Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The chemical and enzymatic properties of 26 analogues of riboflavin are presented. These analogues include both endo- and exocyclically substituted isoalloxazines with redox potentials from -370 to -128 mV. Physical and chemical data such as the electronic absorption spectra, pKas, and redox potentials of the analogues are presented and are discussed with respect to preferred tautomeric and resonance forms. Like riboflavin, most of the analogues are shown to be catalytic oxidants of dihydro-5-deazaflavins. Analogue binding to egg white binding apoprotein has been quantitated and serves to determine the origins of binding site specificity for this protein. Nearly all of the analogues that possess D-ribityl groups are found to be processed to the FAD level by the flavokinase/FAD synthetase system of Brevibacterium ammoniagenes. Most extensively studied are the reactivities of the analogues with the NAD(P)H:flavin oxidoreductase of Beneckea harveyi. Many of the analogues are substrates in this enzymatic redox reaction, and a linear free energy-rate relation (log Vmax vs. E0' of the analogue) is seen that parallels similar relationships in the nonenzymatic oxidation of dihydro-5-deazaflavins. This suggests a common mechanism for the reactions of such diverse flavins as riboflavin, 5-deazariboflavin, and 1-deazariboflavin.
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PMID:Chemical and enzymatic properties of riboflavin analogues. 20 4

A flavokinase preparation from Bacillus subtilis is described which catalyzes the phosphorylation of reduced, but not oxidized, riboflavin. The enzyme is distinguished from other known flavokinases also in having an unusually low Km for the flavin substrate, 50 to 100 nM. ATP is the obligatory phosphate donor; one ATP is utilized for each FMNH2 formed. Mg2+ or Zn2+ is required for the reaction; Co2+ and Mn2+ will substitute, but less effectively. The same enzyme preparation catalyzes the synthesis of FADH2 from FMNH2 and ATP, but not the synthesis of FAD from FMN and ATP. FADH2 is also formed from reduced riboflavin, presumably by sequential flavokinase and FAD synthetase action. Zn2+ cannot replace Mg2+ in FADH2 formation. The reverse reaction, formation of FMN from FAD, occurs only with reduced FAD, giving rise to FMNH2, and is dependent on the presence of inorganic pyrophosphate. The enzyme thus appears to be an FADH2 pyrophosphorylase. The two enzymatic activities, flavokinase and FADH2 pyrophosphorylase, although not separated during the purification procedure, are distinguished by differences in metal ion specificity, in concentration dependence for ATP (apparent Km for ATP = 300 microM for FADH2 synthesis and 6.5 microM for flavokinase), and in the inhibitory effects of riboflavin analogues.
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PMID:Flavokinase and FAD synthetase from Bacillus subtilis specific for reduced flavins. 22 20

A steady-state kinetic analysis with evaluation of product inhibition was accomplished with purified rat liver flavokinase and FAD synthetase. For flavokinase, Km values were calculated as approximately 11 microM for riboflavin and 3.7 microM for ATP. Ki values were calculated for FMN as 6 microM against riboflavin and for ZnADP as 120 microM against riboflavin and 23 microM against ZnATP. From the inhibition pattern, the flavokinase reaction followed an ordered bi bi mechanism in which riboflavin binds first followed by ATP; ADP is released first followed by FMN. For FAD synthetase, Km values were calculated as 9.1 microM for FMN and 71 microM for MgATP. Ki values were calculated for FAD as 0.75 microM against FMN and 1.3 microM against MgATP and for pyrophosphate as 66 microM against FMN. The product inhibition pattern suggests the FAD synthetase reaction also followed an ordered bi bi mechanism in which ATP binds to enzyme prior to FMN, and pyrophosphate is released from enzyme before FAD. Comparison of Ki values with physiological concentrations of FMN and FAD suggests that the biosynthesis of FAD is most likely regulated by this coenzyme as product at the stage of the FAD synthetase reaction.
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PMID:Probable reaction mechanisms of flavokinase and FAD synthetase from rat liver. 215 58

Substrate specificity and product inhibition have been evaluated by using purified rat liver FAD synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2), obtained by an improved purification protocol with optimized flavin affinity chromatography. FMN analogues studied fall into three general classifications: those with substitution on the pyrimidinoid ring and nitrogen replacement, those with substitution on the benzenoid ring, and those with N(10) side chain modifications. Substitutions on the pyrimidinoid ring and replacement of nitrogens have the greatest influence on binding to enzyme and FAD formation. When the hydrogen-bonding capacity of the NH group at position 3 is blocked or removed by substitution, such FMN analogues do not act as substrates or inhibitors of the enzyme. Substitutions on the benzenoid ring by small groups seem to be tolerated, while larger groups inhibit binding. Length of the N(10) side chain is optimal with five carbons and has greatest affinity for the natural ribityl side chain. Affinity matrices show similar binding characteristics in that the N(3)-(carboxymethyl)riboflavin-agarose does not bind enzyme, while agaroses linked to the flavin N(10) side chain provide varying degrees of purification. The C = O group at position 2, the NH group at position 3, and a five-carbon side chain at the N(10) position seem to be most crucial for flavin substrate binding to enzyme. Nucleoside triphosphates other than ATP do not act as substrates or inhibitors when sufficient Mg2+ is present. Products of the reaction, FAD and PPi, act as inhibitors against both ATP and FMN.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Substrate specificity and variables affecting efficiency of mammalian flavin adenine dinucleotide synthetase. 255 3

