Fluorine-19 NMR studies on the mechanism of riboflavin synthase. Synthesis of 6-(trifluoromethyl)-7-oxo-8-(D-ribityl)lumazine and 6-(trifluoromethyl)-7-methyl-8-(D-ribityl)lumazine

Abstract

The reactions of hexafluoropropene oxide (19), methyl trifluoropyruvate (21), and l,l,l-trifluorobutane-2,3-dione (45) with a series of ortho diamines were investigated as an approach to the synthesis of Muoromethyl-substituted quinoxalinones and lumazines. 6-(Trifluoromethyl)-7-oxo-8-(~-ribityl)lumazine (1 1) was synthesized by reaction of methyl trifluoropyruvate (21) with 5-amino-6-(~-ribitylamino)-2,4( lH,3H)-pyrimidinedione (3) hydrochloride and utilized as a 19F NMR probe of the light riboflavin synthase of Bacillus subtilis. The fluorolumazine 11 was found to be an inhibitor of the enzyme with an inhibition constant KI = 55 pM. Equilibrium dialysis experiments indicated the binding of six molecules of 11 per enzyme molecule, corresponding to one molecule bound at each of the three donor and three acceptor sites of the enzyme. The apparent dissociation constants KD were approximately 4 and 112 pM. The enzyme-bound ligand gave rise to several broad I9F NMR signals which were shifted to low field. The bound ligand 11 could be displaced from the enzyme by the enzyme product, riboflavin (2), and the product analog, 5-nitroso-6-(ribitylamino)-2,4(lH,3H)-pyrimidinedione (56). 6-(Trifluoromethyl)-7-methyl-8-(~-ribityl)lumazine (13) was synthesized by reaction of the hydrochloride salt of 3 with l,l,l-trifluorobutane-2,3-dione (45). Three molecules of 13 can be bound relatively tightly per mole of riboflavin synthase, i.e., one ligand molecule per protein subunit. The inhibition constant KI was determined to be 75 pM, while dissociation constants of 17 and 70 pM were determined by equilibrium dialysis and '9F NMR, respectively.The bound ligand 13 could also be displaced by riboflavin and product analog 56. A scheme for the catalytic cycle of riboflavin synthase is proposed.Riboflavin synthase (E.C. 2.5.1.9.) catalyzes an interesting dismutation reaction in which two molecules of 6,7-dimethyl-8-(~-ribityl)lumazine (1) form one molecule of riboflavin (2) and one molecule of the pyrimidinedione 3.1-3 During this reaction, substrate bound at the donor /OH /OH1 2 HQe,, OH R' H::q 3site of the enzyme donates a four-carbon unit to substrate bound at the acceptor site. The mechanism of the reaction is thought to proceed as outlined in Scheme I.4p5 In this process, the anion 5 formed from deprotonation of the C-7 methyl group of 1 at the acceptor site of the enzyme adds to the imine group of 4, which is formed by addition of an unidentified nucleophile to C-7 of 1 bound at the donor site of the enzyme. Various suggestions for the identity of the nucleophile have been offered, including a nucleophilic group from the enzyme: the 2'-hydroxyl or 3'-Purdue University.* Technische Universitiit Munchen.0022-3263/92/ l957-5630$03.00/0 hydroxyl of the ribityl side chain,'^^ and watereg A j3elimination involving cleavage of the N-5 to C-6 bond of the lumazine moiety of 6 at the donor site yields the alkene 7. A 1,g-elimination involving loss of a proton from the C-6 methyl group of the lumazine moiety bound at the acceptor site and "...

Structure of Lumazine Protein, an Optical Transponder of Luminescent Bacteria

“…Thus, 19 F and 13 C NMR studies on lumazine protein in complex with various ligands invariably show single signal sets for the bound ligand, 21,22,26 whereas similar studies with riboflavin synthase show multiple signal sets in line with the fact that the six ligandbinding domains of the enzyme are all topologically non-equivalent. 21,22,27,28 A recent study has also shown that bulky single-site replacements at the Nterminal ligand-binding site of lumazine protein result in major changes of ligand affinity and of the chemical shift values of bound ligands (riboflavin or 6,7-dimethyl-8-ribityllumazine). 18 On the other hand, similar replacements of topologically equivalent residues at the C-terminal domain had hardly any effect on ligand binding and chemical shift values of the bound ligands.…”

Cloning, Sequencing, Mapping and Hyperexpression of the ribC Gene Coding for Riboflavin Synthase of Escherichia coli

“…"IF-NMR studies with these analogs suggested that the trimeric riboflavin synthase of B. subtilis has a non-symmetric structure conducive to somewhat different environments for ligands bound at the different subunits of the homotrimer. Moreover, the studies suggested a major conformational transition of the protein in the course of the catalytic cycle (Cushman et al, 1992).…”

“…Cushman and his coworkers have prepared several trifluoromethyl derivatives of 6,7-dimethyl-8-ribityllumazine, which are thought to mimic reaction-pathway intermediates of riboflavin synthase (Cushman et al, 1991(Cushman et al, , 1992(Cushman et al, , 1993. "IF-NMR studies with these analogs suggested that the trimeric riboflavin synthase of B. subtilis has a non-symmetric structure conducive to somewhat different environments for ligands bound at the different subunits of the homotrimer.…”

Riboflavin Synthase of Escherichia coli

“…Evidence for two different types of binding sites had been obtained earlier by studies with riboflavin synthase from B. subtilis and 6-trifluoromethyl-7-oxo-8-ribityllumazine (16,28), but the 19 F NMR spectra produced by Compound 5 in this case not only differed substantially from those shown in Fig. 4, but also gave the molar ratio of 1:1 of bound ligand per subunit, indicating that only one type of binding site could bind the ligand.…”

“…Protein Perturbation Experiments-In the absence of protein structural data, we decided to perform protein perturbation studies to characterize the interaction of the mutant proteins with a ligand that binds to the catalytic site of riboflavin synthase. The fluorinated Compounds 5 and 6 have been used extensively in ligand binding studies with riboflavin synthase and lumazine synthase of Bacillus subtilis (17,20,28,29). Fluorine substitution favors the covalent hydration of the pteridine ring system to such an extent that the diasteromeric Compounds 5 and 6 are not subject to epimerization.…”