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. |
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Download this section (compressed HTML)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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