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PubMed1989

Potentiation of the antiviral activity of poly r(A-U) by riboflavin, FAD and FMN.

The role of riboflavin (RFN), FAD or FMN in modulating the antiviral activity of poly r(A-U) was examined by the human foreskin fibroblast-vesicular stomatitis virus bioassay in which the concentrations of poly r(A-U) was fixed at 0.1 mM or 0.2 mM while the riboflavin, FAD or FMN concentration was varied to produce variable RFN (or FAD or FMN)/ribonucleotide ratios ranging from 1/16 to 2/1. Riboflavin, FAD and FMN tested individually did not exhibit any antiviral activity, while poly r(A-U) alone exhibited antiviral activity. When poly r(A-U) was combined with riboflavin, FAD or FMN, the antiviral activity was potentiated seven- to twelve-fold at RFN (or FAD or FMN)/ribonucleotide ratios in the region of 1/4.

Cytopathogenic Effect, Viral↗

PubMed2004

Crystal structure of chorismate synthase: a novel FMN-binding protein fold and functional insights.

Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate to chorismate in the shikimate pathway, which represents an attractive target for discovering antimicrobial agents and herbicides. Chorismate serves as a common precursor for the synthesis of aromatic amino acids and many aromatic compounds in microorganisms and plants. Chorismate synthase requires reduced FMN as a cofactor but the catalyzed reaction involves no net redox change. Here, we have determined the crystal structure of chorismate synthase from Helicobacter pylori in both FMN-bound and FMN-free forms. It is a tetrameric enzyme, with each monomer possessing a novel "beta-alpha-beta sandwich fold". Highly conserved regions, including several flexible loops, cluster together around the bound FMN to form the active site. The unique FMN-binding site is formed largely by a single subunit, with a small contribution from a neighboring subunit. The isoalloxazine ring of the bound FMN is significantly non-planar. Our structure illuminates the essential functional roles played by the cofactor.

Amino Acid Sequence↗

PubMed2006

A second FMN binding site in yeast NADPH-cytochrome P450 reductase suggests a mechanism of electron transfer by diflavin reductases.

NADPH-cytochrome P450 reductase transfers two reducing equivalents derived from a hydride ion of NADPH via FAD and FMN to the large family of microsomal cytochrome P450 monooxygenases in one-electron transfer steps. The mechanism of electron transfer by diflavin reductases remains elusive and controversial. Here, we determined the crystal structure of truncated yeast NADPH-cytochrome P450 reductase, which is functionally active toward its physiological substrate cytochrome P450, and discovered a second FMN binding site at the interface of the connecting and FMN binding domains. The two FMN binding sites have different accessibilities to the bulk solvent and different amino acid environments, suggesting stabilization of different electronic structures of the reduced flavin. Since only one FMN cofactor is required for function, a hypothetical mechanism of electron transfer is discussed that proposes shuttling of a single FMN between these two sites coupled with the transition between two semiquinone forms, neutral (blue) and anionic (red).

Amino Acid Sequence↗

PubMed2003

The effect of temperature on FMN absorption spectra in rigid poly(vinyl alcohol) matrices.

Electronic absorption spectra of flavomononucleotide (FMN) in poly(vinyl alcohol) films (PVA) were measured over the concentrations ranging from 6.9 x 10(-4) to 6.8 x 10(-1) M and temperatures from 263 to 338 K. The FMN absorption spectra measurements performed at room temperature have shown two ranges of different changes as a function of dye concentration. For concentrations c<10(-1) M (range I) the spectra exhibited regular changes showing an isosbestic point, which evidences the equilibrium between monomers and dimers. However, for range II (c>1.05 x 10(-1) M) the FMN absorption spectra occurred to be almost independent of concentration and they nearly overlapped with the dimer spectrum (within the error limit). Temperature measurements have shown that the FMN absorption spectra in PVA are stable over a wide temperature range. The mean distances between FMN molecules in PVA films are calculated. For maximal concentrations (from the range II), they are below 13.1 A, whereas the mean dimensions of FMN monomers and dimers are 15.8 and 21.1 A, respectively, which indicates that the orientation of dimers and monomers in the PVA film cannot be random at high concentrations. Molecules are partly ordered, adopting approximately parallel orientation, which is in agreement with the calculations of dimer structure by molecular modelling method (MMM).

