Mitochondrial, magnesium-dependent homotrimeric enzyme of the HpcH/HpaI aldolase family (citrate lyase beta subunit-like subfamily) that clears reactive acyl-CoA thioesters from the mitochondrial matrix. It hydrolyses (S)-malyl-CoA to malate and free CoA, and cleaves (3S)-citramalyl-CoA to pyruvate and acetyl-CoA. Both substrates are inhibitors of the vitamin B12-dependent enzyme methylmalonyl-CoA mutase, so removing them protects the mutase and its adenosylcobalamin cofactor; CLYBL is accordingly classed as a metabolite-repair enzyme rather than a biosynthetic one. Citramalyl-CoA arises from itaconate, an antimicrobial and immunomodulatory metabolite made by activated macrophages, whereas malyl-CoA is a continuously generated side product of promiscuous citric acid cycle enzymes. Which of the two substrates dominates physiologically is debated: the itaconate route is inducible and largely confined to activated myeloid cells, while CLYBL itself is expressed in the mitochondria of all mammalian tissues, and cells lacking CLYBL accumulate malyl-CoA without accumulating itaconyl-CoA yet still lose adenosylcobalamin. The purified enzyme also catalyses the Claisen-condensation reverse reactions, malate synthase and beta-methylmalate synthase, but with a specificity constant more than a thousandfold below the lyase reaction and a millimolar KM for glyoxylate; humans have no glyoxylate shunt for such a reaction to serve. A common premature-stop polymorphism (rs41281112, p.Arg259*) abolishes the protein in a few percent of people, who are otherwise healthy but have reduced circulating vitamin B12.
Definition: Catalysis of the reaction (S)-malyl-CoA + H2O = (S)-malate + CoA + H(+).
Justification: The reaction is assigned EC 3.1.2.30 and RHEA:38291, is directly measured for human CLYBL (KM 11 uM, kcat 9.4 per second), and is now proposed to be the enzyme's physiological function. GO currently has no term for it, so the annotation has to fall back on the generic GO:0016289 acyl-CoA hydrolase activity, which is uninformative about the substrate and gives no handle on the metabolite-repair role.
Parent term: acyl-CoA hydrolase activity
Mappings:
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0047777 (S)-citramalyl-CoA lyase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic assertion of the citramalyl-CoA lyase activity, matching the direct human enzymology. Reason: Correct and specific. The family node is seeded by mouse Clybl and by human CLYBL's own experimental annotation; the target appearing in its own WITH/FROM is the expected marker that the node has experimental grounding on this gene. Supporting Evidence: PMID:29056341 we report that CLYBL operates as a citramalyl-CoA lyase in mammalian cells |
| GO:0106064 regulation of cobalamin catabolic process | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic assertion that CLYBL modulates the rate at which cobalamin is destroyed. Reason: The parent term is directionally neutral and is therefore not wrong. It is however less informative than it could be - CLYBL removes the acyl-CoA species that inactivate the B12 cofactor, so it decreases cobalamin catabolism, and GO:0106122 negative regulation of cobalamin catabolic process would say so. Left as ACCEPT rather than MODIFY because the assertion was placed at a PAINT node whose tree has not been inspected here; the directional issue is raised instead on the GO:0106121 annotations, which do state a direction. |
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: Root-level catalytic activity assigned from the InterPro HpcH/HpaI aldolase signature. Reason: Correct but carries no information; the specific reactions CLYBL catalyses are established by direct enzymology and are already annotated. Proposed replacements: acyl-CoA hydrolase activity (S)-citramalyl-CoA lyase activity |
| GO:0004474 malate synthase activity | IEA GO_REF:0000120 | MODIFY | Summary: Malate synthase (EC 2.3.3.9, RHEA:18181) assigned electronically from the EC/Rhea mapping that UniProt carries for CLYBL. Reason: The activity is genuinely measurable in vitro, but it is a promiscuous reverse reaction of the lyase/thioesterase active site rather than the enzyme's function. Shen et al. measured a specificity constant more than 1000-fold lower than for the citramalyl-CoA lyase reaction, and Strittmatter et al. report kcat 0.12 per second with a KM for glyoxylate of 3.6 mM - a millimolar operating point in the mitochondrial matrix. Humans also lack a glyoxylate shunt, so there is no pathway for a malate synthase to serve. The directly measured physiological reactions are the (S)-malyl-CoA thioesterase and the (3S)-citramalyl-CoA lyase. Proposed replacements: acyl-CoA hydrolase activity (S)-citramalyl-CoA lyase activity Supporting Evidence: PMID:29056341 The specificity constant (kcat/KM) for the citramalyl-CoA lyase activity is >1000 fold higher than the forward malate/methylmalate/citramalate synthase activity |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | ACCEPT | Summary: Mitochondrial localisation from the UniProt subcellular location vocabulary. Reason: Correct; CLYBL carries a cleaved mitochondrial targeting sequence and is detected in the mitochondria of all mammalian organs examined. |
