820 demonstrated adenosylcobalamin cofactor requirement). The binding site annotation at His543 indicates the axial binding residue for the Co atom of the cobalamin cofactor.
|
|
GO:0046872
metal ion binding
|
IEA
GO_REF:0000120 |
ACCEPT |
Summary: This annotation is correct but could be more specific. Ecm binds cobalt indirectly through the adenosylcobalamin (vitamin B12) cofactor. The cobalt ion is coordinated by His543 as the axial ligand.
Reason: While this annotation is somewhat general, it is accurate. The metal ion binding occurs through coordination of the cobalt center of the cobalamin cofactor. UniProt indicates the binding site at position 543 coordinating the Co ligand part of adenosylcob(III)alamin. Given that GO:0031419 (cobalamin binding) is also annotated, which is more specific, this general term provides complementary but redundant information. However, it is not incorrect and can be retained.
|
|
GO:0019681
acetyl-CoA assimilation pathway
|
IDA
PMID:25448820 Ethylmalonyl coenzyme A mutase operates as a metabolic contr... |
NEW |
Summary: Ecm is a key enzyme in the ethylmalonyl-CoA pathway, which serves as the acetyl-CoA assimilation pathway in M. extorquens. This organism lacks isocitrate lyase and uses this alternative pathway to convert acetyl-CoA to glyoxylate. Knockout studies show that ecm mutants cannot grow on C1 or C2 compounds, and growth can be rescued by glyoxylate addition, confirming Ecm's essential role in this pathway.
Reason: GO:0019681 (acetyl-CoA assimilation pathway) is directly applicable to Ecm's function. UniProt states: "Is involved in the ethylmalonyl-CoA pathway for acetyl-CoA assimilation required for M.extorquens growth on one- and two-carbon compounds." Disruption phenotype studies (PMID:8704985) show loss of ability to convert acetyl-CoA into glyoxylate, which is rescued by glyoxylate addition. This provides strong evidence for involvement in the acetyl-CoA assimilation pathway.
|
Q: Is there any evidence that Ecm can also use methylmalonyl-CoA as a substrate, even if with lower efficiency, or is it strictly specific for ethylmalonyl-CoA?
Q: Are there other organisms with ethylmalonyl-CoA pathway that also lack a specific GO term for this activity?
Experiment: Purify recombinant Ecm and measure kinetic parameters (Km, kcat) for both (2R)-ethylmalonyl-CoA and (R)-methylmalonyl-CoA substrates. Compare catalytic efficiency to determine substrate specificity and whether there is any activity toward methylmalonyl-CoA.
Hypothesis: Ecm has strict substrate specificity for ethylmalonyl-CoA over methylmalonyl-CoA
Type: enzyme kinetics
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template_variables:
organism: METEA
gene_id: ecm
gene_symbol: ecm
uniprot_accession: Q49115
protein_description: 'RecName: Full=Ethylmalonyl-CoA mutase {ECO:0000303|PubMed:25448820};
EC=5.4.99.63 {ECO:0000269|PubMed:25448820};'
gene_info: Name=ecm {ECO:0000303|PubMed:25448820}; Synonyms=meaA {ECO:0000303|PubMed:8868443};
OrderedLocusNames=MexAM1_META1p0180;
organism_full: Methylorubrum extorquens (strain ATCC 14718 / DSM 1338 / JCM 2805
/ NCIMB 9133 / AM1) (Methylobacterium extorquens).
protein_family: Belongs to the methylmalonyl-CoA mutase family.
protein_domains: Acid_CoA_mut_C. (IPR006159); Cbl-dep_enz_cat. (IPR016176); Cobalamin-bd.
(IPR006158); Cobalamin-bd_sf. (IPR036724); MeMalonylCoA_mutase_a/b_cat. (IPR006099)
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citation_count: 22
BEFORE YOU BEGIN RESEARCH: You MUST verify you are researching the CORRECT gene/protein. Gene symbols can be ambiguous, especially for less well-characterized genes from non-model organisms.
DO NOT PROCEED WITH RESEARCH ON A DIFFERENT GENE. Instead:
- State clearly: "The gene symbol 'ecm' is ambiguous or literature is limited for this specific protein"
- Explain what you found (e.g., "Found extensive literature on a different gene with the same symbol in a different organism")
- Describe the protein based ONLY on the UniProt information provided above
- Suggest that the protein function can be inferred from domain/family information
Please provide a comprehensive research report on the gene ecm (gene ID: ecm, UniProt: Q49115) in METEA.
The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.
You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.
We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.
We are interested in where in or outside the cell the gene product carries out its function.
We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.
Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.
Question: You are an expert researcher providing comprehensive, well-cited information.
Provide detailed information focusing on:
1. Key concepts and definitions with current understanding
2. Recent developments and latest research (prioritize 2023-2024 sources)
3. Current applications and real-world implementations
4. Expert opinions and analysis from authoritative sources
5. Relevant statistics and data from recent studies
Format as a comprehensive research report with proper citations. Include URLs and publication dates where available.
Always prioritize recent, authoritative sources and provide specific citations for all major claims.
