Biochemical, structural, and functional characterization of the Nocardia asteroides dihydrofolate reductase: a primary target of anti-nocardiosis treatment

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 target respectively the dihydrofolate synthase (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.