| GO:0016289 acyl-CoA hydrolase activity | IEA GO_REF:0000116 | ACCEPT | Summary: Rhea-derived assignment corresponding to RHEA:38291, (S)-malyl-CoA + H2O = (S)-malate + CoA + H(+) (EC 3.1.2.30). Reason: This is the reaction now proposed to be CLYBL's physiological function. The generic term understates it - a specific (S)-malyl-CoA hydrolase term does not exist and is proposed in proposed_new_terms - but the assignment itself is right. |
| GO:0047777 (S)-citramalyl-CoA lyase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment of EC 4.1.3.25 / RHEA:22612, consistent with the direct enzymology. Reason: Correct and specific. |
| GO:0106064 regulation of cobalamin catabolic process | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA-generated restatement of the cobalamin link. Reason: Directionally neutral parent term; correct but under-specific, as for the IBA. |
| GO:0106121 positive regulation of cobalamin catabolic process | IEA GO_REF:0000107 | MODIFY | Summary: Orthology transfer from mouse Clybl of a directional regulation term whose sign appears to be inverted. Reason: CLYBL clears itaconyl-CoA/citramalyl-CoA and malyl-CoA, the species that inactivate the adenosylcobalamin cofactor of methylmalonyl-CoA mutase. It is loss of CLYBL that increases cobalamin destruction - people homozygous for the rs41281112 null allele have reduced circulating B12, and CLYBL-null cells lose adenosylcobalamin. The gene product therefore decreases, not increases, cobalamin catabolism. GO:0106122 negative regulation of cobalamin catabolic process exists and currently carries no annotations at all in QuickGO, while GO:0106121 is annotated essentially only to CLYBL and its orthologues, which is consistent with the term pair having been created for this gene and the wrong sign having been selected. Flagged for a GO curator rather than treated as settled. Proposed replacements: negative regulation of cobalamin catabolic process |
| GO:0106121 positive regulation of cobalamin catabolic process | IDA PMID:29056341 The Human Knockout Gene CLYBL Connects Itaconate to Vitamin ... | MODIFY | Summary: Direct experimental basis for the cobalamin link, but annotated with what appears to be the wrong direction. Reason: The underlying experiment is sound and is not disputed here - Shen et al. showed that CLYBL loss causes a cell-autonomous defect in mitochondrial B12 metabolism and that itaconyl-CoA inactivates the B12 cofactor of methylmalonyl-CoA mutase. What is questioned is the direction of the derived regulation term - CLYBL protects the cofactor, so it negatively regulates cobalamin catabolism. Same reasoning as the orthology-transferred copy of this term. Proposed replacements: negative regulation of cobalamin catabolic process Supporting Evidence: PMID:29056341 We report that CLYBL loss leads to a cell-autonomous defect in the mitochondrial B12 metabolism and that itaconyl-CoA is a cofactor-inactivating, substrate-analog inhibitor of the mitochondrial B12-dependent methylmalonyl-CoA mutase (MUT). PMID:29056341 Our work reveals an unanticipated consequence of exposure to itaconate: B12 inactivation |
| GO:0005739 mitochondrion | IDA PMID:24334609 CLYBL is a polymorphic human enzyme with malate synthase and... | ACCEPT | Summary: Direct demonstration that the protein resides and acts in mitochondria. Reason: Core location; all of CLYBL's substrates and its target enzyme MMUT are matrix species. Supporting Evidence: PMID:24334609 The protein is expressed in the mitochondria of all mammalian organs, with highest expression in brown fat and kidney. |
| GO:0016289 acyl-CoA hydrolase activity | IDA PMID:40108300 CLYBL averts vitamin B(12) depletion by repairing malyl-CoA. | ACCEPT | Summary: Direct measurement of (S)-malyl-CoA thioesterase activity, the reaction Griffith et al. assign as CLYBL's physiological function. Reason: Core molecular function. The activity was already noticed qualitatively by Shen et al. in 2017 and has now been quantified (KM 11 uM, kcat 9.4 per second), placing it on a par with the citramalyl-CoA lyase reaction and roughly two orders of magnitude above the malate synthase reaction. Unlike itaconate, malyl-CoA is generated continuously by promiscuous citric acid cycle enzymes in every cell, which fits CLYBL's ubiquitous expression and the fact that the human null allele lowers B12 outside any inflammatory context. The generic acyl-CoA hydrolase term is the most specific one available; a dedicated (S)-malyl-CoA hydrolase term is proposed. Supporting Evidence: PMID:40108300 Here we discover that CLYBL additionally functions as a metabolite repair enzyme for malyl-CoA, a side product of promiscuous citric acid cycle enzymes. PMID:29056341 We also found that CLYBL has a thioesterase activity and hydrolyzes malyl-CoA to malate and free CoASH, but we did not quantify this activity in detail. |
