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PubMed2005

Chromophore exchange in the LOV2 domain of the plant photoreceptor phototropin1 from oat.

Phototropins are a family of plant photoreceptors mediating blue light responses such as phototropism, leaf expansion, chloroplast relocation, and stomatal opening. Characteristic for phototropins are two LOV domains which, when expressed in heterologous systems, each carry a single flavin mononucleotide (FMN) chromophore. Here we describe removal of FMN from the LOV2 domain of Avena sativa using a hydrophobic matrix and successful incorporation of flavin adenine dinucleotide (FAD), riboflavin, and 5'-malonyl-riboflavin into the resulting apoprotein; 5-deaza-FMN was not incorporated under the applied conditions. The chromoproteins reconstituted with the various flavins showed absorption spectra and photocycle almost identical to those of the native LOV2 domain and that reconstituted with FMN except for the kinetics: LOV2-riboflavin and LOV2-5'-malonyl-riboflavin showed more rapid regeneration in the dark. LOV2-FAD can be hydrolyzed to LOV2-FMN with phosphodiesterase, indicating that the adenosine part extrudes from the protein. Together with the data from the X-ray structure (Crosson, S., and Moffat, K. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 2995-3000), the results allow us to decide which of the chromophore-protein interactions are essential for the reconstitution process.

Avena↗
PubMed1972

Purification and properties of the flavine-stimulated anaerobic L- -glycerophosphate dehydrogenase of Escherichia coli.

The anaerobic l-alpha-glycerophosphate (l-alpha-GP) dehydrogenase of Escherichia coli was purified approximately 40-fold. The activity of the dehydrogenase, although not affected by the addition of pyridine nucleotides, was stimulated three- to fourfold by flavine adenine dinucleotide (K(m) about 10(-7)m) and up to 10-fold by flavine mononucleotide (K(m) about 10(-4)m). Maximal activity of the enzyme was found only in the combined presence of saturating concentrations of both flavines (stimulation by a factor of 10 to 15). The dependence of the rate of the reaction on the concentration of l-alpha-GP was complex in the presence of both flavines, but in the presence of flavine adenine dinucleotide alone the kinetics were of the Michaelis-Menten type with the K(m) for l-alpha-GP being about 10(-4)m. The product of the reaction was identified as dihydroxyacetone phosphate, and the molecular weight of the dehydrogenase was estimated to be 80,000 +/- 10,000. Phenazine methosulfate, menadione and ferricyanide served as artificial acceptors for the dehydrogenase. The enzyme was sensitive to iodoacetate, p-chloromercuribenzoate, and N-ethymaleimide.

Ammonium Sulfate↗
PubMed1999

Debromination of (alpha-bromoiso-valeryl)urea catalysed by rat blood.

(Alpha-bromoiso-valeryl) urea, a sedative or hypnotic, is metabolized to (3-methylbutyryl)urea by reductive debromination. This study was designed to evaluate the role of blood in the debromination of (alpha-bromoiso-valeryl) urea. Rat blood containing an electron donor had significant debrominating activity toward (alpha-bromoiso-valeryl)urea. This debromination proceeded by enzymatic and non-enzymatic processes which required both NADH (or NADPH) and flavin mononucleotide (FMN), under anaerobic conditions. The debrominating activity was sensitive to inhibition by carbon monoxide, and the pH optimum was 8.5. When FMN was replaced by flavin adenine dinucleotide (FAD) or riboflavin, similar results were obtained. The optimum concentration of flavins was 10(-4) M. The reductive debromination was also mediated by rat erythrocytes, but not by plasma. When the blood or erythrocytes were boiled, the debrominating activity was not abolished, but was enhanced, suggesting that the activity arises from the haemoglobin in erythrocytes, and haemoglobin had debrominating activity when supplemented with both a reduced pyridine nucleotide and a flavin. Furthermore, haematin had significant debrominating activity in the presence of these cofactors. The activity of haematin was also observed with the photochemically reduced form of FMN. The results imply that the debromination proceeds in two steps--enzymatic or non-enzymatic reduction of a flavin such as FAD, FMN or riboflavin by NADPH or NADH, then non-enzymatic reductive debromination of (alpha-bromoiso-valeryl)urea to (3-methylbutyryl)urea catalysed by the haem group of rat haemoglobin in the presence of the reduced flavin.