The effect of riboflavin status on hepatic activities of the relatively substrate-specific flavokinase and FAD synthetase and the relatively nonspecific FMN phosphatase and FAD pyrophosphatase was investigated in weanling, male Sprague-Dawley rats fed 0, 5, 15, 30 and 60 micrograms riboflavin/15 g diet for 1, 3 and 5 weeks. Flavokinase activity was determined by using [14C]riboflavin and ATP as substrates and measuring product [14C]FMN after incubation and separation by high performance liquid chromatography. Similarly, FAD synthetase activity was determined by using [3H]ATP and FMN and quantitating [3H]FAD formed. Flavokinase activities among all groups were similar after only 1 week of feeding experimental diets; by 3 weeks, activities were depressed to about 60% of normal in animals that received suboptimal riboflavin; by 5 weeks, activity of rats fed riboflavin-free diet was further decreased to about 40% of normal. FAD synthetase activities were unaffected by riboflavin status at 1 and 3 weeks; however, at 5 weeks, activities were moderately decreased to 85, 65 and 52% of normal with rats which had received 15, 5 and 0 microgram riboflavin/15 g diet, respectively. FMN phosphatase and FAD pyrophosphatase activities decreased with age, but were not influenced by riboflavin status at any period. Overall results indicate the effect of increasing severity of riboflavin deficiency is greater with flavokinase, which is physiologically rate-limiting in the biosynthesis of flavocoenzymes, than with FAD synthetase.
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PMID:Effect of riboflavin status on hepatic activities of flavin-metabolizing enzymes in rats. 613 98

Three flavin derivatives modified at the 2'-position of the flavin N-10 ribityl side chain were synthesized: arabinoflavin, 2'-F-2'-deoxyarabinoflavin, and 2'-deoxyriboflavin. These were converted to the FAD level with FAD synthetase. Apoproteins of lipoamide dehydrogenase, glutathione reductase, and mercuric reductase, a family of flavoprotein oxidoreductases, were reconstituted with these flavins. Significant reduction of the catalytic activities was observed with the modified enzymes. During anaerobic reduction of the modified enzymes with substrate or dithiothreitol, decreased thermodynamic stability of the two-electron reduced enzyme forms (EH2) and the accumulation of the four-electron reduced forms (EH4) noted. This effect was more pronounced in case of arabino-FAD-reconstituted enzymes than with the other two. It was found that NAD+ binding influences the interaction between the flavin and the reduced disulfide in the 2'-F-arabino-FAD-lipoamide dehydrogenase, presumably by altering the relative oxidation-reduction potentials. 19F NMR data were obtained for different forms of the 2'-F-arabino-FAD-lipoamide dehydrogenase, which suggest marked conformational changes from one form to the other. The 19F NMR data for the oxidized forms of all three 2'-F-arabino-FAD proteins suggest that the fluorine experiences very similar chemical environments at the active sites.
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PMID:Chemical modification of the N-10 ribityl side chain of flavins. Effects on properties of flavoprotein disulfide oxidoreductases. 749 74

The cloning of a bifunctional FAD synthetase gene, which shows flavokinase and FMN adenylyltransferase activities, from Corynebacterium ammoniagenes was tried by hybridization with synthetic DNAs corresponding to the N-terminal amino acid sequence. The cloned PstI-digested 4.4 x 10(3)-base (4.4-kb) fragment could not express the FAD synthetase activity in E. coli, but could increase the two activities by the same factor of about 20 in C. ammoniagenes. The FAD-synthetase-gene-amplified C. ammoniagenes cells were applied to the production of FAD from FMN or riboflavin. The productivity of FAD from FMN was increased four to five times compared with the parent strain, and reached a 90% molar yield. The productivity of FAD from riboflavin was increased about eight times, with a 50% molar yield. The addition of Zn2+ to the reaction mixtures for the conversion from riboflavin to FAD brought about the specific inhibition of adenylyl-transferase activity and resulted in the accumulation of FMN.
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PMID:Cloning of FAD synthetase gene from Corynebacterium ammoniagenes and its application to FAD and FMN production. 776 13

