# Small Molecules as Alternate Substrates for 3‐Methylglutaconylation

## TL;DR

This study shows that small molecules with amino groups can react with a specific metabolite byproduct, potentially reducing its harmful effects in metabolic disorders.

## Contribution

The study demonstrates that primary amine-containing small molecules can act as alternate substrates for 3MGC anhydride, offering a novel biochemical mechanism.

## Key findings

- Primary amine-containing molecules like glycine and glutathione reduce 3MGCylated BSA signal intensity in a concentration-dependent manner.
- N-acetylglucosamine and choline do not show this effect, indicating specificity for free amino groups.
- 3MGC anhydride can acylate small molecules with primary amines, suggesting a new pathway for metabolite detoxification.

## Abstract

The leucine catabolism pathway intermediate, trans‐3‐methylglutaconyl (3MGC) CoA, is susceptible to a series of non‐enzymatic reactions that generate organic acid waste products and protein 3MGCylation. These reactions proceed when inborn errors of metabolism (IEM) in HMGCL or AUH lead to enzyme deficiencies. When trans‐3MGC‐CoA levels rise, a portion of this metabolite pool isomerizes to cis‐3MGC‐CoA, forming a diastereomer that is capable of spontaneous intramolecular cyclization, yielding 3MGC anhydride and free CoA. 3MGC anhydride can undergo hydrolysis to 3MGC acid or react with protein lysine residues to 3MGCylate proteins. The present study was designed to examine the ability of small molecules to react with 3MGC anhydride. An antibody directed against 3MGC was employed in vitro experiments designed to assess the ability of candidate biomolecules to attenuate the immunoblot signal intensity of 3MGCylated bovine serum albumin (BSA). When trans‐3MGC‐CoA was incubated in the presence of glycine, glucosamine, ethanolamine, or glutathione, each of these free amino group‐containing molecules, but not N‐acetylglucosamine or choline, induced a concentration‐dependent decrease in 3MGCylated BSA immunoblot signal intensity. It is concluded that 3MGC anhydride can react with primary amine‐containing metabolites to acylate them.