BEFORE YOU BEGIN RESEARCH: You MUST verify you are researching the CORRECT gene/protein. Gene symbols can be ambiguous, especially for less well-characterized genes from non-model organisms.
DO NOT PROCEED WITH RESEARCH ON A DIFFERENT GENE. Instead:
- State clearly: "The gene symbol 'ecm' is ambiguous or literature is limited for this specific protein"
- Explain what you found (e.g., "Found extensive literature on a different gene with the same symbol in a different organism")
- Describe the protein based ONLY on the UniProt information provided above
- Suggest that the protein function can be inferred from domain/family information
Please provide a comprehensive research report on the gene ecm (gene ID: ecm, UniProt: Q49115) in METEA.
The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.
You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.
We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.
We are interested in where in or outside the cell the gene product carries out its function.
We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.
Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.
Plan
- Verify identity and nomenclature for UniProt Q49115 (ecm/meaA) in Methylorubrum extorquens AM1, and ensure the gene symbol matches the ethylmalonyl‑CoA mutase (EMCP) enzyme. (Completed)
- Collect and summarize primary function (reaction, substrates/products, cofactor dependence), pathway role in EMCP and serine‑cycle glyoxylate regeneration, and distinctions from methylmalonyl‑CoA mutase. (Completed)
- Summarize regulation/flux control, genetic/phenotypic evidence (overexpression and deletion), and cellular localization inference. (Completed)
- Highlight recent developments (2023–2024), applications, and quantitative data/statistics. (Completed where supported by gathered evidence; one 2023 item included.)
- Provide a concise artifact table of key facts and references. (Completed)
Comprehensive research report
1) Identity and key definitions
- Nomenclature and organism: The gene ecm (also historically meaA) in Methylorubrum (formerly Methylobacterium) extorquens AM1 encodes ethylmalonyl‑CoA mutase, a core enzyme of the ethylmalonyl‑CoA pathway (EMCP) used by isocitrate lyase–negative methylotrophs for glyoxylate regeneration that feeds the serine cycle (Anthony 2011; Korotkova et al. 2002). The equivalence of ecm and meaA in this organism is explicitly described (Anthony 2011) and meaA mutant phenotypes in AM1 establish its EMCP role (Korotkova et al. 2002) (anthony2011howhalfa pages 13-17, korotkova2002glyoxylateregenerationpathway pages 6-8).
- Enzyme class and reaction: Ethylmalonyl‑CoA mutase catalyzes the intramolecular rearrangement of (2R)‑ethylmalonyl‑CoA to (2S)‑methylsuccinyl‑CoA; it is mechanistically distinct from methylmalonyl‑CoA mutase (which interconverts methylmalonyl‑CoA and succinyl‑CoA) (Anthony 2011; foundational EMCP biochemistry: Erb et al. 2007) (anthony2011howhalfa pages 13-17, good2015metaboliccontrolof pages 13-20). This mutase requires coenzyme B12 (adenosylcobalamin), classifying it as a cobalamin‑dependent mutase (Anthony 2011) (anthony2011howhalfa pages 13-17).
2) Biological role and pathway integration
- Pathway context: The EMCP converts acetyl‑CoA and CO2 into glyoxylate and succinate to replenish the serine cycle in organisms lacking the glyoxylate shunt; pathway resolution and unique steps, including Ecm, were established by Erb et al. (2007) and earlier methylotrophy work in AM1 (Korotkova et al. 2002) (good2015metaboliccontrolof pages 13-20, korotkova2002glyoxylateregenerationpathway pages 6-8). Within EMCP, upstream steps generate (2S)-ethylmalonyl‑CoA via crotonyl‑CoA carboxylase/reductase; an epimerase supplies the (2R) isomer that Ecm converts to methylsuccinyl‑CoA, continuing toward propionyl‑/succinyl‑CoA and glyoxylate regeneration (Anthony 2011; Good 2015) (anthony2011howhalfa pages 13-17, good2015metaboliccontrolof pages 13-20).
- Interplay with serine cycle: The EMCP’s glyoxylate output must be tightly balanced due to glyoxylate toxicity at millimolar levels; Ecm sits at a key node that modulates flux to match downstream assimilation capacity in the serine‑cycle network (Good 2015) (good2015metaboliccontrolof pages 46-53).
3) Regulation, flux control, and genetic evidence
- Flux control point: Multiple experimental switchover studies show that Ecm is a metabolic control point. During a succinate→ethylamine shift in AM1, cells exhibited a ~9 h growth lag with accumulation of ethylmalonyl‑derived degradation products; ecm transcripts and Ecm activity rose by ~9 h, ethylmalonyl‑CoA fell, and growth resumed. Overexpressing ecm prevented ethylmalonyl‑CoA accumulation, shortened the lag, and yielded faster growth, directly demonstrating that Ecm limits EMCP flux under these conditions (Good 2015) (good2015metaboliccontrolof pages 41-46, good2015metaboliccontrolof pages 74-83). In the same context, a minimal Ecm activity of ~12 nmol·min⁻¹·mg⁻¹ was estimated to support growth (Good 2015) (good2015metaboliccontrolof pages 74-83).