Animals↗
PubMed1995

Structural refinement of the non-fluorescent flavoprotein from Photobacterium leiognathi at 1.60 A resolution.

The crystallographically-determined structure of the non-fluorescent flavoprotein (NFP) from Photobacterium leiognathi, a homolog of the bacterial luciferase subunits, has been refined to a conventional R-factor [formula: see text] of 0.175 using synchrotron data between 10.0 and 1.60 A resolution. The molecular structure is a homodimer of beta/alpha domains, the monomer having structural similarities to (beta alpha)8 barrel proteins. However, one beta-strand and three alpha-helices of a typical (beta alpha)8 domain are not present in the NFP structure. The refined structure of NFP consists of the 228 amino acid polypeptide, 191 water molecules, a sulfate ion, and two flavin mononucleotides (FMNs) each with a covalently-attached myristate (C14 fatty acid). Both flavin adducts are well-ordered and have exceptional electron density for both the FMN and the myristate moieties. Each flavin mononucleotide-myristate adduct is characterized by a stereospecific linkage (the S enantiomer) between C-6 of the flavin isoalloxazine ring and the C-3' atom of the fatty acyl chain. The stereospecific nature of this flavin-fatty acid linkage suggests that it is the result of an enzyme-catalyzed reaction, most likely the bioluminescence reaction itself. The myristate chains are buried from solvent in hydrophobic pockets in the interior of the protein. Four amino acid side-chains of the NFP polypeptide have been modeled with alternate conformations. Five of the protein's seven alpha-helices have classical C-capping boxes. NFP is dimeric and many of the extensive contacts at the dimer interface are mediated by hydrogen-bonded water molecules as well as by hydrophobic interactions. One of the myristate acyl chains sits between NFP monomers and contributes a significant portion of the hydrophobic interactions at the NFP dimer interface.

Crystallography↗
PubMedDate not supplied

[Enzymes involved in the metabolism of flavin nucleotides in Streptomyces olivaceus].

The work was aimed at studying enzymes involved in the metabolism of flavin nucleotides, namely, riboflavin kinase (EC 2.7.1.26) and FAD pyrophosphorylase (EC 2.7.7.2), as well as flavin mononucleotide hydrolysis by acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) in Streptomyces olivaceus actively producing vitamin B12. No correlation could be established between changes in the activity of the above enzymes during the culture growth and the qualitative composition of flavins. The enzyme activity was assayed using, as an enzyme preparation, both intact cells and a cell-free extract obtained by disintegrating the mycelium with different techniques. The screening effect of phosphatases exerted when the activity of riboflavin kinase was assayed could be partly eliminated by adding sodium fluoride to the incubation medium. The localisation of the above enzymes in the cytoplasm is discussed.

Acid Phosphatase↗
PubMed2004

Biochemical characterization of StyAB from Pseudomonas sp. strain VLB120 as a two-component flavin-diffusible monooxygenase.

Pseudomonas sp. VLB120 uses styrene as a sole source of carbon and energy. The first step in this metabolic pathway is catalyzed by an oxygenase (StyA) and a NADH-flavin oxidoreductase (StyB). Both components have been isolated from wild-type Pseudomonas strain VLB120 as well as from recombinant Escherichia coli. StyA from both sources is a dimer, with a subunit size of 47 kDa, and catalyzes the enantioselective epoxidation of CC double bonds. Styrene is exclusively converted to S-styrene oxide with a specific activity of 2.1 U mg(-1) (k(cat) = 1.6 s(-1)) and K(m) values for styrene of 0.45 +/- 0.05 mM (wild type) and 0.38 +/- 0.09 mM (recombinant). The epoxidation reaction depends on the presence of a NADH-flavin adenine dinucleotide (NADH-FAD) oxidoreductase for the supply of reduced FAD. StyB is a dimer with a molecular mass of 18 kDa and a NADH oxidation activity of 200 U mg(-1) (k(cat) [NADH] = 60 s(-1)). Steady-state kinetics determined for StyB indicate a mechanism of sequential binding of NADH and flavin to StyB. This enzyme reduces FAD as well as flavin mononucleotide and riboflavin. The NADH oxidation activity does not depend on the presence of StyA. During the epoxidation reaction, no formation of a complex of StyA and StyB has been observed, suggesting that electron transport between reductase and oxygenase occurs via a diffusing flavin.

