and Adelbert Bacher

Biosynthesis of riboflavin: lumazine synthase and riboflavin synthase.

Publisher Summary This chapter discusses the biosynthesis of riboflavin. 6,7-Dimethyl-8-ribityllumazine is developed as the direct biosynthetic precursor of riboflavin. The lumazine derivative is converted to riboflavin by the enzyme riboflavin synthase. The reaction involves the transfer of a four-carbon unit between two identical substrate molecules in an unusual dismutation reaction. The lumazine is biosynthesized from 5-amino-6-ribitylamino-2,4 (1 H ,3 H )-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate by the enzyme lumazine synthase. The riboflavin synthases of Bacillus subtilis and Escherichia coli are homotrimeric molecules. Members of the Bacillaceae also form a large multimeric enzyme complex with lumazine synthase and riboflavin synthase activity. This complex accounts for 12–40% of the total riboflavin synthase activity in different members of the Bacillaceae. This enzyme complex has been designated “heavy riboflavin synthase.” The protein consists of 60 β subunits (lumazine synthase) that form an icosahedral capsid containing 3 α subunits (riboflavin synthase). The structure of this complex is presented in the chapter.

BIOSYNTHESIS OF RIBOFLAVIN : 3,4-DIHYDROXY-2-BUTANONE-4-PHOSPHATE SYNTHASE

Publisher Summary The riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, is formed by condensation of 5-amino-6-ribitylamino-2,4(1 H ,3 H )-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate. The structure of the carbohydrate was established relatively recently. Ribulose 5-phosphate serves as substrate for the formation of 3,4-dihydroxy-2-butanone 4-phosphate catalyzed by the enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase. The enzyme catalyzes the release of carbon-4 of ribulose 5-phosphate as formate, which is accompanied by a complex rearrangement reaction conducive to the formation of the product 3,4-dihydroxy-2-butanone 4-phosphate from carbon atoms 1, 2, 3, and 5 of the substrate. 3,4-Dihydroxy-2-butanone-4-phosphate synthase requires Mg 2+ , and the enzyme reaction can be stopped by adding ethylenediaminetetraacetic acid (EDTA). For detection, the enzyme product is converted enzymatically to 6,7-dimethyl-8-ribityllumazine or riboflavin, which can be determined by fluorescence-monitored high-performance liquid chromatography (HPLC). Lumazine synthase or the lumazine synthase/riboflavin synthase complex is required for the assay and can be prepared using the method described in the chapter.

Lumazine proteins from photobacteria: localization of the single ligand binding site to the N-terminal domain.

Abstract Lumazine protein is believed to serve as an optical transponder in bioluminescence emission by certain marine bacteria. Sequence arguments suggest that the protein comprises two similarly folded riboflavin synthase-type domains, but earlier work also suggested that only one domain binds 6,7-dimethyl-8-ribityllumazine (DMRL). We show that the replacement of serine-48 or threonine-50 in the N-terminal domain of lumazine protein of Photobacterium leiognathi modulates the absorbance and fluorescence properties of bound DMRL or riboflavin. Moreover, the replacement of these amino acids is accompanied by reduced ligand affinity. Replacement of serine-48 by tryptophan shifts the 13C NMR signal of the 6-methyl group in bound DMRL upfield by 2.9 ppm as compared to the wild-type protein complex. Replacement of threonine-50 causes a downfield shift of approximately 20 ppm for the 15N NMR signal of N-5, as well as an upfield shift of 3 ppm for the 13C NMR signal of C-7 in bound DMRL, respectively. The replacement of the topologically equivalent serine-144 and proline-146 in the C-terminal domain had no significant impact on optical properties, chemical shifts and apparent binding constants of bound DMRL. These data show that the N-terminal domain is the unique site for ligand binding in lumazine protein.