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PubMed1983

Phosphates of riboflavin and riboflavin analogs: a reinvestigation by high-performance liquid chromatography.

Phosphoric acid esters of riboflavin can be easily separated by reverse-phase high-performance liquid chromatography using eluants of 0.1 M ammonium formate in aqueous methanol. Commercial FMN preparations contained seven different flavin phosphates; the content of riboflavin 5'-phosphate was 70-75% and is in agreement with previous studies. Millimole amounts of crude FMN can be processed by preparative HPLC. The method permits the preparation of greater than 99%-pure 5'-FMN. The following compounds were isolated in pure form and their structures determined: riboflavin 4'-phosphate, riboflavin 3'-phosphate, riboflavin 4',5'-diphosphate; riboflavin 3',4'-diphosphate, and riboflavin 3',5'-diphosphate. The latter compound binds tightly to apoflavodoxin from Megasphaera elsdenii (KD = 9.7 X 10(-9) M). The bound flavin has high catalytic activity, thus representing a novel type of FMN analog. A wide variety of structural analogs of FMN can be obtained in pure form by preparative HPLC.

Apoproteins↗
PubMed1984

Enzymology of the response of the carpal tunnel syndrome to riboflavin and to combined riboflavin and pyridoxine.

Differential enzymic analyses of the erythrocyte glutamic-oxaloacetic transaminase and the erythrocyte glutathione reductase of a patient with a 3-yr history of the carpal tunnel syndrome (CTS) revealed high deficiencies of both vitamin B-6 and riboflavin as based on approximately equal to 30% levels of the specific activities of these enzymes. Riboflavin for 5 months caused nearly complete disappearance of the CTS and caused no change in the specific activity of erythrocyte glutamic-oxaloacetic transaminase. Combined riboflavin and pyridoxine treatment increased (P less than 0.001) the specific activities of erythrocyte glutathione reductase and erythrocyte glutamic-oxaloacetic transaminase to normal levels with total disappearance of the CTS. Objectively, the strength of pinch of both hands increased (P less than 0.001) on treatment with riboflavin and further increased (P less than 0.001) on the combined treatment. For the first time, a significant riboflavin deficiency has been found to be related to CTS. Riboflavin therapy was effective biochemically, subjectively, and objectively, and riboflavin and pyridoxine were even more effective when concomitantly administered.

Aspartate Aminotransferases↗
PubMed1998

Riboflavin and vitamin B-6 intakes and status and biochemical response to riboflavin supplementation in free-living elderly people.

Free-living elderly people aged > or = 65 y were recruited to assess riboflavin and vitamin B-6 intakes and status and the effect of riboflavin supplementation on biochemical indicators of these 2 vitamins. The status of riboflavin (erythrocyte glutathione reductase activation coefficient; EGRAC) and vitamin B-6 (plasma pyridoxal-5'-phosphate; PLP) were determined in a total sample of 92 subjects, from whom dietary intake data were obtained by using the diet history method (n = 83). Although dietary intakes of both vitamins were considered to be adequate according to current reference values, abnormal EGRAC and plasma PLP values were identified in 49% and 38% of subjects, respectively, with 21% having suboptimal status for both nutrients. A subgroup of subjects from the initial sample (n = 45) was assigned in a double-blind manner to receive either 1.6 or 25 mg riboflavin or placebo daily for 12 wk. In those subjects with a baseline EGRAC or plasma PLP value falling outside the currently accepted threshold value for adequacy, low-dose riboflavin supplementation improved status of the limiting nutrient significantly (P<0.0001 and P = 0.020 for EGRAC and plasma PLP responses, respectively). We conclude that a high proportion of healthy elderly people may have suboptimal status for these nutrients despite apparently adequate dietary intakes. Furthermore, we showed that riboflavin supplementation at physiologic doses corrects biochemical abnormalities of not only EGRAC, but also plasma PLP, confirming the biochemical interdependency of these vitamins and suggesting that riboflavin is the limiting nutrient.

Age Factors↗
PubMed2002

Impaired functioning of thermolabile methylenetetrahydrofolate reductase is dependent on riboflavin status: implications for riboflavin requirements.