Cloning, Molecular↗
PubMed1992

The primary structures of the flavodoxins from two strains of Desulfovibrio gigas. Cloning and nucleotide sequence of the structural genes.

The structural genes coding for the flavodoxin proteins from two different strains of the sulfate-reducing bacteria Desulfovibrio gigas (ATCC 19364/NCIB 9332 and ATCC 29494/DSM 496) have been identified, cloned and the nucleotide sequence established. The protein sequences derived from the gene from each strain share a sequence identity of 66% with regions directly involved in binding the flavin mononucleotide cofactor being the most homologous. Both aromatic residues that flank the flavin isoalloxazine ring in the crystal structure of the flavodoxin from D. vulgaris, i.e., Trp-60 and Tyr-98, are also present in these flavodoxin proteins. These observations stand in contrast to reports that the flavodoxin from Desulfovibrio gigas contains a single tryptophan residue which is located distant from the flavin binding site. Therefore, the FMN binding site of this flavodoxin is not distinct from the other Desulfovibrio flavodoxins in this regard.

Amino Acid Sequence↗
PubMed2002

Structures of nitroreductase in three states: effects of inhibitor binding and reduction.

The crystal structure of the nitroreductase enzyme from Enterobacter cloacae has been determined for the oxidized form in separate complexes with benzoate and acetate inhibitors and for the two-electron reduced form. Nitroreductase is a member of a group of enzymes that reduce a broad range of nitroaromatic compounds and has potential uses in chemotherapy and bioremediation. The monomers of the nitroreductase dimer adopt an alpha+beta fold and together bind two flavin mononucleotide prosthetic groups at the dimer interface. In the oxidized enzyme, the flavin ring system adopts a strongly bent (16 degrees ) conformation, and the bend increases (25 degrees ) in the reduced form of the enzyme, roughly the conformation predicted for reduced flavin free in solution. Because free oxidized flavin is planar, the induced bend in the oxidized enzyme may favor reduction, and it may also account for the characteristic inability of the enzyme to stabilize the one electron-reduced semiquinone flavin, which is also planar. Both inhibitors bind over the pyrimidine and central rings of the flavin in partially overlapping sites. Comparison of the two inhibitor complexes shows that a portion of helix H6 can flex to accommodate the differently sized inhibitors suggesting a mechanism for accommodating varied substrates.

Crystallography, X-Ray↗
PubMed1968

Microbial degradation of corrinoids. VI. Reduction of hydroxocobalamin by cell-free particles from Pseudomonas rubescens.

Cell-free particles from Pseudomonas rubescens have been shown to reduce hydroxocobalamin to vitamin B(12r). The particles are unable to reduce the B(12r) to B(12s). The reduction of hydroxocobalamin is dependent upon reduced nicotinamide adenine dinucleotide and is stimulated by flavin adenine dinucleotide. Cobinamide and diaquocobinamide were reduced at 25 and 10%, respectively, of the rate of hydroxocobalamin. Cyanocobalamin, coenzyme B(12), pseudovitamin B(12), and diaquopseudocobalamin were not reduced. Reduced nicotinamide adenine dinucleotide phosphate and flavin mononucleotide were not active. Diaphorase and xanthine oxidase activity were not present in the particulate fraction.

Cell-Free System↗
PubMed2002

White collar-1, a DNA binding transcription factor and a light sensor.

