The Human Knockout Gene CLYBL Connects Itaconate to Vitamin B12

Abstract

Summary CLYBL encodes a ubiquitously expressed mitochondrial enzyme, conserved across all vertebrates, whose cellular activity and pathway assignment are unknown. Its homozygous loss is tolerated in seemingly healthy individuals, with reduced circulating B12 levels being the only and consistent phenotype reported to date. Here, by combining enzymology, structural biology and activity-based metabolomics we report that CLYBL operates as a citramalyl-CoA lyase in mammalian cells. Cells lacking CLYBL accumulate citramalyl-CoA, an intermediate in the C5-dicarboxylate metabolic pathway that includes itaconate, a recently identified human antimicrobial metabolite and immunomodulator. 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-analogue inhibitor of the mitochondrial B12-dependent methylmalonyl-CoA mutase (MUT). Our work de-orphans the function of human CLYBL and reveals that a consequence of exposure to the immunomodulatory metabolite itaconate is B12 inactivation.

Itaconate: an emerging determinant of inflammation in activated macrophages

“…Itaconyl‐CoA is an intermediate of itaconate catabolism. It has been reported that itaconate inactivates vitamin B12 in the absence of CLYBL . Mechanistically, knockout of CLYBL promotes the accumulation of itaconyl‐CoA, which directly causes vitamin B12 degradation.…”

“…It has been reported that itaconate inactivates vitamin B12 in the absence of CLYBL. 18 Mechanistically, knockout of CLYBL promotes the accumulation of itaconyl-CoA, which directly causes vitamin B12 degradation. Severe deficiency of vitamin B12 is a common cause of anemia and neurological impairment.…”

“…[15][16][17] More recently, Shen et al showed that itaconate is converted to itaconyl-CoA and then to citramalyl-CoA, which is further catabolized by CLYBL to generate pyruvate and acetyl-CoA, fueling the TCA cycle in LPS-treated RAW 264.7 cells. 18 This provides a route of itaconate catabolism during macrophage activation ( Figure 1a). How itaconate is converted to itaconyl-CoA and whether other catabolic approaches exist, however, are currently unclear.…”

Discovery of a novel stereospecific β-hydroxyacyl-CoA lyase/thioesterase shared by three metabolic pathways inMycobacterium tuberculosis

“…There is only one cluster that includes metazoan sequences, that of the itaconate-detoxifying CLYBL. [25] The Rv2498c cluster is actinobacterial-specific and contains only uncharacterised sequences (seventy-two in UnitProt). A variety of enzymatic activities has been described for PF03328 members, but the majority fall into two classes as it is evident from the tree: aldehyde-lyases (EC:4.1.2) and oxo-acid-lyases (EC:4.1.3); Rv2498c belongs to the latter.…”

“…We showed for the first time that Mtb can dissimilate itaconate, a hallmark macrophage metabolite synthesised after lipopolysaccharide activation during host inflammatory response to fight infection and also linked to Vitamin B12 deficiency. [25, 35, 36]. Itaconate is present in some prokaryotes, but its exact physiological function remains unclear.…”