BACKGROUND: Methylenetetrahydrofolate reductase (MTHFR; EC 1.7.99.5) supplies the folate needed for the metabolism of homocysteine. A reduction in MTHFR activity, as occurs in the homozygous state for the 677C-->T (so-called thermolabile) enzyme variant (TT genotype), is associated with an increase in plasma total homocysteine (tHcy). OBJECTIVE: In vitro studies suggest that the reduced activity of thermolabile MTHFR is due to the inappropriate loss of its riboflavin cofactor. We investigated the hypothesis that MTHFR activity in the TT genotype group is particularly sensitive to riboflavin status. DESIGN: We studied tHcy and relevant B-vitamin status by MTHFR genotype in a cross-sectional study of 286 healthy subjects aged 19-63 y (median: 27 y). The effect of riboflavin status was examined by dividing the sample into tertiles of erythrocyte glutathionine reductase activation coefficient, a functional index of riboflavin status. RESULTS: Lower red blood cell folate (P = 0.0001) and higher tHcy (P = 0.0082) concentrations were found in the TT group than in the heterozygous (CT) or wild-type (CC) groups. However, these expected relations in the total sample were driven by the TT group with the lowest riboflavin status, whose mean tHcy concentration (18.09 micromol/L) was almost twice that of the CC or CT group. By contrast, adequate riboflavin status rendered the TT group neutral with respect to tHcy metabolism. CONCLUSIONS: The high tHcy concentration typically associated with homozygosity for the 677C-->T variant of MTHFR occurs only with poor riboflavin status. This may have important implications for governments considering new fortification policies aimed at the prevention of diseases for which this genotype is associated with increased risk.

Adult↗
PubMed2003

The structure of the N-terminal domain of riboflavin synthase in complex with riboflavin at 2.6A resolution.

Riboflavin synthase of Escherichia coli is a homotrimer with a molecular mass of 70 kDa. The enzyme catalyzes the dismutation of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine, affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The N-terminal segment (residues 1-87) and the C-terminal segment (residues 98-187) form beta-barrels with similar fold and a high degree of sequence similarity. A recombinant peptide comprising amino acid residues 1-97 forms a dimer, which binds riboflavin with high affinity. Here, we report the structure of this construct in complex with riboflavin at 2.6A resolution. It is demonstrated that the complex can serve as a model for ligand-binding in the native enzyme. The structure and riboflavin-binding mode is in excellent agreement with structural information obtained from the native enzyme from Escherichia coli and riboflavin synthase from Schizosaccharomyces pombe. The implications for the binding specificity and the regiospecificity of the catalyzed reaction are discussed.

Amino Acid Sequence↗
PubMed1995

Riboflavin biosynthesis in Saccharomyces cerevisiae. Cloning, characterization, and expression of the RIB5 gene encoding riboflavin synthase.

Saccharomyces cerevisiae has a monofunctional riboflavin synthase that catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine. We have isolated the gene encoding this enzyme from a yeast genomic library by functional complementation of a mutant, rib5-10, lacking riboflavin synthase activity. Deletion of the chromosomal copy of RIB5 led to riboflavin auxotrophy and loss of enzyme activity. Intragenic complementation between point and deletion mutant alleles suggested that the encoded protein (Rib5p) assembles into a multimeric complex and predicted the existence of a discrete functional domain located at the N terminus. Nucleotide sequencing revealed a 714-base pair open reading frame encoding a 25-kDa protein. Rib5p was purified to apparent homogeneity by a simple procedure. The specific activity of the enzyme was enriched 8500-fold. The N-terminal sequence of the purified enzyme was identical to the sequence predicted from the nucleotide sequence of the RIB5 gene. Initial structural characterization of riboflavin synthase by gel filtration chromatography and both nondenaturing pore limit and SDS-polyacrylamide gel electrophoresis showed that the enzyme forms a trimer of identical 25-kDa subunits. The derived amino acid sequence of RIB5 shows extensive homology to the sequences of the alpha subunits of riboflavin synthase from Bacillus subtilis and other prokaryotes. In addition, the sequence also shows internal homology between the N-terminal and the C-terminal halves of the protein. Taken together, these results suggest that the Rib5p subunit contains two structurally related (substrate-binding) but catalytically different (acceptor and donator) domains.

Base Sequence↗
PubMed1978

Biosynthesis of riboflavin in Bacillus subtilis: function and genetic control of the riboflavin synthase complex.