Blue light regulates many physiological processes in fungi, but their photoreceptors are not known. In Neurospora crassa, all light responses depend on the Per-Arnt-Sim (PAS) domain-containing transcription factor white collar-1 (wc-1). By removing the WC-1 light, oxygen, or voltage domain, a specialized PAS domain that binds flavin mononucleotide in plant phototropins, we show that light responses are abolished, including light entrainment of the circadian clock. However, the WC-1-mediated dark activation of frq remains normal in this mutant, and the circadian clock can be entrained by temperature. Furthermore, we demonstrate that the purified Neurospora WC-1-WC-2 protein complex is associated with stoichiometric amounts of the chromophore flavin-adenine dinucleotide. Together, these observations suggest that WC-1 is the blue-light photoreceptor for the circadian clock and other light responses in Neurospora.

Amino Acid Sequence↗
PubMed1997

Differential stabilization of the three FMN redox forms by tyrosine 94 and tryptophan 57 in flavodoxin from Anabaena and its influence on the redox potentials.

Flavodoxins are electron transfer proteins that carry a noncovalently bound flavin mononucleotide molecule as the redox-active center. The redox potentials of the flavin nucleotide are profoundly altered upon interaction with the protein. In Anabaena flavodoxin, as in many flavodoxins, the flavin is sandwiched between two aromatic residues (Trp57 and Tyr94) thought to be implicated in the alteration of the redox potentials. We have individually replaced these two residues by each of the other aromatic residues, by alanine and by leucine. For each mutant, we have determined the redox potentials and the binding energies of the oxidized FMN--apoflavodoxin complexes. From these data, the binding energies of the semireduced and reduced complexes have been calculated. Comparison of the binding energies of wild-type and mutant flavodoxins at the three redox states suggests that the interaction between Tyr94 and FMN stabilizes the apoflavodoxin--FMN complex in all redox states. The oxidized and semireduced complexes are, however, more strongly stabilized than the reduced complex, making the semiquinone/hydroquinone midpoint potential more negative in flavodoxin than in unbound FMN. Trp57 also stabilizes all redox forms of FMN, thus cooperating with Tyr94 in strong FMN binding. On the other hand, Trp57 seems to slightly destabilize the semireduced complex relative to the oxidized one. Finally, we have observed that reduction of mutants lacking Trp57 is slow relative to that of wild-type or mutants lacking Tyr94, which suggests that Trp57 could play a role in the kinetics of flavodoxin redox reactions.

Anabaena↗
PubMed1996

Biochemical characterization of NfsA, the Escherichia coli major nitroreductase exhibiting a high amino acid sequence homology to Frp, a Vibrio harveyi flavin oxidoreductase.

We identified the nfsA gene, encoding the major oxygen-insensitive nitroreductase in Escherichia coli, and determined its position on the E. coli map to be 19 min. We also purified its gene product, NfsA, to homogeneity. It was suggested that NfsA is a nonglobular protein with a molecular weight of 26,799 and is associated tightly with a flavin mononucleotide. Its amino acid sequence is highly similar to that of Frp, a flavin oxidoreductase from Vibrio harveyi (B. Lei, M. Liu, S. Huang, and S.-C. Tu, J. Bacteriol. 176:3552-3558, 1994), an observation supporting the notion that E. coli nitroreductase and luminescent-bacterium flavin reductase families are intimately related in evolution. Although no appreciable sequence similarity was detected between two E. coli nitroreductases, NfsA and NfsB, NfsA exhibited a low level of the flavin reductase activity and a broad electron acceptor specificity similar to those of NfsB. NfsA reduced nitrofurazone by a ping-pong Bi-Bi mechanism possibly to generate a two-electron transfer product.

Amino Acid Sequence↗
PubMed1987

Mechanisms underlying the differential effects of ethanol on the bioavailability of riboflavin and flavin adenine dinucleotide.

