Characterization of Wild-type and a Novel Mutant of Arylamine N-acetyltransferase (NAT) from Mycobacterium Marinum as a Model for NAT from Mycobacterium Tuberculosis

Arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes linked to antibiotic resistance in Mycobacterium tuberculosis (MTB ), making them promising targets for therapeutic intervention. This research used Mycobacterium marinum’s NAT homolog as a model to evaluate the structural and functional effects of a site-directed missense mutation, T109Q, situated near the active site. The mutation substitutes threonine, a minor polar residue, with glutamine, a larger polar amino acid, which may influence substrate binding and catalytic performance. Both wild-type and T109Q mutant NAT proteins were produced in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) and ion exchange chromatography. SDS-PAGE and Western blotting were used to measure expression and solubility, with the wild-type protein outperforming the mutant in terms of both. Protein concentrations were measured using the Bradford and UV absorbance as-says. Enzymatic activity was assessed using acetyl-CoA and either isoniazid or hydralazine as substrates. The wild-type protein catalyzed isoniazid acetylation, but the mutant had no detectable activity for either drug. Thermal shift experiments revealed that the mutant was less thermally stable than the wild-type, indicating that folding or structural integrity had been disrupted. These results show that the T109Q mutation drastically reduced NAT expression, solubility, stability, and enzymatic performance, most likely due to disruption of active-site dynamics. The findings highlight NAT’s structural vulnerability to changes around its catalytic core and give mechanistic insights that might influence future inhibitor design targeting MTB NAT (Areej Abuhammad et al., 2010).