Two riboflavin synthase activities (heavy and light) have been observed in earlier studies with Bacillus subtilis. The heavy enzyme is a complex of one molecule of light enzyme (consisting of three alpha subunits) and approximately 60 beta subunits (A. Bacher, R. Bauer, U. Eggers, H. Harders, and H. Schnepple, p. 729--732, in T. P. Singer (ed.), Flavins and Flavoproteins, Elsevier, Amsterdam, 1976). The formation of alpha and beta subunits is coordinately controlled. Mutants apparently deficient in beta subunits were isolated as riboflavin requires after mutagenesis of B. subtilis with ICR 191. The mutants could grow with diacetyl instead of riboflavin. Growth with diacetyl was associated with the accumulation of substantial amounts of the riboflavin precursor, 6,7-dimethyl-8-(D-ribityl)lumazine. It follows that the mutants are deficient in an enzyme activity required for the formation of the lumazine from the pyrimidine precursor. We conclude that heavy riboflavin synthase is a bifunctional enzyme. The riboflavin synthase activity is mediated by the alpha subunits, whereas the beta subunits are necessary for an earlier biosynthetic step.

Bacillus subtilis↗
PubMed1984

Riboflavin status of adolescents in southern China. Average intake of riboflavin and clinical findings.

The average riboflavin intake of 11 200 schoolchildren, aged 12-19 years, in Guangdong Province, China, was measured. Food intake was measured by weighing, and riboflavin intake was calculated by means of food tables. The average intake of riboflavin was 0.45 mg/day. In addition, clinical observations were made in 1313 adolescents in the dietary survey. The findings were consistent with the low intake of riboflavin. The observed clinical signs of riboflavin deficiency were scrotal dermatitis (7.9% of boys), angular stomatitis (5.8% of boys, 2.7% of girls), cheilosis (8.0% of boys, 5.6% of girls) and magenta tongue (36.0% of boys, 40.8% of girls). Corneal vascularization was found only in two of the 1313 children. Scrotal lesions resolved within three to six days after the oral administration of riboflavin (15 mg/day); the resolution of tongue and lip signs progressed more slowly.

Adolescent↗
PubMed1978

Aldosterone stimulation of riboflavin incorporation into rat renal flavin coenzymes and the effect of inhibition by riboflavin analogues on sodium reabsorption.

This study was designed to investigate a possible relationship between the effect of aldosterone upon urinary electrolytes and the incorporation of [(14)C]riboflavin into renal [(14)C]flavin mononucleotide (FMN) and [(14)C]flavin adenine dinucleotide (FAD). Adrenalectomized Sprague-Dawley rats that weighed between 185 and 210 g were pretreated with 15 mug/100 g body wt dexamethasone intraperitoneally. 16 h later they were administered aldosterone (1.5 mug/100 g body wt) and [(14)C]riboflavin (5.0 muCi/200 g body wt). The urethra of each rat was ligated, and the rats were sacrificed by decapitation 3 h later. The urine was aspirated from the bladders of each rat and analyzed for total Na(+) and K(+) excretion while the kidneys were removed and the formation of [(14)C]FMN and [(14)C]FAD was determined for each kidney. There was a significant increase in the formation of renal [(14)C]FMN and [(14)C]FAD (27.3 and 14.4%, respectively) after aldosterone treatment. Aldosterone significantly decreased the excretion of Na(+) by 50%, and increased that of K(+) by 55%. To determine if the increased incorporation of [(14)C]riboflavin into renal [(14)C]FMN and [(14)C]FAD was an important intermediary step in the aldosterone-induced alterations in urinary Na(+) and K(+), the riboflavin analogues 7,8-dimethyl-10-formylmethyl isoalloxazine or 7,8-dimethyl-10-(2'-hydroxyethyl) isoalloxazine were given to the animals i.p. to diminish the conversion of riboflavin to FMN by competitively inhibiting the enzyme flavokinase (EC 2.7.1.26). These analogues were found to significantly counteract the decreased urinary excretion of Na(+) as a result of aldosterone from 26+/-9% to 124+/-58% (SEM) with a dose-related response when administered from 10 to 25 mug/100 g body wt. The same doses of the analogues that significantly increased the urinary output of Na(+) when administered simultaneously with aldosterone also significantly decreased the formation of renal [(14)C]FMN from 15+/-4 to 38+/-3% when compared with the effects of aldosterone alone. The analogues exerted no significant effect on the increased urinary excretion of K(+) by aldosterone. The analogues alone had no influence on urinary Na(+) and K(+) output or the formation of renal [(14)C]FMN and [(14)C]FAD at the dose levels that we investigated. These data strongly suggest that the enhanced synthesis of renal FMN and FAD may be a causative factor in the increased reabsorption of Na(+) as a result of aldosterone; and, consequently, riboflavin analogues may function as a novel class of antimineralocorticoids.

Aldosterone↗
PubMed1988

Enhancement of adriamycin-induced mortality during riboflavin administration and riboflavin deficiency in rats.

