19F NMR Ligand Perturbation Studies on 6,7-Bis(trifluoromethyl)-8-ribityllumazine-7-hydrates and the Lumazine Synthase Complex of Bacillus s ubtilis. Site-Directed Mutagenesis Changes the Mechanism and the Stereoselectivity of the Catalyzed Haloform-Type Reaction

• Published in: Journal of organic chemistry 2001-06, Vol.66 (11), p.3811-3819
• Main Authors: Scheuring, Johannes, Kugelbrey, Karl, Weinkauf, Sevil, Cushman, Mark, Bacher, Adelbert, Fischer, Markus
• Format: Article
• Language: English
• ISSN: 0022-3263; 1520-6904
• DOI: 10.1021/jo001739u

The riboflavin synthase/lumazine synthase complex of Bacillus subtilis catalyzes the last two steps in riboflavin biosynthesis. The protein comprises a capsid of 60 β subunits with lumazine synthase activity and a core of three α subunits with riboflavin synthase activity. The β subunits catalyze the 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 recombinant lumazine 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 separated signals of free and bound ligand could be obtained. An unusually large shift difference of 8 ppm was observed between the 7-trifluoromethyl signals of free and bound ligand for epimer B of 11 and 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-diastereomer epimer 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 enhanced reaction rate. From a model of the epimers in the active site of the protein, the main function of the side chain of F22 seems to be to keep the substrate ring in the correct position. H88 is in a position 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, initiated by hydrogen bonding of the 7-trifluorormethyl group and the guanidinium group of the arginine residue.