Chronic alcoholism is associated with a high prevalence of riboflavin deficiency. Experiments were designed in an animal model to determine whether ethanol alters selectively the absorption of riboflavin and flavin adenine dinucleotide (FAD), the predominant dietary form of the vitamin. Rats received by gavage a liver homogenate to which either [14C]riboflavin or [14C]FAD was added with either ethanol or isocaloric sucrose solutions. Ethanol markedly diminished the bioavailability of [14C]FAD to a greater degree than that of [14C]riboflavin. Corroboration of an ethanol-impaired intraluminal hydrolysis of FAD was provided by using everted jejunal segments and measuring mucosal uptake of [14C]riboflavin together with nonradiolabeled FAD. In subsequent studies with mucosal cell extracts, ethanol markedly inhibited activities of FAD pyrophosphatase and flavin mononucleotide (FMN) phosphatase. These findings suggest that dietary sources of riboflavin (FMN and FAD) are not absorbed as well in the presence of ethanol than are vitamin preparations containing riboflavin, which is utilized more readily.

Acetaldehyde↗
PubMed1983

Purification and properties of dihydroorotate oxidase from Crithidia fasciculata and Trypanosoma brucei.

Dihydroorotate oxidases have been highly purified from the parasitic protozoa Crithidia fasciculata and Trypanosoma brucei. The Crithidia enzyme was purified 4200-fold from a crude soluble protein extract in four steps. The protein is a dimer as judged from the native (Mr 60 000) and subunit (Mr 32 700) molecular weights. The purified enzyme exhibits a characteristic flavin electronic spectrum, and each mole of native dimer contains 1.0 mol of tightly bound flavin mononucleotide. Under anaerobic conditions, the flavin chromophore is reduced upon addition of L-dihydroorotate. In air-saturated buffer, the enzyme catalyzes the conversion of L-dihydroorotate to orotate with concomitant reduction of equimolar amounts of molecular oxygen to hydrogen peroxide. A variety of low molecular weight oxidants (e.g., quinones or ferricyanide) may replace oxygen as the electron acceptor during catalysis. The dihydroorotate oxidase of T. brucei was purified 1400-fold to apparent homogeneity by a highly similar isolation procedure. The estimated native (Mr 62 000) and subunit (Mr 30 500) molecular weights indicated a dimeric protein comparable in size to the enzyme from Crithidia. These results suggest that dihydroorotate oxidation is mediated by flavoprotein oxidases in these parasitic protozoa rather than by pterin-linked hydroxylases as recently proposed [Kidder, G. W., & Nolan, L.L. (1973) Biochem. Biophys. Res. Commun. 53, 929-936; Gutteridge, W. E., Dave, D., & Richards, W. H. G. (1979) Biochim. Biophys. Acta 582, 390-401].

Animals↗
PubMed1995

Intrauterine vitamin B2 uptake of preterm and full-term infants.

Intrauterine uptake of vitamin B2 in preterm and full-term infants was examined. Factors of influence on vitamin supply were considered. Forty-four women and their infants were included in the study. Fetal vitamin uptake was calculated as arteriovenous concentration gradient in cord plasma times umbilical plasma flow. Concentration of vitamin B2 (free riboflavin and flavocoenzymes) was determined by high performance liquid chromatography of placental tissue and blood plasma (maternal vein, umbilical artery, umbilical vein). Flavocoenzymes were analyzed as flavin mononucleotide after acid hydrolysis of flavin adenine dinucleotide. Umbilical plasma flow was measured using pulsed Doppler sonography. Both free riboflavin and flavocoenzymes were transferred from the maternal plasma to the umbilical vein, but only free riboflavin was accumulated (approximately 1:4 for preterm and full-term infants, respectively). Flavocoenzyme concentration was higher in the umbilical vein than in the umbilical artery (p < 0.05). This indicated a median uptake of flavocoenzymes of 1.5 nmol/min.kg in preterm infants and 0.4 nmol/min.kg in full-term infants (preterm versus full-term, p < 0.01). Fetal vitamin supply depended on umbilical plasma flow and on maternal vitamin status (the latter was shown only in full-term infants). No dependence on placental vitamin concentration was observed (p > 0.05). Concentration of free riboflavin was higher in umbilical artery than in umbilical vein (p < 0.05). This indicated a release of free riboflavin from fetal tissues independent of gestational age (0.4 nmol/min.kg, preterm; 0.2 nmol/min.kg, full-term; p > 0.05).