- Overexpression metabolomics: Targeted metabolomics of ecm overexpression strains revealed decreased 3‑hydroxybutyryl‑CoA and altered mesaconate and propionyl‑CoA dynamics consistent with increased EMCP throughput, supporting the control‑point conclusion (Good 2015) (good2015metaboliccontrolof pages 96-103).
- Deletion phenotype and functional rescue: Δecm mutants cannot grow on methanol, establishing ecm as essential for methylotrophic growth; growth is rescued by external glyoxylate (5 mM), consistent with the EMCP’s role as the glyoxylate‑regenerating route in AM1 (Sonntag et al. 2015) (sonntag2015highlevelproductionof pages 204-209). This aligns with broader EMCP use in AM1 as an alternative to the absent glyoxylate shunt (Sonntag et al. 2015) (sonntag2015highlevelproductionof pages 94-95).
- Cellular localization: Ecm functions in central carbon metabolism and EMCP in bacteria; while direct localization data were not identified here, EMCP enzymes in AM1 are soluble metabolic enzymes and Ecm is inferred to be cytosolic based on pathway biochemistry and experimental overexpression in the cytosolic milieu (inference; see growth and metabolite dynamics upon plasmid‑based overexpression) (good2015metaboliccontrolof pages 41-46, good2015metaboliccontrolof pages 96-103).
4) Substrate specificity and distinction from methylmalonyl‑CoA mutase
- Specific substrate and stereochemistry: Ethylmalonyl‑CoA mutase operates on (2R)‑ethylmalonyl‑CoA to form (2S)‑methylsuccinyl‑CoA, whereas methylmalonyl‑CoA mutase catalyzes the methylmalonyl‑CoA ↔ succinyl‑CoA step later in the EMCP/TCA interface; these are distinct cobalamin‑dependent mutases with different physiological roles and substrates (Anthony 2011) (anthony2011howhalfa pages 13-17). Earlier AM1 labeling and mutant work mapped the mutase step to precede decarboxylation, with accumulation of methylsuccinyl‑derived acids in mutants (Korotkova et al. 2002) (korotkova2002glyoxylateregenerationpathway pages 6-8).
5) Recent developments and expert perspectives (prioritizing 2023–2024)
- Coordination of EMCP and glyoxylate cycle: In Paracoccus denitrificans Pd1222, RamB links EMCP status to expression of the glyoxylate cycle and binds EMCP CoA‑esters, revealing a metabolite‑sensing regulatory axis between two acetyl‑CoA assimilation routes (Kremer et al., Applied and Environmental Microbiology; published June 15, 2023; https://doi.org/10.1128/aem.00238-23). This supports a general principle of EMCP‑linked regulation in Alphaproteobacteria relevant to AM1’s control logic (kremer2023functionaldegeneracyin pages 1-2).
- Biotechnological applications leveraging EMCP/Ecm: EMCP engineering in AM1 enabled high‑level EMCP‑derived dicarboxylic acid production and clarified constraints such as cobalt (cobalamin) availability and PHB pathway interplay (Sonntag et al. 2015) (sonntag2015highlevelproductionof pages 94-95). Ecm overexpression has been used to alleviate flux limitations and improve growth transitions, a strategy applicable to production hosts (Good 2015) (good2015metaboliccontrolof pages 41-46).
6) Data and statistics
- Growth control by Ecm: ~9 h growth lag during succinate→ethylamine transition in wild type; ecm overexpression shortens the lag and ultimately yields higher growth rates (Good 2015) (good2015metaboliccontrolof pages 41-46, good2015metaboliccontrolof pages 74-83). Minimal Ecm activity estimated: ~12 nmol·min⁻¹·mg⁻¹ to support observed growth (Good 2015) (good2015metaboliccontrolof pages 74-83).
- Essentiality and rescue: Δecm is methanol‑negative; growth restored by 5 mM glyoxylate supplementation (Sonntag et al. 2015) (sonntag2015highlevelproductionof pages 204-209).
7) Notes on gene model and domains
- Gene symbol ambiguity: In AM1, ethylmalonyl‑CoA mutase is encoded by ecm, historically also referred to as meaA; these denote the same functional gene in this organism (Anthony 2011; Korotkova et al. 2002) (anthony2011howhalfa pages 13-17, korotkova2002glyoxylateregenerationpathway pages 6-8).
- Protein family and cofactor: Ecm belongs to the cobalamin‑dependent mutase family; explicit cobalamin dependence is supported by biochemical descriptions (Anthony 2011) (anthony2011howhalfa pages 13-17). Specific domain architecture and the precise ordered locus tag (e.g., MexAM1_META1p0180) were not recovered in the gathered literature excerpts; these are consistent with database annotations but are not directly cited here.
Current applications and implementations
- AM1 as a cell factory: EMCP provides a rich set of CoA‑ester intermediates amenable to rerouting for dicarboxylic acids and other chemicals; strain engineering (including modulation of cobalt supply and PHB pathway, and employing thioesterases) enables high titers (Sonntag et al. 2015) (sonntag2015highlevelproductionof pages 94-95).
- Control‑point engineering: ecm overexpression serves as a practical lever to increase EMCP throughput during dynamic conditions (Good 2015) (good2015metaboliccontrolof pages 41-46).
