Role of non‐enzymatic chemical reactions in 3‐methylglutaconic aciduria

Abstract

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.

Characterization of trans-3-Methylglutaconyl CoA-Dependent Protein Acylation

“…Previous studies investigating AUH activity employed Tris as the buffering agent [ 9 , 10 , 11 , 12 ]. However, knowledge that trans -3MGC CoA is susceptible to isomerization/cyclic anhydride formation suggests that cis -3MGC anhydride may react with the free amino group on Tris to yield 3MGCylated Tris as a product and, thereby, attenuate 3MGCylated BSA signal intensity.…”

The causal relationship between serum metabolites and the risk of psoriasis: a Mendelian randomization and meta-analysis study

“…Therefore, individuals with psoriasis should limit their intake of ultra-processed foods to prevent the potential risk increase associated with high levels of catechol sulfate metabolites. Dysregulation of 3-methylglutaconic acid metabolism has been noted in conditions such as 3-methylglutaconic aciduria and various congenital metabolic defects where mitochondrial energy metabolism is impaired ( 44 , 45 ). Elevated levels of 3-methylglutaconic acid detected in various inherited metabolic diseases could lead to dysfunctions in the nervous system, heart, liver, and other organs ( 66 , 67 ).…”

Diversion of Acetyl CoA to 3-Methylglutaconic Acid Caused by Discrete Inborn Errors of Metabolism

“…The resulting cyclic anhydride is reactive and can be hydrolyzed to yield the dead-end organic acid, cis -3MGC acid, or it can undergo nucleophilic attack by protein lysine side chain amino groups to form a covalent adduct ( Figure 4 ). Based on this non-enzymatic reaction sequence, a potential route from trans -3MGC CoA to cis -3MGC acid exists [ 22 ]. Not only does this reaction sequence provide an explanation for the presence of cis -3MGC acid in urine of subjects with primary and secondary 3MGC aciduria, it also explains how AUH’s equilibrium constant [ 16 ], which strongly favors hydration over dehydration, is overcome.…”

“…The hydrolysis of cis -3MGC anhydride exclusively generates cis -3MGC acid, thereby providing a molecular explanation for the occurrence of cis -3MGC acid in urine of subjects with 3MGC aciduria. In the case of protein 3MGCylation, in vitro studies [ 22 ] revealed that, although cis -3MGC anhydride displays a preference for hydrolytic cleavage versus acylation, considerable protein 3MGCylation also occurs. The fact that Young et al [ 17 ] detected 3MGCylated bovine serum albumin (BSA) following incubation of trans -3MGC CoA with BSA provides compelling evidence in support of the non-enzymatic phase of the acetyl CoA diversion pathway.…”

“…Studies with SIRT4-knockout mice [ 25 ] revealed the role of this enzyme in removal of 3MGC- adducts from substrate proteins. Although it has been reported [ 26 ] that trans -3MGC- is the acyl moiety bound to protein lysine side chains, evidence now exists that it is actually the cis -diastereomer of 3MGC- that forms an amide linkage with substrate proteins [ 22 ].…”