Factors Affecting Non-Enzymatic Protein Acylation by trans -3-Methylglutaconyl Coenzyme A

The leucine catabolism pathway intermediate, -3-methylglutaconyl (3MGC) CoA, is considered to be the precursor of 3MGC acid, a urinary organic acid associated with specific inborn errors of metabolism (IEM). -3MGC CoA is an unstable molecule that can undergo a sequence of non-enzymatic chemical reactions that lead to either 3MGC acid or protein 3MGCylation. Herein, the susceptibility of -3MGC CoA to protein 3MGCylation was investigated. -3MGC CoA was generated through the activity of recombinant 3-methylcrotonyl CoA carboxylase (3MCCCase). Following enzyme incubations, reaction mixtures were spin-filtered to remove 3MCCCase. The recovered filtrates, containing -3MGC CoA, were then incubated in the presence of bovine serum albumin (BSA). Following this, sample aliquots were subjected to α-3MGC IgG immunoblot analysis to probe for 3MGCylated BSA. Experiments revealed a positive correlation between -3MGC CoA incubation temperature and 3MGCylated BSA immunoblot signal intensity. A similar correlation was observed between incubation time and 3MGCylated BSA immunoblot signal intensity. When -3MGC CoA hydratase (AUH) was included in incubations containing -3MGC CoA and BSA, 3MGCylated BSA immunoblot signal intensity decreased. Evidence that protein 3MGCylation occurs in vivo was obtained in studies with liver-specific 3-hydroxy-3-methylglutaryl (HMG) CoA lyase knockout mice. Therefore, -3MGC CoA is a reactive, potentially toxic metabolite, and under normal physiological conditions, lowering -3MGC CoA levels via AUH-mediated hydration to HMG CoA protects against aberrant non-enzymatic chemical reactions that lead to protein 3MGCylation and 3MGC acid production.