Respiratory defective mutants of Saccharomyces cerevisiae previously assigned to complementation group G178 are characterized by an abnormally low ratio of FAD/FMN in mitochondria. A nuclear gene, designated FLX1, was selected from a yeast genomic library, based on its ability to confer wild-type growth properties to a representative G178 mutant. Genetic evidence has confirmed that the flavin nucleotide imbalance of G178 mutants is caused by mutations in FLX1. The sequence of FLX1 is identical to a reading frame recently reported to be present on yeast chromosome IX (GenBank Z47047). The sequence and tripartite repeat structure of the FLX1 product (Flx1p) indicate it is a member of a protein family consisting of mitochondrial substrate and nucleotide carriers. In yeast, FAD synthetase is present in the soluble cytoplasmic protein fraction but not in mitochondria. Riboflavin kinase, the preceding enzyme in flavin biosynthesis, is present in both subcellular fractions. The absence of FAD synthetase in mitochondria implies that FAD is imported from the cytoplasm. The lower concentration of mitochondrial FAD in flx1 mutants suggests that Flx1p is involved in flavin transport, a role that is also supported by biochemical evidence indicating more efficient flux of FAD across mitochondrial membrane vesicles prepared from wild-type strains than membrane vesicles from flx1 mutants.
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PMID:FLX1 codes for a carrier protein involved in maintaining a proper balance of flavin nucleotides in yeast mitochondria. 863 63

Previous control studies carried out in children showed that respiratory infection alters riboflavin metabolism and leads to excessive urinary losses of the vitamin. In order to understand the nature of biochemical changes in riboflavin metabolism during respiratory infection, a study was carried out using the mouse as the experimental model, and Klebsiella pneumoniae as the infective organism. Mice were fed on either a low (0.5 mg/kg)- or high (13.3 mg/kg)-riboflavin semi-synthetic diet. Infection resulted in a 5-6-fold higher excretion of riboflavin in the urine of mice fed on the low-riboflavin diet. Higher erythrocyte FAD levels and lower liver FAD levels were also observed during infection. Of the four enzymes involved in the synthesis and breakdown of the flavin coenzymes studied, the activity of hepatic flavokinase (ATP: riboflavin 5'-phosphotransferase; EC 2.7.1.26) was significantly lower, and that of FAD synthetase (ATP: FMN adenylyltransferase; EC 2.7.7.2) was higher during riboflavin restriction and infection. The activity of FMN (acid) phosphatase (EC 3.1.3.2) was unchanged, whereas FAD (nucleotide) pyrophosphatase (EC 3.6.1.9) activity was significantly higher both with the low-riboflavin diet and during infection. Thyroid hormone is known to modulate flavokinase activity and, hence, thyroid status was assessed. Plasma triiodothyronine (T3) levels were not affected, but thyroxine levels were lower in the mice fed on the low-riboflavin diet. However, plasma T3 was significantly lower during infection, suggesting a mechanistic role for the hormone in the reduction of flavokinase activity.
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PMID:Flavin metabolism during respiratory infection in mice. 888 17

A high potential analog of riboflavin with a cyano function at the 8-position was synthesized by employing novel reaction conditions, starting from 8-amino-riboflavin. This was converted to the FAD level with FAD synthetase. The reduced 8-CN-riboflavin, unlike normal reduced flavin, has a distinctive absorption spectrum with two distinctive peaks in the near ultraviolet region. The oxidation-reduction potential of the new flavin was determined to be -50 mV, approximately 160 mV more positive than that of normal riboflavin. The 8-CN-riboflavin and 8-CN-FMN were found to be photoreactive and need to be protected from exposure to light. However such complications were not encountered with protein-bound flavins. The apoproteins of flavodoxin and Old Yellow Enzyme (OYE) were reconstituted with the 8-CN-FMN and apoDAAO was reconstituted with 8-CN-FAD. Spectral properties of the enzyme-bound neutral and anionic semiquinones were determined from these reconstituted proteins. In the case of 8-CN-FMN-OYE I, it was shown that the comproportionation reaction of a mixture of reduced and oxidized enzyme bound flavin is very rapid, compared with the same reaction with native protein, resulting in approximately 100% thermodynamically stable anionic semiquinone. In the case of 8-CN-OYE I, it was shown that the rate of reduction of the enzyme bound flavin by NADPH is approximately 40 times faster, and the rate of reoxidation of reduced enzyme bound flavin by oxygen is an order of magnitude slower than with the normal FMN enzyme. This is in accord with the high oxidation-reduction potential of the flavin, which thermodynamically stabilizes the reduced enzyme.
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PMID:Synthesis and properties of 8-CN-flavin nucleotide analogs and studies with flavoproteins. 953 83


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