- Broader regulatory insight: The RamB study in P. denitrificans (2023) suggests a generalizable strategy where EMCP metabolites regulate alternate anaplerotic routes, informing control strategies in AM1 (Kremer et al. 2023; https://doi.org/10.1128/aem.00238-23) (kremer2023functionaldegeneracyin pages 1-2).
Expert opinions and analysis
- Foundational perspective: The EMCP filled long‑standing gaps in understanding C1/C2 growth in methylotrophs, with Ecm among the “new” key steps; its B12 dependence and distinct substrate scope underscore specialized control within central metabolism (Anthony 2011) (anthony2011howhalfa pages 13-17).
- Control theory in AM1: Ecm’s positioning near the EMCP’s convergence point makes it an effective buffer and control valve for glyoxylate regeneration, preventing toxic accumulation while enabling rapid resetting of metabolic flux after substrate shifts (Good 2015) (good2015metaboliccontrolof pages 46-53, good2015metaboliccontrolof pages 41-46).
Embedded reference table
| Topic | Key finding (1–2 sentences) | Organism/Context | Source (authors, year) | Journal | URL | Publication date/month |
|---|---|---|---|---|---|---|
| Identity & catalytic reaction | Ethylmalonyl-CoA mutase (ecm/meaA) catalyzes the rearrangement of (2R)-ethylmalonyl-CoA to (2S)-methylsuccinyl-CoA and is distinct from methylmalonyl-CoA mutase; the reaction is cobalamin (adenosylcobalamin) dependent. (anthony2011howhalfa pages 13-17, good2015metaboliccontrolof pages 13-20) | Methylorubrum (Methylobacterium) extorquens AM1 and other EMCP-containing bacteria | Anthony C. (2011); Erb TJ et al. (2007) | Science Progress; PNAS | https://doi.org/10.3184/003685011x13044430633960; https://doi.org/10.1073/pnas.0702791104 | Jun 2011; Jun 2007 |
| Role/pathway context in EMCP and glyoxylate regeneration | Ecm is a pathway-unique enzyme in the ethylmalonyl-CoA pathway (EMCP) that helps convert acetyl-CoA + CO2 into glyoxylate/succinate, thereby regenerating glyoxylate to feed the serine cycle in isocitrate-lyase–negative methylotrophs. (good2015metaboliccontrolof pages 13-20, korotkova2002glyoxylateregenerationpathway pages 6-8) | M. extorquens AM1; general EMCP context | Erb TJ et al. (2007); Korotkova N. et al. (2002) | PNAS; Journal of Bacteriology | https://doi.org/10.1073/pnas.0702791104; https://doi.org/10.1128/jb.184.6.1750-1758.2002 | Jun 2007; Mar 2002 |
| Flux control/regulation & overexpression effects | Ecm functions as a metabolic control point in the EMCP: overexpression accelerates consumption of ethylmalonyl-CoA, shortens growth lag during succinate→ethylamine switches, and can increase growth rate by relieving a bottleneck. (good2015metaboliccontrolof pages 41-46, good2015metaboliccontrolof pages 96-103) | Experimental switchover assays in M. extorquens AM1 | Good N. (2015) (study reporting Ecm control-point and overexpression experiments) | (reported study, 2015) | N/A | 2015 (reported experiments) |
| Deletion phenotype & glyoxylate rescue | Δecm deletion strains cannot grow on methanol (EMCP essential for methylotrophy); growth is restored by adding glyoxylate (5 mM), indicating the block is in glyoxylate regeneration. (sonntag2015highlevelproductionof pages 94-95, sonntag2015highlevelproductionof pages 204-209) | M. extorquens AM1 gene-deletion experiments | Sonntag F. et al. (2015) | Applied Microbiology and Biotechnology | https://doi.org/10.1007/s00253-015-6418-3 | Feb 2015 |
| EMCP vs glyoxylate cycle coordination (RamB) | In related alphaproteobacteria (Paracoccus denitrificans), the transcription factor RamB links EMCP metabolite levels to glyoxylate-cycle expression, showing genetic/metabolic coordination between the two acetyl-CoA assimilation routes. (kremer2023functionaldegeneracyin pages 1-2) | Paracoccus denitrificans Pd1222 (model for functional degeneracy) | Kremer K. et al. (2023) | Applied and Environmental Microbiology | https://doi.org/10.1128/aem.00238-23 | Jun 2023 |
| Pangenome prevalence / pathway architecture | The EMCP occurs broadly among type II methylotrophs as an alternative to the glyoxylate shunt; EMCP typically comprises ~12 enzymes often encoded at dispersed loci rather than a single operon. (good2015metaboliccontrolof pages 13-20, sonntag2015highlevelproductionof pages 94-95) | Type II methylotrophs including M. extorquens AM1 | Erb TJ et al. (2007); Sonntag F. et al. (2015) | PNAS; Applied Microbiology and Biotechnology | https://doi.org/10.1073/pnas.0702791104; https://doi.org/10.1007/s00253-015-6418-3 | Jun 2007; Feb 2015 |
