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The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate. The imidazole ring of GTP is hydrolytically opened, yielding a 4,5-diaminopyrimidine that is converted to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction, and dephosphorylation. Condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate affords 6,7-dimethyl-8-ribityllumazine. Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is recycled in the biosynthetic pathway. Two reaction steps in the biosynthetic pathway catalyzed by 3,4-dihydroxy-2-butanone 4-phosphate synthase and riboflavin synthase are mechanistically very complex. The enzymes of the riboflavin pathway are potential targets for antibacterial agents.

Publisher Summary The riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, is formed by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-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 Mg2+, 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.

Publisher Summary This chapter describes the early steps in the biosynthesis of riboflavin. The first committed step is the conversion of guanosine-5'-triphosphate (GTP) to 2,5-diamino-6-ribosylamino-4(3 H )-pyrimidinone 5'-phosphate by the release of formate and pyrophosphate. The reaction is catalyzed by GTP cyclohydrolase II, which was first isolated from Escherichia coli ( E. coli ). The GTP cyclohydrolase lI of Bacillus subtilis ( B. subtilis )is a bifunctional enzyme that can also catalyze the formation of 3,4-dihydroxy-2-butanone 4-phosphate from ribulose 5-phosphate. The product of GTP cyclohydrolase II is converted to 5-amino-6-ribitylamino-2,4(1 H ,3 H )-pyrimidinedione 5'-phosphate by deamination of the pyrimidine ring and reduction of the glycosidic side chain. Both reaction steps are catalyzed by bifunctional proteins in E. coli and B. subtilis that are specified by the genes ribD and the ribG in the respective microorganisms. The deamination of the pyrimidine precedes the reduction of the side chain. This chapter describes the GTP cyclohydrolase II of E. coli and the bifunctional deaminase/reductase of E. coil and B. subtilis .