Biosynthesis of Riboflavin (Flavocoenzymes)


Flavocoenzymes are essential cofactors for catalysis of a wide variety of redox reactions. Moreover, they are involved in numerous other physiological processes involving light sensing, bioluminescence, circadian time-keeping and DNA repair.
Vitamin B2 (riboflavin) is the universal precursor of flavocoenzymes. Riboflavin is biosynthesized in plants and in many bacteria. Vegetables and milk are major sources of the vitamin in human nutrition. Ruminants can derive vitamin B2 from their intestinal flora. The daily recommended allowance for vitamin B2 is 1.8 mg. Although the flavocoenzymes are absolutely indispensable in all cellular organisms, symptoms of riboflavin deficiency are rarely observed in man. However, latent riboflavin deficiency may be relatively common, especially in women and adolescents – especially in developmental countries.

The compound is manufactured in relatively large quantity (about 4000 metric tons per year) for use as a vitamin in human and animal nutrition and as a colorant, and biotechnological aspects were an important driving form for studies on its biosynthesis which extend over a period of more than five decades. In fact, the manufacture of the vitamin by fermentation has by now essentially replaced chemical synthesis.

The biosynthesis of one riboflavin molecule requires one molecule of GTP (1) and two molecules of ribulose 5-phosphate (6). The imidazole ring of GTP is hydrolytically opened by the GTP cyclohydrolase II (A), yielding a 4,5-diaminopyrimidine that is converted to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (4)) by a sequence of deamination (B), side chain reduction (C), and dephosphorylation (D). Condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (5) with 3,4-dihydroxy-2-butanone 4-phosphate (7) obtained from ribulose 5-phosphate affords 6,7-dimethyl-8-ribityllumazine (8). This reaction is catalysed by the 6,7-dimethyl-8-ribityllumazine synthase (F). Dismutation of the lumazine derivative (riboflavin synthase; G) yields riboflavin (9) and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (5), which is recycled in the biosynthetic pathway.
Flavoenzymes contain flavin nucleotides as redox cofactors, either riboflavin monophosphate (FMN; 10) or flavin adenine dinucleotide (FAD; 11). All organisms are able to convert the precursor, riboflavin, into the active flavin nucleotide cofactors. The formation of FAD depends on the sequential utilisation of two molecules of ATP in reactions that first involve the phosphorylation of riboflavin to form FMN by riboflavin kinase (H) and then the adenylation of the latter to form FAD, a reaction catalysed by FAD synthetase (I).

Enzymes from plants and microorganisms (yeast, eubacteria, archaea) involved in this pathway, their reaction mechanisms and their structures are mainly topics in the riboflavin division. Among this, both biophysical and biochemical methods are employed.








Structure and function of lumazine protein


A number of fluorescent proteins (e.g. lumazine protein, yellow fluorescent protein, blue fluorescent protein) have been found to serve as optical transponders in luminescent photobacteria. They are brought to an excited state by Förster transfer from bacterial luciferase. Their overall contribution to bioluminescence is an increase in quantum yield and a shift of the emission spectrum.
A non-covalently bound molecule of 6,7-dimethyl-8-ribityllumazine serves as fluorophore of the lumazine protein, the emitter protein of Photobacterium leiognathi.

Modulation of the binding site by site directed mutagenesis in conjunction with the spectroscopic techniques (multinuclear NMR, EPR, ENDOR, fluorescence spectroscopy and X-ray structure analysis) is expected to provide structural as well as dynamic information on protein-ligand interaction and its role for luminescence amplification by enhanced fluorescence quantum yield.