| Biotech applications using EMCP intermediates | EMCP-derived CoA-esters can be diverted to produce C5-dicarboxylic acids and other products; engineering (e.g., thioesterase variants, PHB-negative strains, cobalt modulation) increases production of EMCP-derived dicarboxylic acids. (sonntag2015highlevelproductionof pages 94-95, good2015metaboliccontrolof pages 96-103) | M. extorquens AM1 strain engineering and metabolic engineering contexts | Sonntag F. et al. (2015); Good N. (2015) | Applied Microbiology and Biotechnology; (reported study, 2015) | https://doi.org/10.1007/s00253-015-6418-3 | Feb 2015; 2015 |
| Application in M. extorquens engineering (glycolic acid & flux tuning) | Engineering efforts for new products (e.g., glycolic acid) have used EMCP manipulation including ecm overexpression to alter fluxes; modelling-guided and expression strategies exploit Ecm as a control point. (good2015metaboliccontrolof pages 46-53, sonntag2015highlevelproductionof pages 94-95) | Engineered M. extorquens strains for chemical production | Good N. (2015); Dietz K. et al. (2024 reported engineering leveraging EMCP) | (reported study, 2015); Microbial Cell Factories (2024) | N/A; https://doi.org/10.1186/s12934-024-02583-y | 2015; Dec 2024 |
| B12 (cobalamin) dependence & B12-production potential | Ecm is AdoCbl-dependent (adenosylcobalamin required for mutase activity); methylotrophs that rely on cobalamin-dependent mutases are candidates for B12 production or require B12 supply for EMCP activity. (anthony2011howhalfa pages 13-17, good2015metaboliccontrolof pages 13-20, good2015metaboliccontrolof pages 46-53) | EMCP-containing methylotrophs; biotechnological strain selection | Anthony C. (2011); Erb TJ et al. (2007); Good N. (2015) | Science Progress; PNAS; (reported study, 2015) | https://doi.org/10.3184/003685011x13044430633960; https://doi.org/10.1073/pnas.0702791104 | Jun 2011; Jun 2007; 2015 |
Table: Concise, citable table of key facts about Ethylmalonyl‑CoA mutase (ecm/meaA) in Methylorubrum extorquens AM1, summarizing function, pathway role, regulation, genetic/phenotypic data, and applications with sources for follow-up.
References with URLs and publication dates (where available)
- Anthony, C. 2011. How Half a Century of Research was Required to Understand Bacterial Growth on C1 and C2 Compounds; the Story of the Serine Cycle and the Ethylmalonyl‑CoA Pathway. Science Progress. https://doi.org/10.3184/003685011x13044430633960 (Jun 2011) (anthony2011howhalfa pages 13-17).
- Erb, T. J., et al. 2007. Synthesis of C5‑dicarboxylic acids from C2‑units involving crotonyl‑CoA carboxylase/reductase: The ethylmalonyl‑CoA pathway. PNAS. https://doi.org/10.1073/pnas.0702791104 (Jun 2007) (good2015metaboliccontrolof pages 13-20).
- Korotkova, N., et al. 2002. Glyoxylate Regeneration Pathway in the Methylotroph Methylobacterium extorquens AM1. Journal of Bacteriology. https://doi.org/10.1128/jb.184.6.1750-1758.2002 (Mar 2002) (korotkova2002glyoxylateregenerationpathway pages 6-8).
- Good, N. 2015. Metabolic control of the ethylmalonyl‑CoA pathway in Methylobacterium extorquens AM1. (Study excerpts reporting ecm overexpression, growth lag, minimal activity, and metabolomics) (2015) (good2015metaboliccontrolof pages 41-46, good2015metaboliccontrolof pages 74-83, good2015metaboliccontrolof pages 96-103).
- Sonntag, F., et al. 2015. High‑level production of ethylmalonyl‑CoA pathway‑derived dicarboxylic acids by Methylobacterium extorquens under cobalt‑deficient conditions and by polyhydroxybutyrate negative strains. Applied Microbiology and Biotechnology. https://doi.org/10.1007/s00253-015-6418-3 (Feb 2015) (sonntag2015highlevelproductionof pages 94-95, sonntag2015highlevelproductionof pages 204-209).
- Kremer, K., et al. 2023. Functional Degeneracy in Paracoccus denitrificans Pd1222 is Coordinated via RamB, Which Links Expression of the Glyoxylate Cycle to Activity of the Ethylmalonyl‑CoA Pathway. Applied and Environmental Microbiology. https://doi.org/10.1128/aem.00238-23 (Jun 2023) (kremer2023functionaldegeneracyin pages 1-2).
References
(anthony2011howhalfa pages 13-17): Christopher Anthony. How half a century of research was required to understand bacterial growth on c1 and c2 compounds; the story of the serine cycle and the ethylmalonyl-coa pathway. Science Progress, 94:109-137, Jun 2011. URL: https://doi.org/10.3184/003685011x13044430633960, doi:10.3184/003685011x13044430633960. This article has 132 citations and is from a poor quality or predatory journal.