Adriamycin-treated rats were monitored for survivorship while consuming a normal diet adequate in riboflavin, a normal diet and receiving daily high-dose injections of riboflavin-5'-phosphate (flavin mononucleotide, FMN), or a riboflavin-deficient diet. Each animal was compared to a corresponding pair-fed, saline-treated control. In Adriamycin-treated rats fed the normal chow diet alone, survivorship declined within 7 days and remained constant after 12 days to about 50%. Adriamycin-treated rats consuming the normal diet and injected with FMN initially showed similar survivorship; however, after 20 days survival fell to 14%. Adriamycin-treated, riboflavin-deficient rats showed within 5 days a precipitous decline in survivorship which leveled to 5%. These results suggest that during Adriamycin treatment, proper riboflavin nutriture may be a crucial determinant of survival.

Animals↗
PubMed2006

Biomass recycling from a riboflavin cultivation with B. subtilis: lysis, extract production and testing as substrate in riboflavin cultivation.

Autolysis of riboflavin-producing B. subtilis can be induced by pH, lack of carbon source, and the buffer system. Stress factors like temperature shift or oxygen dearth enhance the autolysis process. After cultivation of a riboflavin-producing strain, the pH of the whole culture broth was adjusted to 6.5-7.5. At a temperature of 40 degrees C, autolysis started after 1 h. Adding a defined amount of commercially available endo- and exo-proteases enhanced both auto- and proteo-lysis. Optimization of endo- and exo-protease concentrations and of the time increased the degree of proteolysis. Additionally, the amount of DNA and Protein trapped in the riboflavin crystals could be significantly reduced by autolysis. After autolysis, the cultivation broth was centrifuged and the supernatant was cross-flow filtrated with a cut off of 10 kDa. Using this autolysate instead of yeast extract as a medium component for riboflavin production with B. subtilis, a riboflavin yield of 77% was obtained in comparison with the standard cultivation on yeast extract.

Bacillus subtilis↗
PubMed2005

Determination of riboflavin by high-performance liquid chromatography with riboflavin-depleted urine as calibration and control matrix.

A simple method, exposure to natural-light, was developed to remove riboflavin from urine to enhance its use as the biological matrix for the preparation of calibration and control samples. Riboflavin-depleted urine containing less than 1 ng/ml of riboflavin was used to validate a high-performance liquid chromatography with fluorescence detection method for the determination of urinary riboflavin. The linearity of the assay (r2=0.999) was acceptable over the range of 10-5000 ng/ml. The intra-assay and inter-assay CVs were 3.3% and 9%, respectively. Subsequent stability studies found that urine riboflavin was stable for up to 6 months at 4 or -20 degrees C.

Adult↗
PubMed2003

Crystal structure of Schizosaccharomyces pombe riboflavin kinase reveals a novel ATP and riboflavin-binding fold.

The essential redox cofactors riboflavin monophosphate (FMN) and flavin adenine dinucleotide (FAD) are synthesised from their precursor, riboflavin, in sequential reactions by the metal-dependent riboflavin kinase and FAD synthetase. Here, we describe the 1.6A crystal structure of the Schizosaccharomyces pombe riboflavin kinase. The enzyme represents a novel family of phosphoryl transferring enzymes. It is a monomer comprising a central beta-barrel clasped on one side by two C-terminal helices that display an L-like shape. The opposite side of the beta-barrel serves as a platform for substrate binding as demonstrated by complexes with ADP and FMN. Formation of the ATP-binding site requires significant rearrangements in a short alpha-helix as compared to the substrate free form. The diphosphate moiety of ADP is covered by the glycine-rich flap I formed from parts of this alpha-helix. In contrast, no significant changes are observed upon binding of riboflavin. The ribityl side-chain might be covered by a rather flexible flap II. The unusual metal-binding site involves, in addition to the ADP phosphates, only the strictly conserved Thr45. This may explain the preference for zinc observed in vitro.

Adenosine Diphosphate↗
PubMed1981

Further studies of the riboflavin-binding immunoglobulin IgGGar. Resolution into fractions of different riboflavin content and aspects of reassembly.

