Objectives/Goals: The specialized riboflavin (vitamin B 2 ) biosynthetic enzymes are targets for novel antibiotics. This project provides direct evidence for the chemical mechanism of Mycobacterium tuberculosis lumazine synthase, the penultimate enzyme of the riboflavin production and target for therapeutic intervention. Methods/Study Population: Lumazine synthase catalyzes the condensation of 3,4-dihydroxy-2-butanone-4-phosphate and 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione forming 6,7-dimethyl-8-ribityllumazine. To determine the chemical mechanism of Mycobacterium tuberculosis lumazine synthase, we performed transient state kinetics to monitor the formation and disappearance of intermediates and products over time. These traces indicated time points to flash-freeze the reaction to isolate intermediates that are analyzed with 13C-NMR to determine the chemical structure. Time resolved X-ray crystallography is used as a complementary method to determine the structures of the reaction intermediates in the context of the active site. Results/Anticipated Results: The NMR data provide direct evidence of the chemical mechanism of lumazine synthase. Complementary crystal structures of lumazine synthase with intermediates bound to the active site will corroborate the chemical mechanism determined by NMR and model key residues in the active site that facilitate catalysis. These data will inform steps of the chemical mechanism that can be targeted for therapeutic intervention. Discussion/Significance of Impact: This work informs drug design to address the growing threat of antibiotic resistance. The riboflavin biosynthetic enzymes do not have human homologs, so drugs designed to specifically target these enzymes will minimize off target effects.
Hoffpauir et al. (2026) studied this question.