| Abstract
| - The riboflavin synthase/lumazine synthase complex of Bacillus subtilis catalyzes the last two stepsin riboflavin biosynthesis. The protein comprises a capsid of 60 β subunits with lumazine synthaseactivity and a core of three α subunits with riboflavin synthase activity. The β subunits catalyzethe formation of 6,7-dimethyl-8-ribityllumazine (3) from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (1) and 3,4-dihydroxy-2-butanone 4-phosphate (2). Complexes of recombinantlumazine synthase (β60 capsids) with 6-trifluoromethyl-7-oxo-8-ribityllumazine (10) as well as 7S-or 7R-6,7-bistrifluoromethyl-8-ribityllumazine hydrate (11) were studied by 19F NMR spectroscopy.Despite the large molecular weight of approximately 960 kDa of the protein, spectra with separatedsignals of free and bound ligand could be obtained. An unusually large shift difference of 8 ppmwas observed between the 7-trifluoromethyl signals of free and bound ligand for epimer B of 11and the enzyme. The signal is sensitive to the replacement of amino acid residues F22 and H88.Lumazine synthase catalyzes the elimination of the 7-trifluoromethyl group of R-diastereomerepimer A in a haloform-like reaction. The elimination reaction is also catalyzed by F22 mutants.The H88R mutant displays an opposite stereoselectivity for epimer B and a greatly enhancedreaction rate. From a model of the epimers in the active site of the protein, the main function ofthe side chain of F22 seems to be to keep the substrate ring in the correct position. H88 is in aposition suited to act as proton acceptor in both the physiological as well as the haloform reaction.A different mechanism of the haloform-reaction is proposed in the case of the H88R mutant, initiatedby hydrogen bonding of the 7-trifluorormethyl group and the guanidinium group of the arginineresidue.
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