| GO:0110052 toxic metabolite repair | IDA PMID:29056341 The Human Knockout Gene CLYBL Connects Itaconate to Vitamin ... | ACCEPT | Summary: Clearance of citramalyl-CoA/itaconyl-CoA, which otherwise poison the B12 cofactor of methylmalonyl-CoA mutase. Reason: Core biological process, and the framing that reconciles both substrate models - whether the offending species is derived from itaconate or from a citric acid cycle side reaction, CLYBL's job is to destroy it before it damages MMUT. Supporting Evidence: PMID:29056341 Cells lacking CLYBL accumulate citramalyl-CoA, an intermediate in the C5-dicarboxylate metabolic pathway that includes itaconate |
| GO:0110052 toxic metabolite repair | IDA PMID:40108300 CLYBL averts vitamin B(12) depletion by repairing malyl-CoA. | ACCEPT | Summary: Clearance of malyl-CoA, a continuously generated side product of promiscuous citric acid cycle enzymes and a potent methylmalonyl-CoA mutase inhibitor. Reason: Core biological process. This annotation is the one that best matches CLYBL's housekeeping expression pattern and the human loss-of-function phenotype. Supporting Evidence: PMID:40108300 We found that CLYBL knockout cells, accumulating malyl-CoA but not itaconyl-CoA, show decreased levels of adenosylcobalamin and that malyl-CoA is a more potent inhibitor of methylmalonyl-CoA mutase than itaconyl-CoA. |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | ACCEPT | Summary: High-throughput mitochondrial proteomics places CLYBL in the mitochondrion. Reason: Consistent with all focused studies. |
| GO:0000287 magnesium ion binding | IDA PMID:29056341 The Human Knockout Gene CLYBL Connects Itaconate to Vitamin ... | ACCEPT | Summary: One Mg(2+) ion per subunit, resolved in the crystal structure and required for catalysis. Reason: Genuine catalytic cofactor binding, part of the core activity rather than an incidental metal contact. |
| GO:0004474 malate synthase activity | IDA PMID:29056341 The Human Knockout Gene CLYBL Connects Itaconate to Vitamin ... | MODIFY | Summary: Malate synthase activity re-measured with purified protein, and found to be >1000-fold less efficient than the citramalyl-CoA lyase reaction of the same enzyme. Reason: The activity is genuinely measurable in vitro, but it is a promiscuous reverse reaction of the lyase/thioesterase active site rather than the enzyme's function. Shen et al. measured a specificity constant more than 1000-fold lower than for the citramalyl-CoA lyase reaction, and Strittmatter et al. report kcat 0.12 per second with a KM for glyoxylate of 3.6 mM - a millimolar operating point in the mitochondrial matrix. Humans also lack a glyoxylate shunt, so there is no pathway for a malate synthase to serve. The directly measured physiological reactions are the (S)-malyl-CoA thioesterase and the (3S)-citramalyl-CoA lyase. Proposed replacements: acyl-CoA hydrolase activity (S)-citramalyl-CoA lyase activity Supporting Evidence: PMID:29056341 The specificity constant (kcat/KM) for the citramalyl-CoA lyase activity is >1000 fold higher than the forward malate/methylmalate/citramalate synthase activity |
| GO:0047777 (S)-citramalyl-CoA lyase activity | IDA PMID:29056341 The Human Knockout Gene CLYBL Connects Itaconate to Vitamin ... | ACCEPT | Summary: Direct demonstration, by enzymology plus activity-based metabolomics in CLYBL-null cells, that CLYBL cleaves (3S)-citramalyl-CoA to pyruvate and acetyl-CoA. Reason: Core molecular function. This remains correct after the 2025 reassignment - Griffith et al. add malyl-CoA repair rather than removing the lyase activity, and both reactions converge on protecting the B12 cofactor of methylmalonyl-CoA mutase. What has changed is which substrate is thought to dominate in a typical tissue. Supporting Evidence: PMID:29056341 we report that CLYBL operates as a citramalyl-CoA lyase in mammalian cells |
| GO:0070207 protein homotrimerization | IDA PMID:29056341 The Human Knockout Gene CLYBL Connects Itaconate to Vitamin ... | KEEP AS NON CORE | Summary: The crystal structure shows CLYBL to be a homotrimer. Reason: A real structural property, but an assembly state rather than a biological process the enzyme performs. |
