Aberrant Biochemistry of a Leucine Metabolism Intermediate
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Authors
Jennings, Elizabeth
Issue Date
2024
Type
Dissertation
Language
en_US
Keywords
3-methylglutaconic aciduria , 3-methylglutaconyl CoA , HMG CoA lyase deficiency , inborn errors of metabolism , protein acylation
Alternative Title
Abstract
Urinary organic acids are often associated with inborn errors of metabolism (IEM) or other disease states. For example, 3-methylglutaconic (3MGC) acid is excreted in IEMs associated with leucine degradation pathway enzyme deficiencies (primary 3MGC aciduria). Mutations in 3-hydroxy-3-methylglutaryl (HMG) CoA lyase (HMGCL) or 3MGC CoA hydratase (AUH) cause an accumulation of the upstream pathway intermediate, trans-3MGC CoA, the precursor of 3MGC acid. Alternatively, in secondary 3MGC aciduria, 3MGC acid excretion is associated with IEMs that affect mitochondrial energy metabolism. In these disorders, a previously unknown biosynthetic route termed the “acetyl CoA diversion pathway” leads to production of trans-3MGC CoA and excretion of 3MGC acid. Studies have shown that trans-3MGC CoA is an intrinsically unstable chemical entity that is susceptible to non-enzymatic isomerization to cis-3MGC CoA. Once produced, cis-3MGC CoA can undergo intramolecular cyclization, yielding 3MGC anhydride plus free CoA. The anhydride is reactive and has at least two potential fates including 1) hydrolysis to form 3MGC acid or 2) reaction with lysine side chain amino groups to covalently 3MGCylate proteins. In chapter 2, the reaction catalyzed by the leucine degradation pathway enzyme trans-3MGC CoA hydratase (AUH) was investigated. AUH-mediated dehydration of HMG CoA produces trans-3MGC CoA, which, in the presence of bovine serum albumin (BSA) leads to 3MGCylation of BSA. A polyclonal antibody directed against 3MGC moieties was generated and utilized in immunoblot experiments designed to detect protein 3MGCylation. In chapter 3, the effect of temperature and time on the non-enzymatic reaction sequence initiated by trans-3MGC CoA was examined. When AUH was included in incubations containing trans-3MGC CoA and BSA, the extent of BSA 3MGCylation was attenuated, indicating AUH activity protects against the aberrant non-enzymatic reaction sequence that leads to protein 3MGCylation / 3MGC acid production. Finally, evidence that protein 3MGCylation occurs in vivo was obtained in HMGCL knockout mice. In the studies presented in chapter 4, the metabolic origin of another organic acid derived from trans-3MGC CoA, 3-methylglutaric (3MG) acid, was investigated. Whereas these experiments provided preliminary evidence that 3MG CoA is formed by reduction of the double bond in trans-3MGC CoA, further studies are required to validate the underlying hypothesis. In summary, studies in this dissertation provide novel information regarding the biochemical origins of 3MGC acid and 3MG acid. This enhanced understanding of the metabolic origins of these organic acids is likely to improve their utility as disease biomarkers.