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3‐Methylglutaconic (3MGC) aciduria is a common phenotypic feature of a growing number of inborn errors of metabolism. “Primary” 3MGC aciduria is caused by deficiencies in leucine pathway enzymes while “secondary” 3MGC aciduria results from inborn errors of metabolism that impact mitochondrial energy production. The metabolic precursor of 3MGC acid is trans‐3MGC CoA, an intermediate in the leucine catabolism pathway. Gas chromatography‐mass spectrometry (GC‐MS) analysis of commercially available trans‐3MGC acid yielded a mixture of cis and trans isomers while 1H‐NMR spectroscopy of trans‐3MGC acid at 25°C provided no evidence for the cis isomer. When trans‐3MGC acid was incubated under conditions used for sample derivatization prior to GC‐MS (but with no trimethylsilane added), 1H‐NMR spectroscopy provided evidence of trans to cis isomerization. Incubation of trans‐3MGC acid at 37°C resulted in time‐dependent isomerization to cis‐3MGC acid. Cis‐3MGC acid behaved in a similar manner except that, under identical incubation conditions, less isomerization occurred. In agreement with these experimental results, molecular modeling studies provided evidence that the energy minimized structure of cis‐3MGC acid is 4 kJ/mol more stable than that for trans‐3MGC acid. Once generated in vivo, trans‐3MGC acid is proposed to isomerize via a mechanism involving π electron delocalization with formation of a resonance structure that permits bond rotation. The data presented are consistent with the occurrence of both diastereomers in urine samples of subjects with 3MGC aciduria.

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3‐Methylglutaconic (3MGC) aciduria occurs in numerous inborn errors associated with compromised mitochondrial energy metabolism. In these disorders, 3MGC CoA is produced de novo from acetyl CoA in three steps with the final reaction catalysed by 3MGC CoA hydratase (AUH). In in vitro assays, whereas recombinant AUH dehydrated 3‐hydroxy‐3‐methylglutaryl (HMG) CoA to 3MGC CoA, free CoA was also produced. Although HMG CoA is known to undergo non‐enzymatic intramolecular cyclisation, forming HMG anhydride and free CoA, the amount of free CoA generated increased when AUH was present. To test the hypothesis that the AUH‐dependent increase in CoA production is caused by intramolecular cyclisation of 3MGC CoA, gas chromatography—mass spectrometry analysis of organic acids was performed. In the absence of AUH, HMG CoA was converted to HMG acid while, in the presence of AUH, 3MGC acid was also detected. To determine which 3MGC acid diastereomer was formed, immunoblot assays were conducted with 3MGCylated BSA. In competition experiments, when α‐3MGC IgG was preincubated with trans‐3MGC acid or cis‐3MGC acid, the cis diastereomer inhibited antibody binding to 3MGCylated BSA. When an AUH assay product mix served as competitor, α‐3MGC IgG binding to 3MGCylated BSA was also inhibited, indicating cis‐3MGC acid is produced in incubations of AUH and HMG CoA. Thus, non‐enzymatic isomerisation of trans‐3MGC CoA drives AUH‐dependent HMG CoA dehydration and explains the occurrence of cis‐3MGC acid in urine of subjects with 3MGC aciduria. Furthermore, the ability of cis‐3MGC anhydride to non‐enzymatically acylate protein substrates may have deleterious pathophysiological consequences.