Affiliation:
1. Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 89557, USA
2. Medical Genetics Service, Department of Pediatrics and Research Center, CHU Sainte-Justine and Université de Montréal, Montreal, QC H3T 1C5, Canada
Abstract
The leucine catabolism pathway intermediate, trans-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). trans-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 trans-3MGC CoA to protein 3MGCylation was investigated. trans-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 trans-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 trans-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 trans-3MGC CoA hydratase (AUH) was included in incubations containing trans-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, trans-3MGC CoA is a reactive, potentially toxic metabolite, and under normal physiological conditions, lowering trans-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.
Funder
US National Institutes of Health
Alice and Fred Ottoboni endowed trust