Structural Superimposition

Abstact

Nocardiosis is a human infectious disease caused by several species of Nocardia and primarily affecting the skin, lungs and central nervous system. The first-line treatment is based on cotrimoxazole, combining trimethoprim and sulfamethoxazole. These two drugs respectively target the dihydrofolate reductase (DHFR) and the dihydropteroate synthase (DHPS) involved in the essential folate synthesis pathway. The occurrence of drug resistance to these two drugs is however frequent. While the molecular mechanisms of trimethoprim resistance are well documented in other bacteria, they remain poorly explored and documented in Nocardia. This is partly because few biochemical, structural or genetic studies have been conducted on DHFR from this genus. In this study, we report the biochemical and structural characterization of DHFR from Nocardia asteroides (DHFR(Nad)). We show that overexpression of DHFR(Nad) in N. asteroides confers strong resistance to trimethoprim. We recombinantly expressed and purified active DHFR(Nad) and determined its inhibition constant for trimethoprim. We solved the crystal structure of DHFR(Nad) bound to trimethoprim at high- resolution. Further, biochemical studies of mutant DHFR variants pinpointed the role of important residues for trimethoprim binding and drug-resistance.