Adult↗
PubMed2003

Energy transfer evidence for in vitro and in vivo complexes of Vibrio harveyi flavin reductase P and luciferase.

Conservation of energetically "expensive" metabolites is facilitated by enzymatic intra- and intermolecular channeling mechanisms. Our previous in vitro kinetic studies indicate that Vibrio harveyi reduced nicotinamide adenine dinucleotide phosphate-flavin mononucleotide (NADPH-FMN) oxidoreductase flavin reductase P (FRP) can transfer reduced riboflavin 5'-phosphate (FMNH2) to bacterial luciferase by direct channeling. However, no evidence has ever been reported for such an FMNH2 channeling between these two enzymes in vivo. The formation of a donor-acceptor enzyme complex, stable or transient, is mandatory for direct metabolite channeling between two enzymes regardless of details of the transfer mechanisms. In this study, we have obtained direct evidence of in vitro and in vivo FRP-luciferase complexes that are functionally active. The approach used is a variation of a technique previously described as Bioluminescence Resonance Energy Transfer. Yellow fluorescence protein (YFP) was fused to FRP to generate an active FRP-YFP fusion enzyme, which emits fluorescence peaking at 530 nm. In comparison with the normal 490 nm bioluminescence, an additional 530 nm component was observed in both the in vitro bioluminescence from the coupled reaction of luciferase and FRP-YFP and the in vivo bioluminescence from frp gene-negative V. harveyi cells that expressed FRP-YFP. This 530 nm bioluminescence component was not detected in a control in which a much higher level of YFP was present but not fused to FRP. Such findings indicate an energy transfer from the exited emitter of luciferase to the FRP component of the luciferase-FRP-YFP complex. Hence, the formation of an active complex of luciferase and FRP-YFP was detected both in vitro and in vivo.

Bacterial Proteins↗
PubMed1983

Riboflavin metabolism in the single lens of rat.

To study the mode of riboflavin metabolism in the lens in vivo, the uptake of 14C-labeled riboflavin and synthesis of ester forms from 14C-riboflavin were assayed in the single whole lens of the rats. 14C-riboflavin uptake increased in proportion to the substrate concentration in the incubation medium, reaching a plateau within 60 min. At 4 degrees C the uptake was lowest, at 30, 35 and 40 degrees C it was similar. Neither uncoupler (2,4-dinitrophenol and monoiodoacetate) nor ouabain inhibited the uptake. Within 15 min, approximately 35% of transported 14C-riboflavin was converted to ester forms of riboflavin; this ratio remained constant until 120 min of incubation. Characteristically, 2-3% of 14C-riboflavin was bound to lens protein. 2,4-Dinitrophenol or monoiodoacetate inhibited the synthesis of ester forms of riboflavin, but ouabain did not. No synthesis of ester forms of riboflavin was observed in the lens capsule with epithelium. These results suggest that two enzymes, flavokinase and flavin adenine dinucleotide (FAD) pyrophosphorylase which convert riboflavin to flavin mononucleotide and FAD, respectively, exist in the lens of the rat. These enzymes may participate in riboflavin uptake and riboflavin may pass through the lens capsule by simple diffusion without undergoing a conformational change and then may be metabolized to ester forms of riboflavin with subsequent partial binding to lens protein.

Animals↗
PubMed1995

Crystal structure of NADH oxidase from Thermus thermophilus.

The crystal structures of the flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) containing isoforms of NADH oxidase from Thermus thermophilus have been determined by isomorphous and molecular replacement and refined to 2.3 A and 1.6 A resolution with R-values of 18.5% and 18.6% respectively. The structure of the homodimeric enzyme consists of a central 4-stranded antiparallel beta-sheet covered by helices, a more flexible domain formed by two helices, and a C-terminal excursion connecting the subunits. The active sites are located in a deep cleft between the subunits. The binding site of the flavin cofactor lacks the common nucleotide binding fold and is different from the FMN binding site found in flavodoxins.

Binding Sites↗