Substrate Channeling in the Lumazine Synthase/Riboflavin Synthase Complex of Bacillus subtilis(*)

The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 β subunits surrounding a core of three α subunits. The β subunits catalyze the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (PYR) with 3,4-dihydroxy-2-butanone 4-phosphate (DHB) yielding 6,7-dimethyl-8-ribityllumazine. This intermediate is converted to riboflavin by the α subunits via an unusual dismutation. The second product of this reaction is PYR, which is also a substrate of the β subunits and can be recycled in the catalytic process. Sigmoidal kinetics would be expected for the formation of riboflavin from PYR and DHB and are indeed observed with mixtures of artifactual β60 capsids and α subunit trimers. In contrast, the formation of riboflavin from PYR and DHB by the native α3β60 is characterized by a finite initial rate, which is similar to the rate of lumazine formation. Most notably, the rate of riboflavin formation has its maximum value at t = 0 and decreases dramatically after the consumption of PYR and DHB despite the presence of transiently formed lumazine. These data suggest that a significant fraction of DHB is converted to riboflavin by substrate channeling, which is conducive to an improved overall catalytic rate of riboflavin formation at low substrate concentrations. The channel is leaky, and the intermediate lumazine is therefore transiently accumulated in the bulk solution. The partitioning factor relating the direct formation of riboflavin via substrate channeling and the formation of transient 6,7-dimethyl-8-ribityllumazine increases at low concentrations of the substrates PYR and DHB and has a maximum value at pH 7.5. Channeling appears to result from the compartmentalization of the α subunits inside the icosahedral β subunit capsid whose catalytic sites are located close to the inner capsid surface.