Lumazine synthase catalyzes the penultimate step in riboflavin biosynthesis and is therefore essential for the existence of life as we know it on earth. It catalyzes the condensation of 5-amino-6-(D-ribitylamino)-2,4-(lH,3W)pyrimidinedionewith the novel carbohydrate, L-3,4-dihydroxy-2-butanone-4-phosphate, to form the immediate precursor to riboflavin, 6,7-dimethyl-8-(Dribityl) lumazine. A hypothetical mechanism for the lumazine synthase-catalyzed reaction has been proposed that involves initial attack of the 5-amino group of the pyrimidinedione on the ketone of the carbohydrate to generate a Schiff base intermediate. Phosphate elimination then affords an enol which tautomerizes to a ketone. Intramolecular nucleophilic attack of the ribitylamino group on the ketone would then provide a carbinolamine that dehydrates to yield the final product. Recent developments in our understanding of how lumazine synthase functions have resulted from crystallographic analysis of complexes formed from substrate, intermediate phosphate, and product analogs. The present research project will focus on the mechanistic details of the conversion of the phosphate to the carbinolamine intermediate. The approach will involve the design and synthesis of metabolically stable analogs of the hypothetical reaction intermediates. These enzyme probes will be crystallized with lumazine synthase and very high resolution structures will be obtained through the refinement of their crystal structures by REDOR NMR. The distances from certain atoms of the ligands to 15N nuclei of the protein will be determined to within 0.2 A, and these distances will be used in a series of distance-restrained molecular dynamics simulations to obtain a binding model for each ligand. This will afford a series of snapshots that can be linked to provide a movie of the detailed structural changes that occur in the active site during catalysis. These techniques are general and could be applied to other enzymes as well. In addition to ligand modification, site-directed mutagenesis will be performed so that the mechanistic interpretation can be aided by protein modification as well as through manipulation of the substrate structure. The recombinant mutant proteins will be expressed in E. coli, and uniformly labeled 15N-labeled enzyme will be made to be used in the REDOR NMR studies. The standard thermodynamic and kinetic parameters of the new enzyme inhibitors will be obtained, including inhibition constants and dissociation constants (tfd), as well as ATis, Ks, and kcat. Ligand displacement studies will be performed, and binding stochiometry determined. These experiments will be carried out on both heavy riboflavin synthase as well as hollow lumazine synthase beta60, capsids. A rapid screening assay for lumazine synthase inhibitors will also be performed in the Pi's laboratory involving the displacement of riboflavin from the active site of pentameric Schizosoccharomyces pombe lumazine synthase. The development of antibiotic resistance by pathogenic microorganisms is one of the major problems presently complicating the successful chemotherapy of infectious diseases. Many of the existing antibiotics are becoming useless, and there is therefore an urgent medical need for new and effective antibiotics. Since humans obtain riboflavin from dietary sources while pathogenic microorganisms are required to synthesize their own riboflavin, the inhibition of the biosynthesis of riboflavin provides a rational strategy for the design and synthesis of new antibiotics.
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