Dimerization↗

PubMed2006

Identification of the C=O stretching vibrations of FMN and peptide backbone by 13C-labeling of the LOV2 domain of Adiantum phytochrome3.

Phototropin, a blue-light photoreceptor in plants, has two FMN-binding domains named LOV1 and LOV2. We previously observed temperature-dependent FTIR spectral changes in the C=O stretching region (amide-I vibrational region of the peptide backbone) for the LOV2 domain of Adiantum phytochrome3 (phy3-LOV2), suggesting progressive structural changes in the protein moiety (Iwata, T., Nozaki, D., Tokutomi, S., Kagawa, T., Wada, M., and Kandori, H. (2003) Biochemistry 42, 8183-8191). Because FMN also possesses two C=O groups, in this article, we aimed at assigning C=O stretching vibrations of the FMN and protein by using 13C-labeling. We assigned the C(4)=O and C(2)=O stretching vibrations of FMN by using [4,10a-13C2] and [2-13C] FMNs, respectively, whereas C=O stretching vibrations of amide-I were assigned by using 13C-labeling of protein. We found that both C(4)=O and C(2)=O stretching vibrations shift to higher frequencies upon the formation of S390 at 77-295 K, suggesting that the hydrogen bonds of the C=O groups are weakened by adduct formation. Adduct formation presumably relocates the FMN chromophore apart from its hydrogen-bonding donors. Temperature-dependent amide-I bands are unequivocally assigned by separating the chromophore bands. The hydrogen bond of the peptide backbone in the loop region is weakened upon S390 formation at low temperatures, while being strengthened at room temperature. The hydrogen bond of the peptide backbone in the alpha-helix is weakened regardless of temperature. On the other hand, structural perturbation of the beta-sheet is observed only at room temperature, where the hydrogen bond is strengthened. Light-signal transduction by phy3-LOV2 must be achieved by the progressive protein structural changes initiated by the adduct formation of the FMN.

Adiantum↗

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↗

PubMed1998

Modulation of the redox potentials of FMN in Desulfovibrio vulgaris flavodoxin: thermodynamic properties and crystal structures of glycine-61 mutants.

Mutants of the electron-transfer protein flavodoxin from Desulfovibrio vulgaris were made by site-directed mutagenesis to investigate the role of glycine-61 in stabilizing the semiquinone of FMN by the protein and in controlling the flavin redox potentials. The spectroscopic properties, oxidation-reduction potentials, and flavin-binding properties of the mutant proteins, G61A/N/V and L, were compared with those of wild-type flavodoxin. The affinities of all of the mutant apoproteins for FMN and riboflavin were less than that of the wild-type apoprotein, and the redox potentials of the two 1-electron steps in the reduction of the complex with FMN were also affected by the mutations. Values for the dissociation constants of the complexes of the apoprotein with the semiquinone and hydroquinone forms of FMN were calculated from the redox potentials and the dissociation constant of the oxidized complex and used to derive the free energies of binding of the FMN in its three oxidation states. These showed that the semiquinone is destabilized in all of the mutants, and that the extent of destabilization tends to increase with increasing bulkiness of the side chain at residue 61. It is concluded that the hydrogen bond between the carbonyl of glycine-61 and N(5)H of FMN semiquinone in wild-type flavodoxin is either absent or severely impaired in the mutants. X-ray crystal structure analysis of the oxidized forms of the four mutant proteins shows that the protein loop that contains residue 61 is moved away from the flavin by 5-6 A. The hydrogen bond formed between the backbone nitrogen of aspartate-62 and O(4) of the dimethylisoalloxazine of the flavin in wild-type flavodoxin is absent in the mutants. Reliable structural information was not obtained for the reduced forms of the mutant proteins, but if the mutants change conformation when the flavin is reduced to the semiquinone, to facilitate hydrogen bonding between N(5)H and the carbonyl of residue 61, then the change must be different from that known to occur in wild-type flavodoxin.