| GO:0000287 magnesium ion binding | IDA PMID:24334609 CLYBL is a polymorphic human enzyme with malate synthase and... | ACCEPT | Summary: Mg(2+) dependence confirmed by enzyme kinetics on the recombinant protein. Reason: Genuine catalytic cofactor binding, part of the core activity rather than an incidental metal contact. |
| GO:0004474 malate synthase activity | IDA PMID:24334609 CLYBL is a polymorphic human enzyme with malate synthase and... | MODIFY | Summary: The original de-orphaning assignment of CLYBL as a malate/beta-methylmalate synthase. Reason: The activity is genuinely measurable in vitro, but it is a promiscuous reverse reaction of the lyase/thioesterase active site rather than the enzyme's function. Shen et al. measured a specificity constant more than 1000-fold lower than for the citramalyl-CoA lyase reaction, and Strittmatter et al. report kcat 0.12 per second with a KM for glyoxylate of 3.6 mM - a millimolar operating point in the mitochondrial matrix. Humans also lack a glyoxylate shunt, so there is no pathway for a malate synthase to serve. The directly measured physiological reactions are the (S)-malyl-CoA thioesterase and the (3S)-citramalyl-CoA lyase. The measurement itself is not disputed and the paper's own framing is appropriately cautious about physiological relevance. Proposed replacements: acyl-CoA hydrolase activity (S)-citramalyl-CoA lyase activity Supporting Evidence: PMID:24334609 We report that CLYBL encodes a malate/Ξ²-methylmalate synthase, converting glyoxylate and acetyl-CoA to malate, or glyoxylate and propionyl-CoA to Ξ²-methylmalate. |
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Download this section (compressed HTML)Q: Is the direction of GO:0106121 positive regulation of cobalamin catabolic process correct for CLYBL? CLYBL destroys the acyl-CoA species that inactivate adenosylcobalamin, and its loss is what lowers circulating B12, which argues for GO:0106122 negative regulation of cobalamin catabolic process. The negative-regulation term currently has no annotations at all, while the positive one is annotated essentially only to CLYBL and its orthologues.
Suggested experts: Vamsi K Mootha, Carole L Linster
Q: Which substrate dominates CLYBL flux in a resting, non-inflamed tissue - malyl-CoA from promiscuous citric acid cycle chemistry, or citramalyl-CoA from itaconate? The two models predict different tissue and stimulus dependence for the B12 phenotype.
Suggested experts: Carole L Linster, Vamsi K Mootha
Q: What enzyme actually generates mitochondrial (S)-malyl-CoA in vivo, and at what flux? The repair model requires a continuous source, but the side reaction responsible has not been pinned to a specific citric acid cycle enzyme.
Suggested experts: Carole L Linster
Q: Should EC 2.3.3.9 (malate synthase) and EC 2.3.3.- (beta-methylmalate synthase) be retained on UniProt Q8N0X4 as function-level claims, given a specificity constant more than 1000-fold below the lyase reaction, a millimolar KM for glyoxylate, and the absence of a glyoxylate shunt in humans?
Experiment: Measure malyl-CoA, itaconyl-CoA, citramalyl-CoA, adenosylcobalamin and methylmalonic acid in primary cells (e.g. fibroblasts and unstimulated monocytes) from rs41281112 homozygotes and matched controls, then repeat after LPS/IFN-gamma stimulation to induce ACOD1 and itaconate. If the itaconate model is dominant the B12/MMA defect should be largely stimulation-dependent; if the malyl-CoA model is dominant it should be present at baseline.
Hypothesis: Malyl-CoA, not itaconyl/citramalyl-CoA, is the species whose accumulation causes the B12 phenotype of CLYBL loss-of-function humans.
Type: Targeted acyl-CoA metabolomics in human primary cells
Experiment: Use the CLYBL crystal structure to design active-site variants that retain (S)-malyl-CoA thioesterase and (3S)-citramalyl-CoA lyase activity but lose the glyoxylate-condensation reactions, then test whether such a variant rescues adenosylcobalamin levels in CLYBL-null cells. Rescue by a synthase-dead protein would show the condensation chemistry is dispensable.
Hypothesis: The malate synthase activity of CLYBL is a promiscuous side reaction that can be separated from the physiological thioesterase/lyase activity.
Type: Structure-guided separation-of-function mutagenesis with cellular rescue
Experiment: Quantify the turnover of labelled cobalamin (and of the adenosylcobalamin bound to MMUT specifically) in isogenic CLYBL wild-type and knockout cells, with and without itaconate or malate loading. A higher cobalamin degradation rate in the knockout would settle the direction of the GO regulation term empirically.
Hypothesis: CLYBL negatively regulates cobalamin catabolism.
Type: Cofactor turnover measurement with stable-isotope-labelled cobalamin
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