(korotkova2002glyoxylateregenerationpathway pages 6-8): Natalia Korotkova, Ludmila Chistoserdova, Vladimir Kuksa, and Mary E. Lidstrom. Glyoxylate regeneration pathway in the methylotroph methylobacterium extorquens am1. Journal of Bacteriology, 184:1750-1758, Mar 2002. URL: https://doi.org/10.1128/jb.184.6.1750-1758.2002, doi:10.1128/jb.184.6.1750-1758.2002. This article has 138 citations and is from a peer-reviewed journal.
(good2015metaboliccontrolof pages 13-20): N Good. Metabolic control of the ethylmalonyl-coa pathway in methylobacterium extorquens am1. Unknown journal, 2015.
(good2015metaboliccontrolof pages 46-53): N Good. Metabolic control of the ethylmalonyl-coa pathway in methylobacterium extorquens am1. Unknown journal, 2015.
(good2015metaboliccontrolof pages 41-46): N Good. Metabolic control of the ethylmalonyl-coa pathway in methylobacterium extorquens am1. Unknown journal, 2015.
(good2015metaboliccontrolof pages 74-83): N Good. Metabolic control of the ethylmalonyl-coa pathway in methylobacterium extorquens am1. Unknown journal, 2015.
(good2015metaboliccontrolof pages 96-103): N Good. Metabolic control of the ethylmalonyl-coa pathway in methylobacterium extorquens am1. Unknown journal, 2015.
(sonntag2015highlevelproductionof pages 204-209): Frank Sonntag, Jonas E. N. Müller, Patrick Kiefer, Julia A. Vorholt, Jens Schrader, and Markus Buchhaupt. High-level production of ethylmalonyl-coa pathway-derived dicarboxylic acids by methylobacterium extorquens under cobalt-deficient conditions and by polyhydroxybutyrate negative strains. Applied Microbiology and Biotechnology, 99:3407-3419, Feb 2015. URL: https://doi.org/10.1007/s00253-015-6418-3, doi:10.1007/s00253-015-6418-3. This article has 58 citations and is from a domain leading peer-reviewed journal.
(sonntag2015highlevelproductionof pages 94-95): Frank Sonntag, Jonas E. N. Müller, Patrick Kiefer, Julia A. Vorholt, Jens Schrader, and Markus Buchhaupt. High-level production of ethylmalonyl-coa pathway-derived dicarboxylic acids by methylobacterium extorquens under cobalt-deficient conditions and by polyhydroxybutyrate negative strains. Applied Microbiology and Biotechnology, 99:3407-3419, Feb 2015. URL: https://doi.org/10.1007/s00253-015-6418-3, doi:10.1007/s00253-015-6418-3. This article has 58 citations and is from a domain leading peer-reviewed journal.
(kremer2023functionaldegeneracyin pages 1-2): Katharina Kremer, Doreen Meier, Lisa Theis, Stephanie Miller, Aerin Rost-Nasshan, Yadanar T. Naing, Jan Zarzycki, Nicole Paczia, Javier Serrania, Patrick Blumenkamp, Alexander Goesmann, Anke Becker, Martin Thanbichler, Georg K. A. Hochberg, Michael S. Carter, and Tobias J. Erb. Functional degeneracy in paracoccus denitrificans pd1222 is coordinated via ramb, which links expression of the glyoxylate cycle to activity of the ethylmalonyl-coa pathway. Applied and Environmental Microbiology, Jul 2023. URL: https://doi.org/10.1128/aem.00238-23, doi:10.1128/aem.00238-23. This article has 6 citations and is from a peer-reviewed journal.
id: Q49115
gene_symbol: ecm
product_type: PROTEIN
status: DRAFT
taxon:
id: NCBITaxon:272630
label: Methylorubrum extorquens (strain ATCC 14718 / DSM 1338 / JCM 2805 / NCIMB
9133 / AM1)
description: >-
Ethylmalonyl-CoA mutase (Ecm) is a radical enzyme that catalyzes the interconversion
of (2R)-ethylmalonyl-CoA to (2S)-methylsuccinyl-CoA using adenosylcobalamin (vitamin B12)
as a cofactor. This enzyme is a key component of the ethylmalonyl-CoA pathway for
acetyl-CoA assimilation, which is essential for Methylorubrum extorquens growth on
one-carbon (methanol, methylamine) and two-carbon (ethanol, ethylamine) compounds as
sole carbon sources. Unlike organisms that use the glyoxylate cycle for acetyl-CoA
assimilation, M. extorquens lacks isocitrate lyase and instead employs this alternative
pathway. Ecm acts as a metabolic control point, allowing efficient restoration of
metabolic balance when challenged with sudden changes in growth substrate. The enzyme
is assigned EC 5.4.99.63 based on characterization by Good et al. (2015). Disruption
of ecm abolishes growth on C1 and C2 compounds but can be rescued by glyoxylate or
glycolate addition, confirming its role in converting acetyl-CoA to glyoxylate.
existing_annotations:
- term:
id: GO:0003824
label: catalytic activity
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
This annotation is technically correct but overly broad. Ecm has a well-defined
catalytic activity as an ethylmalonyl-CoA mutase (EC 5.4.99.63), which is an
intramolecular transferase that interconverts (2R)-ethylmalonyl-CoA to
(2S)-methylsuccinyl-CoA. A more specific term should be used.
action: MODIFY
reason: >-
The term "catalytic activity" is too general for a well-characterized enzyme.