Amino Acid Substitution↗

PubMed2000

Crystal structure of FMN-dependent nitroreductase from Escherichia coli B: a prodrug-activating enzyme.

The FMN-dependent flavoprotein nitroreductase from Escherichia coli B (NTR) is used in cancer chemotherapy to activate a range of prodrugs. The crystal structure of this enzyme has been determined, using molecular replacement methods and refined at 2.06 A resolution. The recombinant 24-kDa enzyme was crystallized in the tetragonal space group P4(1)2(1)2, with unit cell dimensions of a = b = 57.74 A and c = 275.51 A and two molecules in the asymmetric unit. The structure has a final R factor of 20.3% (R(free) = 26.7%), for all data between the resolution ranges of 10-2.06 A, and includes 4453 protein atoms, 230 water molecules, and 2 flavin mononucleotide (FMN) molecules. The functional unit is a homodimer, which forms the asymmetric unit in the crystal structure. The tertiary structures of these two monomers and their subunit interactions are nearly identical. The molecular replacement search model, the crystal structure of the major NAD(P)H:FMN oxidoreductase of Vibrio fisheri (FRase 1), was selected on the basis of its high sequence identity to that of NTR. The final superposition of these two enzymes revealed a very similar overall fold, with variation in the structures focused around surface loops and helices near the FMN cofactor. Helix G is implicated in substrate specificity and is better resolved in the present NTR structure than in the previously reported FRase 1 structure. The FMN binding pocket is also well-resolved, showing the presence of two channels leading into the active site. The amino acid side chains and main chain atoms interacting with the FMN are well-ordered. The structure of the substrate binding pocket has been used to examine substrate specificity and enzyme kinetics for prodrugs used in antibody-directed enzyme prodrug therapy (ADEPT) and gene-directed enzyme prodrug therapy (GDEPT).

Amino Acid Sequence↗

PubMed2000

Dissecting the energetics of the apoflavodoxin-FMN complex.

Many flavoproteins are non-covalent complexes between FMN and an apoprotein. To understand better the stability of flavoproteins, we have studied the energetics of the complex between FMN and the apoflavodoxin from Anabaena PCC 7119 by a combination of site-directed mutagenesis, titration calorimetry, equilibrium binding constant determinations, and x-ray crystallography. Comparison of the strength of the wild type and mutant apoflavodoxin-FMN complexes and that of the complexes between wild type apoflavodoxin and shortened FMN analogues (riboflavin and lumiflavin) allows the dissection of the binding energy into contributions associated with the different parts of the FMN molecule. The estimated contribution of the phosphate is greatest, at 7 kcal mol(-1); that of the isoalloxazine is of around 5-6 kcal mol(-1) (mainly due to interaction with Trp-57 and Tyr-94 in the apoprotein) and the ribityl contributes least: around 1 kcal mol(-1). The stabilization of the complex is both enthalpic and entropic although the enthalpy contribution is dominant. Both the phosphate and the isoalloxazine significantly contribute to the enthalpy of binding. The ionic strength does not have a large effect on the stability of the FMN complex because, although it weakens the phosphate interactions, it strengthens the isoalloxazine-protein hydrophobic interactions. Phosphate up to 100 mM does not affect the strength of the riboflavin complex, which suggests the isoalloxazine and phosphate binding sites may be independent in terms of binding energy. Interestingly, we find crystallographic evidence of flexibility in one of the loops (57-62) involved in isoalloxazine binding.

Apoproteins↗

PubMed1982

Studies on FAD- and FMN-binding domains in NADPH-cytochrome P-450 reductase from rabbit liver microsomes.

