Itaconate is metabolized to 2-hydroxymethylsuccinate through a CoA-independent degradation pathway in mitochondria

The immunometabolite itaconate modulates cellular metabolism and is converted into structurally similar C ₅ dicarboxylates that require advanced analytics to decipher their metabolic fate. Here, we employ high-resolution mass spectrometry and tracing approaches and identify 2-hydroxymethylsuccinate (2HMS) as a previously unrecognized C ₅ dicarboxylate derived from itaconate. 2HMS synthesis occurs during inflammatory responses and upon itaconate treatment, as detected by ¹³ C itaconate tracing. Pathway analysis reveals that methylglutaconyl-CoA hydratase (AUH) drives 2HMS synthesis through a CoA-independent conversion (CIC) pathway. This pathway is distinct from the CoA-dependent conversion (CDC) pathway that generates mesaconate and itaconyl-CoA influencing B ₁₂ -dependent processes. In vivo inflammation studies reveal that adipose tissue prefers CIC to produce 2HMS and liver favors CDC-mediated mesaconate synthesis, highlighting tissue-specific itaconate degradation routes. This study identifies a new branch of itaconate metabolism, provides an analytical framework to resolve C ₅ dicarboxylate networks, and links 2HMS to inflammation and mitochondrial metabolism that might be targeted therapeutically.