Riboflavin synthase

RIBOFLAVINASE RIBOFLAVIN KINASE RIBOFLAVIN SYNTHASE RIBOFLAVINASE RIBOFLAVIN KINASE RIBOFLAVIN SYNTHASE RIBONUCLEASE (or RNase) PSEUDOROTATION DIETHYL PYROCARBONATE RIBONUCLEASE F RIBONUCLEASE II RIBONUCLEASE III Ribonuclease inactivation,... 

New derivatives of 4-amino and 2,4-diaminopteridines have been synthesized and their capability to inhibit neuronal nitric oxide synthase evaluated <99JMC4108>. The synthesis of folic acid multiply labeled with stable isotopes, for bioavailability studies in human nutrition, has been reported <99JCS(P1)1311>, Synthesis and antiviral evaluation of several 6-(methylenecarbomethoxy)pteridine-4,7-diones have been described <99JHC435>. Synthesis and biochemical evaluation of bis(6,7-dimethyl-8-D-ribityllumazines) as potential bisubstrate analog inhibitors of riboflavin synthase have been reported <99JOC4635>. Synthesis and cyclization of novel lumazine-enediyne chimeras have been reported <99H13>. 

Riboflavin synthase catalyzes the dismutation of 8-D-ribityl-6,7-dimethyllumazine to form the flavin ring system and the general features of the mechanism of this reaction have been known for some time. Recent X-ray structural studies of the enzyme from archaeal organisms such as methanobacteria have shown that the chemical mechanism of action is similar to that of enzymes from eubacteria and eukaryotes although the structures of the enzymes differ greatly <2006JBC1224>. 

Figure7.3. Biosynthesis of riboflavin in fungi in bacteria, deamination precedes reduction of sugar. GTP cyclohydrolase, EC 3.5.4.25 and riboflavin synthase, EC 2.5. f.9.

The final step is a dismutation reaction between two molectdes of dimethyl-lumazine, catalyzed by riboflavin synthase, yielding riboflavin and amino-ribitylamino-pyrirnidinedione. Thus latter product can undergo reaction with dihydroxybutanone 4-phosphate to yield dimethyl-ribityllumazine. 

Recently, we screened riboflavin synthase (RiSy) against a small library of -containing ligands. The library encompasses about 400 ligands containing either an aromatic fluorine or a trifluoromethyl moiety. 

Riboflavin synthase catalyzes the disproportionation of two molecules of lumazine to give riboflavin and 39 [34]. This complex reaction also occurs non-enzymatically [35-37]. The mechanism has not yet been fully established. A proposal, consistent with the regiochemistry of the reaction [31,38 -40], with the observation of facile H/D exchange at the C-7 methyl group of lumazine [38, 40 - 42], with the facile nucleophilic addition to C-7 of lumazine [36,43,44], and with NMR studies [45-47], is outlined in Fig. 11 [39-40]. 

It is still unknown how the pyrimidine intermediate 5 is dephosphorylated (reaction VI). However, it is well established that the dephosphorylation product 6 is condensed with 3,4-dihydroxy-2-butanone 4-phosphate (8) by the catalytic action of lumazine synthase (reaction VIII). The carbohydrate substrate 8 is in turn obtained from ribulose phosphate (7) by a complex reaction sequence that is catalyzed by a single enzyme, 3,4-dihydroxy-2-butanone 4-phosphate synthase (reaction VII). As mentioned above, the lumazine 9 is converted to riboflavin (10) by the catalytic action of riboflavin synthase (reaction IX). Ultimately, riboflavin is converted to the coenzymes, riboflavin 5 -phosphate (flavin mononucleotide (FMN), 11) and flavin adenine dinucleotide (FAD, 12) by the catalytic action of riboflavin kinase (reaction X) and FAD synthase (reaction XI). These reaction steps are required in all organisms, irrespective of their acquisition of riboflavin from nutritional sources or by endogenous biosynthesis. 

Since the discovery of riboflavin synthase more than four decades ago, it was understood that all eight carbon atoms of the benzenoid ring of the vitamin are derived from carbon atoms 6a, 6, 7, and 7a of 6,7-dimethyl-... 

Studies in the 1970s revealed the presence of two different proteins with riboflavin synthase activity in B. subtilis. One was a homotrimer of 25 kDa subunits, the other was a complex protein where a homotrimer of the 25 kDa subunits was associated with sixty 16 kDa subunits.More precisely, the trimer of 25 kDa subunits was enclosed in a capsid of sixty 15 kDa subunits with icosahedral 532 symmetry. The complex protein had a mass of about 1 MDa and was designated heavy riboflavin synthase, whereas the homotrimer with a mass of about 75 kDa was designated light riboflavin synthase. The 25 kDa subunits (which were subsequently designated a subunits) were apparently the carriers of the riboflavin synthase activity in both proteins, and the function of 16 kDa subunits that were designated (3 subunits remained unknown for two decades. 