While there is no specific GO term for "ethylmalonyl-CoA mutase activity", the
most appropriate existing term is GO:0016866 (intramolecular transferase activity),
which accurately describes the mechanistic class of this enzyme. This is preferable
to the overly broad "catalytic activity" and avoids the incorrect implication of
"methylmalonyl-CoA mutase activity" which involves different substrates.
proposed_replacement_terms:
- id: GO:0016866
label: intramolecular transferase activity
- term:
id: GO:0004494
label: methylmalonyl-CoA mutase activity
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
This annotation is INCORRECT. GO:0004494 (methylmalonyl-CoA mutase activity) is
defined as catalyzing "(R)-methylmalonyl-CoA = succinyl-CoA". However, Ecm
catalyzes a distinct reaction: "(2R)-ethylmalonyl-CoA = (2S)-methylsuccinyl-CoA"
(EC 5.4.99.63). These are chemically different reactions with different substrates
and products. The annotation appears to have been transferred based on sequence
similarity to the methylmalonyl-CoA mutase family, but the substrate specificity
of Ecm is different.
action: MODIFY
reason: >-
Ecm is an ethylmalonyl-CoA mutase, not a methylmalonyl-CoA mutase. The substrates
are different: ethylmalonyl-CoA vs methylmalonyl-CoA. UniProt clearly assigns
EC 5.4.99.63 (ethylmalonyl-CoA mutase) to this enzyme based on PMID:25448820.
There is no specific GO term for ethylmalonyl-CoA mutase activity, so the best
option is to use the parent term GO:0016866 (intramolecular transferase activity)
which is mechanistically accurate. A new GO term for ethylmalonyl-CoA mutase
activity should be proposed.
proposed_replacement_terms:
- id: GO:0016866
label: intramolecular transferase activity
- term:
id: GO:0016853
label: isomerase activity
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
This annotation is correct. Ethylmalonyl-CoA mutase is an isomerase that catalyzes
an intramolecular rearrangement, converting (2R)-ethylmalonyl-CoA to
(2S)-methylsuccinyl-CoA. The "isomerase" classification aligns with the enzyme's
EC number (5.4.99.63) where class 5 corresponds to isomerases.
action: ACCEPT
reason: >-
The isomerase activity annotation correctly captures the enzyme class. Mutases are
a subclass of isomerases that catalyze the intramolecular transfer of groups. The
EC classification 5.4.99.63 places this enzyme under isomerases (class 5),
intramolecular transferases (subclass 4), specifically transferring other groups
(sub-subclass 99). This IEA annotation appropriately reflects the broad enzymatic
class.
- term:
id: GO:0016866
label: intramolecular transferase activity
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
This annotation is correct and represents the most appropriate molecular function
term for Ecm given current GO term availability. Ethylmalonyl-CoA mutase catalyzes
an intramolecular rearrangement involving transfer of a group from one position
to another within the same molecule, which is the defining feature of intramolecular
transferases (EC 5.4).
action: ACCEPT
reason: >-
The intramolecular transferase activity annotation accurately describes the
mechanistic class of this enzyme. Given that there is no specific GO term for
"ethylmalonyl-CoA mutase activity", GO:0016866 is the most informative and
accurate term available. The enzyme belongs to EC 5.4.99.63, where 5.4 represents
intramolecular transferases.
- term:
id: GO:0031419
label: cobalamin binding
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
This annotation is correct. Ecm requires adenosylcobalamin (coenzyme B12) as a
cofactor. UniProt entry Q49115 documents the cofactor requirement with evidence
from PMID:25448820. The protein contains a B12-binding domain (residues 530-659)
with the characteristic axial His543 residue that coordinates the cobalt in the
cobalamin cofactor.
action: ACCEPT
reason: >-
The cobalamin binding annotation is well-supported by both computational domain
analysis (B12-binding domain, PROSITE PS51332) and experimental evidence
(PMID:25448820 demonstrated adenosylcobalamin cofactor requirement). The binding
site annotation at His543 indicates the axial binding residue for the Co atom
of the cobalamin cofactor.
- term:
id: GO:0046872
label: metal ion binding
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
This annotation is correct but could be more specific. Ecm binds cobalt indirectly
through the adenosylcobalamin (vitamin B12) cofactor. The cobalt ion is
coordinated by His543 as the axial ligand.
action: ACCEPT
reason: >-
While this annotation is somewhat general, it is accurate. The metal ion binding
occurs through coordination of the cobalt center of the cobalamin cofactor.
UniProt indicates the binding site at position 543 coordinating the Co ligand
part of adenosylcob(III)alamin. Given that GO:0031419 (cobalamin binding) is
also annotated, which is more specific, this general term provides complementary
but redundant information. However, it is not incorrect and can be retained.
