Tuberculosis is a disease caused by Mycobacterium tuberculosis and is the leading cause of death from a single infectious pathogen, with a high prevalence in developing countries in Africa and Asia. There still is a need for the development or repurposing of novel therapies to combat this disease owing to the long-term nature of current therapies and because of the number of reported resistant strains. Here, structures of dihydrofolate reductase from M. tuberculosis (MtDHFR), which is a key target of the folate pathway, are reported in complex with four antifolates, pyrimethamine, cycloguanil, diaverdine and pemetrexed, and its substrate dihydrofolate in order to understand their binding modes. The structures of all of these complexes were obtained in the closed-conformation state of the enzyme and a fine structural analysis indicated motion in key regions of the substrate-binding site and different binding modes of the ligands. In addition, the affinities, through K d measurement, of diaverdine and methotrexate have been determined; MtDHFR has a lower affinity (highest K d) for diaverdine than pyrimethamine and trimethoprim, and a very high affinity for methotrexate, as expected. The structural comparisons and analysis described in this work provide new information about the plasticity of MtDHFR and the binding effects of different antifolates.

Targeting Mycobacterium tuberculosis peptidoglycans with β-lactam antibiotics represents a strategy to address increasing resistance to antitubercular drugs. β-Lactams inhibit peptidoglycan synthases such as L,D-transpeptidases, a group of carbapenem-sensitive enzymes that stabilize peptidoglycans through 3 → 3 cross-links. M. tuberculosis encodes five L,D-transpeptidases (Ldt Mt1−5 ), of which Ldt Mt3 is one of the less understood. Herein, we structurally characterized the apo and faropenem-acylated forms of Ldt Mt3 at 1.3 and 1.8 Å resolution, respectively. These structures revealed a fold and catalytic diad similar to those of other Ldts Mt enzymes, supporting its involvement in transpeptidation reactions despite divergences in active site size and charges. The Ldt Mt3 −faropenem structure indicated that faropenem is degraded after Cys-246 acylation, and possibly only a β-OH-butyrate or an acetyl group (C 2 H 3 O) covalently attached to the enzyme remains, an observation that strongly supports the notion that Ldt Mt3 is inactivated by β-lactams. Docking simulations with intact β-lactams predicted key Ldt Mt3 residues that interact with these antibiotics. We also characterized the heat of acylation involved in the binding and reaction of Ldt Mt3 for ten β-lactams belonging to four different classes, and imipenem had the highest inactivation constant. This work provides key insights into the structure, binding mechanisms, and degradation of β-lactams by Ldt Mt3 , which may be useful for the development of additional β-lactams with potential antitubercular activity.

No abstract