Subsequent to the identification of 3,4-dihydroxy-2-butanone 4-phosphate (8) as the 4-carbon precursor of 6,7-dimethyl-8-ribityllumazine in the late 1980s,it was not difficult to show that the j3 subunits catalyzed the condensation of 6 with 8 under formation of the lumazine derivative 9. The heavy riboflavin synthase can now be correctly addressed as a riboflavin synthase/lumazine synthase complex. However, the designation /3 subunit of riboflavin synthase clung tenaciously to the 16 kDa peptide that had ultimately been assigned a function as lumazine synthase (reaction VIII in Figure 1) notably, this somewhat unfortunate nomenclature survives in databases, despite the fact that in most organisms, lumazine synthase and riboflavin synthase (reaction IX in Figure 1) are separate proteins. 

In Bacillaceae, the icosahedral lumazine synthase capsid can trap a homotrimeric riboflavin synthase molecule in the central That unusual complex was originally designated heavy riboflavin... 

Steady-state kinetic analysis afforded evidence for intermediate channeling in the lumazine synthase/ riboflavin synthase complex. Briefly, the conversion of 6,7-dimethyl-8-ribityllumazine molecules that have been newly formed by the lumazine synthase module of the protein complex are more rapidly converted to riboflavin than molecules from the bulk solvent. The topological constraint by the capsid is believed to cause this phenomenon. It has been proposed that the channels along the five-fold axes could serve as port of entry and exit for substrates and products. ... 

Specifically, an alkaline aqueous solution of 6,7-dimethyl-8-ribityllumazine contains a mixture of at least five anionic species, namely the exomethylene-type anion with an open chain structure of the position 8 substituent and two diastereomers each of the 5-ring and 6-ring forms arising by intramolecular reaction of the 2 and 3 hydroxyl groups. The hypothesis of an involvement of one of the tricyclic species as intermediates in the biosynthesis of riboflavin is not supported by more recent data. The exomethylene form appears likely to be a late intermediate in the mechanism of lumazine synthase as documented by stopped flow analysis. Moreover, the exomethylene form (31) has been proposed as an early intermediate of the riboflavin synthase reaction. This is supported by the early observation that riboflavin synthase accelerates proton exchange at the position 7 methyl group. 

While the stereochemically favored addition of a 2 or 3 hydroxyl group to C-8 of 6,7-dimethyl-8-ribityllumazine has been documented in considerable detail, the formation of a position 7 hydrate has only been observed after the introduction of fluorine to increase the electronegativity. Indeed, the bistrifluromethyl-8-ribityllumazine is so stable that the two diastereomeric forms A and B show no evidence of racemization whatsoever. Notably, a hypothetical covalent hydrate of 6,7-dimethyl-8-ribityllumazine has been proposed as an intermediate at the donor site of riboflavin synthase. " ... 

Compound Q can be converted by riboflavin synthase into a mixture of 6,7-dimethyl-8-ribityllumazine, riboflavin, and 5-amino-6-ribitylamino-2,4(l//,3f/)-pyrimidinedione. " This is best explained by the hypothesis that Compound Q is an intermediate of the enzyme-catalyzed reaction than can undergo either a forward reaction affording riboflavin (10) and 6 or a reverse reaction affording 6,7-dimethyl-8-ribityllumazine (9) with similar reaction velocity. Moreover, it was shown by presteady-state kinetic analysis that Compound (X fulfills the criteria for a kinetically competent reaction intermediate. As described in more detail below, it can be deduced from X-ray structure data that the two lumazine components in the pentacyclic adduct 36 must be fused in cis. ... 

The sequence of riboflavin synthase of B. subtilis, the first that became available, was determined at the protein level by Edman sequencing and, independently, by DNA sequencing. "" In line with the hypothesis that the enzyme could provide a pseudo-C2-symmetric environment, it was gratifying to observe that the 215 amino acid peptide shows strong internal sequence homology specifically, 26 amino acids in the internal sequence repeat of the B. subtilis enzyme are identical and 23 are similar (Figure 12). "" ... 

Notably, however, the sequence similarity does not extend to a segment of about 22 amino acids at the C-terminus. Meanwhile, that internal homology has been found in more than hundred riboflavin synthase sequences of bacterial, fungal, or plant origin. It should be noted, however, that the riboflavin synthases of Archaea have no intramolecular sequence similarity (see below). 

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