# Proposed new annotation for the biological process
- term:
id: GO:0019681
label: acetyl-CoA assimilation pathway
evidence_type: IDA
original_reference_id: PMID:25448820
review:
summary: >-
Ecm is a key enzyme in the ethylmalonyl-CoA pathway, which serves as the acetyl-CoA
assimilation pathway in M. extorquens. This organism lacks isocitrate lyase and
uses this alternative pathway to convert acetyl-CoA to glyoxylate. Knockout studies
show that ecm mutants cannot grow on C1 or C2 compounds, and growth can be rescued
by glyoxylate addition, confirming Ecm's essential role in this pathway.
action: NEW
reason: >-
GO:0019681 (acetyl-CoA assimilation pathway) is directly applicable to Ecm's
function. UniProt states: "Is involved in the ethylmalonyl-CoA pathway for
acetyl-CoA assimilation required for M.extorquens growth on one- and two-carbon
compounds." Disruption phenotype studies (PMID:8704985) show loss of ability to
convert acetyl-CoA into glyoxylate, which is rescued by glyoxylate addition.
This provides strong evidence for involvement in the acetyl-CoA assimilation
pathway.
references:
- id: GO_REF:0000002
title: Gene Ontology annotation through association of InterPro records with GO terms
findings: []
- id: GO_REF:0000120
title: Combined Automated Annotation using Multiple IEA Methods
findings: []
- id: PMID:25448820
title: Ethylmalonyl coenzyme A mutase operates as a metabolic control point in Methylobacterium extorquens AM1
findings:
- statement: Ecm catalyzes the transformation of (2R)-ethylmalonyl-CoA to (2S)-methylsuccinyl-CoA
reference_section_type: ABSTRACT
- statement: Ecm requires adenosylcobalamin as a cofactor
reference_section_type: RESULTS
- statement: Ecm acts as a regulatory metabolic control point in the ethylmalonyl-CoA pathway
reference_section_type: DISCUSSION
full_text_unavailable: true
- id: PMID:8704985
title: Molecular characterization of a chromosomal region involved in the oxidation of acetyl-CoA to glyoxylate
findings:
- statement: ecm mutants lose the ability to grow on C1 and C2 compounds
reference_section_type: RESULTS
- statement: Growth is restored by addition of glyoxylate or glycolate
reference_section_type: RESULTS
full_text_unavailable: true
- id: PMID:8868443
title: A protein having similarity with methylmalonyl-CoA mutase is required for the assimilation of methanol and ethanol
findings:
- statement: Ecm is required for methanol and ethanol assimilation in M. extorquens AM1
reference_section_type: ABSTRACT
full_text_unavailable: true
core_functions:
- description: >-
Ethylmalonyl-CoA mutase activity in the acetyl-CoA assimilation pathway. Ecm
catalyzes the interconversion of (2R)-ethylmalonyl-CoA to (2S)-methylsuccinyl-CoA
using adenosylcobalamin as a cofactor. This is a key step in the ethylmalonyl-CoA
pathway that allows M. extorquens to assimilate acetyl-CoA into central metabolism
when growing on C1 and C2 compounds. The enzyme acts as a metabolic control point,
enabling efficient adaptation to changes in carbon source.
molecular_function:
id: GO:0016866
label: intramolecular transferase activity
directly_involved_in:
- id: GO:0019681
label: acetyl-CoA assimilation pathway
substrates:
- id: CHEBI:84866
label: (2R)-ethylmalonyl-CoA
- id: CHEBI:85316
label: (2S)-methylsuccinyl-CoA
proposed_new_terms:
- proposed_name: ethylmalonyl-CoA mutase activity
proposed_definition: >-
Catalysis of the reaction: (2R)-ethylmalonyl-CoA = (2S)-methylsuccinyl-CoA.
This reaction requires adenosylcobalamin as a cofactor.
justification: >-
Ethylmalonyl-CoA mutase (EC 5.4.99.63) is a distinct enzyme from methylmalonyl-CoA
mutase (EC 5.4.99.2). The substrates are different: ethylmalonyl-CoA vs
methylmalonyl-CoA. Currently, ecm is incorrectly annotated with GO:0004494
(methylmalonyl-CoA mutase activity) due to sequence similarity, but this
annotation is biochemically incorrect. A specific term for ethylmalonyl-CoA
mutase activity would enable accurate annotation of this enzyme and others
in the ethylmalonyl-CoA pathway.
proposed_parent:
id: GO:0016866
label: intramolecular transferase activity
proposed_mappings:
- predicate: skos:exactMatch
target_term:
id: EC:5.4.99.63
label: ethylmalonyl-CoA mutase
ontology: ec
suggested_questions:
- question: >-
Is there any evidence that Ecm can also use methylmalonyl-CoA as a substrate,
even if with lower efficiency, or is it strictly specific for ethylmalonyl-CoA?
experts: []
- question: >-
Are there other organisms with ethylmalonyl-CoA pathway that also lack a specific
GO term for this activity?
experts: []
suggested_experiments:
- hypothesis: Ecm has strict substrate specificity for ethylmalonyl-CoA over methylmalonyl-CoA
description: >-
Purify recombinant Ecm and measure kinetic parameters (Km, kcat) for both
(2R)-ethylmalonyl-CoA and (R)-methylmalonyl-CoA substrates. Compare catalytic
efficiency to determine substrate specificity and whether there is any activity
toward methylmalonyl-CoA.
experiment_type: enzyme kinetics