Six sulfhydryl group were determined after complete denaturation of NADPH-cytochrome P-450 reductase; of these, about 5.2 in both the native holoenzyme and FMN-depleted enzyme are accessible to p-hydroxychloromercuribenzoate (pCMB), which may be differentiated as follows: four --SH groups are modified by low concentration of the reagent but are not essentially involved in the catalytic function; additional block of one --SH group at high concentrations of pCMB completely inhibited the reductase activity. The fluorescence quenching of the FAD in the FMN-depleted enzyme was removed after the fifth --SH group was reacted slowly with pCMB. Kinetic and fluorometric analysis indicated that this finally modified --SH group was assumed to be essential for the activity and significantly protected by either 1 mM NADP+ or 2'-AMP against attack by mercurial compounds. A strong negative ellipticity at around 450 nm is clearly decreased upon binding of pCMB to an essential --SH group, while the CD spectra in the near and far UV region show only minor differences during the modification of --SH groups. Removal of the FMN prosthetic group from the native holoprotein results in 1.25-fold greater tryptophan fluorescence with a slight red shift of the emission maximum from 332 to 336 nm, and FMN reconstitution reduces the protein fluorescence quantum yield to approximately that of the holoprotein. Oxidation of tryptophan indol rings of the FMN-depleted enzyme is associated with a loss of FMN binding ability to the protein which causes the inactivation of cytochrome c reductase activity, but ferricyanide reductase activity is not strongly affected by tryptophan modification.

Animals↗

PubMed1983

Hydrolysis of FMN and FAD by alkaline phosphatase of the intestinal brush-border membrane.

Both FMN and FAD were found to be hydrolysed with saturation kinetics by purified alkaline phosphatase (aPase E.C. 3.1.3.1) as well as by a brush-border membrane preparation (BBMp) from rat jejunum. With aPase the KM-value was 11.0 mmole/l when FMN was applied and 4.4 mmole/l when FAD was used. The apparent KM-values with the BBMp were calculated to be 22.9 mmole/l for FMN and 5.7 mmole/l for FAD as substrates. The BBMp contained FMN- and FAD-hydrolysing activity besides that due to the aPase. Regarding the high phosphatase activities associated with the brush-border membrane, it seems unlikely that FMN and FAD penetrate this membrane without being split. The transmural intestinal transport of 14C-riboflavin was tested in vitro in the presence of non-labelled FMN and FAD. The transport rate of the labelled riboflavin was found to be reduced by the coenzymes. It could be concluded that 14C-riboflavin competed with the non-labelled riboflavin released by the phosphatases for the binding sites of a hypothetical transport carrier.

Alkaline Phosphatase↗

PubMed1996

Molecular recognition in the FMN-RNA aptamer complex.

We report on a combined NMR-molecular dynamics calculation approach that has solved the solution structure of the complex of flavin mononucleotide (FMN) bound to the conserved internal loop segment of a 35 nucleotide RNA aptamer identified through in vitro selection. The FMN-RNA aptamer complex exhibits exceptionally well-resolved NMR spectra that have been assigned following application of two, three and four-dimensional heteronuclear NMR techniques on samples containing uniformly 13C, 15N-labeled RNA aptamer in the complex. The assignments were aided by a new through-bond NMR technique for assignment of guanine imino and adenine amino protons in RNA loop segments. The conserved internal loop zippers up through the formation of base-pair mismatches and a base-triple on complex formation with the isoalloxazine ring of FMN intercalating into the helix between a G.G mismatch and a G.U.A base-triple. The recognition specificity is associated with hydrogen bonding of the uracil like edge of the isoalloxazine ring of FMN to the Hoogsteen edge of an adenine at the intercalation site. There is significant overlap between the intercalated isoalloxazine ring and its adjacent base-triple platform in the complex. The remaining conserved residues in the internal loop participate in two G.A mismatches in the complex. The zippered-up internal loop and flanking stem regions form a continuous helix with a regular sugar-phosphate backbone except at a non-conserved adenine, which loops out of the helix to facilitate base-triple formation. Our solution structure of the FMN-RNA aptamer complex is to our knowledge the first structure of an RNA aptamer complex and outlines folding principles that are common to other RNA internal and hairpin loops, and molecular recognition principles common to model self-replication systems in chemical biology.