A previously described human immunoglobulin with unusual flavin-binding activity, IgGGar [Farhangi, M., & Osserman, E. F. (1976) N. Engl. J. Med. 294, 177], is further characterized. The protein can be fractionated into two subpopulations, one of which is nearly completely saturated with riboflavin and one in which the binding sites are largely vacant. Possible differences between these fractions and/or their binding sites are explored. While electrophoretically distinct, the IgGGar-riboflavin complexes possess a basic similarity in the binding sites of both fractions as evidenced by spectroscopic examination. However, an important difference exists in that added riboflavin equilibrates reversibly with the vacant sites of native IgGGar, while the riboflavin in the occupied sites is essentially irreversibly bound. The tight association may be due to an in vivo combination of riboflavin with protein of different conformation than occurs in vitro, such as an incompletely assembled or folded tetramer. Accordingly, in vitro renaturation was examined. Studies of renaturation revealed that the reduced interchain disulfides within a tetramer reoxidize smoothly, although inter-heavy-chain bonds form less readily than inter-heavy-light-chain disulfides. Renaturation of IgGGar, unlike previously studied IgG molecules, does not proceed under conditions in which the protein structure had previously been significantly disrupted. The assembly defect is localized in the inability of the denatured heavy chain to refold into a stable species capable of combining with the light chain.

Binding Sites↗
PubMedDate not supplied

A novel rhodamine-riboflavin conjugate probe exhibits distinct fluorescence resonance energy transfer that enables riboflavin trafficking and subcellular localization studies.

Riboflavin (vitamin B2, RF) is taken up in eukaryotic cells via specialized transport mechanisms. Although RF has fluorescence properties, direct microscopic visualization of RF uptake and trafficking has been complicated by cellular autofluorescence. We describe the synthesis, cellular uptake characteristics, and spectroscopic properties of a novel rhodamine-riboflavin conjugate (RD-RF), including absorption and emission spectra, two-photon excitation spectra, and fluorescence pH dependence. The conjugate has a molar extinction coefficient of 23 670 M(-1) cm(-1) at 545 nm (excitation wavelength) with a fluorescence quantum yield of 0.94. This compound exhibits intramolecular fluorescence resonance energy transfer (FRET). Selective quenching of the FRET signal is observed when RD-RF is bound with high affinity by the chicken riboflavin carrier protein. In addition to the typical rhodamine excitation and emission, FRET provides a secondary signal for conjugate localization and an in situ mechanism for observing riboflavin binding. Solution and in vitro stability determinations indicate that the linkage between riboflavin and rhodamine is stable for the duration of typical pulse--chase and cellular trafficking experiments. The distinct spectroscopic properties of RD-RF together with a comparable affinity for RF-binding proteins render it an excellent tool for the study of RF transport and trafficking in living cells.

Cell Compartmentation↗
PubMed2004

Effects of riboflavin on 6,7-dimethyl-8-ribityllumazine formation in growing cells of a riboflavin-adenine-deficient mutant, Bacillus subtilis.

The effects of riboflavin on the formation of 6,7-dimethyl-8-ribityllumazine (DMRL) were examined using the growing cells of a riboflavin, adenine deficient-mutant, Bacillus subtilis. The administration of low concentrations (50-500 ng/mL) of riboflavin to the basal medium significantly inhibited the formation of DMRL without any change in growth. Especially, the supplementation of more than 250 ng/mL riboflavin completely restricted the formation of DMRL under the experimental conditions. The results clearly indicate that a rigid negative feedback inhibition is operative for the biosynthetic pathway of riboflavin in B. subtilis.

Adenine↗
PubMed1978

[Operon of riboflavin biosynthesis in Bacillus subtilis. XV. A study of mutants related to the initial stages of biosynthesis. The origin of the ribityl chain of the riboflavin molecule].

The incorporation of 14C-labelled guanosine and xanthosine into riboflavin was studied. It is concluded that the ribose mojety of guanosine is converted to the ribityl side chain of riboflavin. Thus the immediate precursor of riboflavin biosynthesis is a guanosine compound. Two classes of the riboflavin-dependent mutants of Bacillus subtilis were studied. They are closely linked to the lysine markers and probably correspond to the initial steps of riboflavin biosynthesis pathway.

Bacillus subtilis↗
PubMedDate not supplied

[Riboflavin transport in cells of riboflavin-dependent yeast mutants].

Riboflavin was transported at a high rate into yeast cells of Pichia guilliermondii and Schwanniomyces occidentalis mutants capable of growth in a medium containing low concentrations of riboflavin, and having multiple susceptibility to some antibiotics and antimetabolites. Sucrose and sodium azide inhibited transport of riboflavin. Other riboflavin dependent mutants of Pichia guilliermondii, Pichia ohmeri, Torulopsis candida, and Saccharomyces cerevisiae, also growing in media containing low concentrations of riboflavin, were not capable of its active transport.

Biological Transport, Active↗