A pentacyclic reaction intermediate of riboflavin synthase

The S41A mutant of riboflavin synthase from Escherichia coli catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a very low r ate. Quenching of presteady-state reaction mixtures with trifluoroacetic ac id afforded a compound with an absorption maximum at 412 nm (pH 1.0) that c an be converted to a mixture of riboflavin and 6,7-dimethyl-8-ribityllumazi ne by treatment with wild-type riboflavin synthase. The compound was shown to qualify as a kinetically competent intermediate of the riboflavin syntha se-catalyzed reaction. Multinuclear NMR spectroscopy, using various C-13- a nd N-15-labeled samples, revealed a pentacyclic structure arising by dimeri zation of 6.7-dimethyl-8-ribityllumazine. Enzyme-catalyzed fragmentation of this compound under formation of riboflavin can occur easily by a sequence of two elimination reactions.