Base Sequence↗

PubMed1981

Reduction of exogenous FMN by isolated rat liver mitochondria. Significance to the mobilization of iron from ferritin.

When FMN is added to rat liver mitochondria or mitoplasts it is reduced at a rate of approx. 0.2 nmol . min-1 . mg-1 protein. Sonicated mitochondria do not reduce exogenous FMN. The reduction depends on drainage of reducing equivalents from the respiratory chain at the level of ubiquinone. The net production of reduced FMN is detectable only at oxygen concentrations less than 4-5 muM. The mitochondrial ubiquinol-FMN oxidoreductase provides a mechanism for the coupling of FMN-reduction to the reductive mobilization of iron from ferritin. The results are discussed in relation to the role of ferritin as a donor of iron to the mitochondria.

Animals↗

PubMed1992

Analysis of the ACP1 gene product: classification as an FMN phosphatase.

The relationship between the ACP1 gene product, an 18kDa acid phosphatase (E.C. 3.1.3.2) postulated to function as a protein tyrosyl phosphatase, and the cellular flavin mononucleotide (FMN) phosphatase has been examined in vitro and by using cultured Chinese hamster ovary (CHO) cells. Kinetic analysis indicated that at pH 6 the acid phosphatase utilized a variety of phosphate monoesters as substrates. While small molecules such as FMN were effectively utilized as substrates (kcat/Km = 7.3 x 10(3) s-1M-1), the tyrosyl phosphorylated form of the adipocyte lipid binding protein was a relatively poor substrate (kcat/Km = 1.7 x 10(-1) s-1M-1) suggesting a role for the phosphatase in flavin metabolism. Fractionation of CHO cell extracts revealed that 90% of the FMN phosphatase activity was soluble and that all of the soluble activity eluted from a Sephadex G-75 column with the acid phosphatase. All of the soluble FMN phosphatase activity was inhibited by immunospecific antibodies directed against the bovine heart ACP1 gene product. These results suggest that the ACP1 gene product functions cellularly not as a protein tyrosyl phosphatase but as a soluble FMN phosphatase.

Acid Phosphatase↗

PubMed2003

Investigation of electrochemical properties of FMN and FAD adsorbed on titanium electrode.

The electrochemical properties (such as the values of the formal potentials, the dependence of the formal potentials on solution pH, the reversibility of the electrochemical process) of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) adsorbed on a titanium electrode were dependent on the electrolyte. The formal potentials of adsorbed FMN and FAD in phosphate, HEPES and PIPES buffers at pH 7 were similar to those for dissolved flavins (-460 to -480 mV vs. SCE) and changed linearly with a slope of about 52 mV per pH unit in the pH region 3 to 8. In TRIS buffer, the formal potentials of adsorbed FMN and FAD were also pH-dependent, however, with invariance in the pH range 4.5 to 5.5. In non-buffered solutions (KCl, LiCl, NaCl, CsCl, CaCl(2), Na(2)SO(4) at different concentrations), the electrochemical behavior of adsorbed FMN and FAD differed from that of dissolved flavins and was dependent on the electrolyte (especially at pH 4.5 and pH 5). Under certain conditions (electrolyte, concentration, pH), a two-step oxidation of FMN could be observed.

Buffers↗

PubMed1977

Purification and properties of the NADH and NADPH specific FMN oxidoreductases from Beneckea harveyi.

The NADH and NADPH specific FMN oxidoreductases from Beneckea harveyi have been purified to homogeneity as judged by single bands on sodium dodecyl sulfate gel electrophoresis. The overall purification for the NADH specific enzyme is 3000-fold and 4000-fold for the NADPH specific enzyme from a crude extract. The final step in the purification procedure is chromatography on a 5'-AMP-Sepharose 4B affinity column which results in approximately a 50-fold purification to a final specific activity of 31 mumol of NADH oxidized min-1 (mg of protein)-1 for the NADH specific FMN reductase. The NADPH specific reductase has been purified to a final specific activity of 51 mumol of NADPH oxidized min-1 (mg of protein)-1 using a NADP agarose affinity column, which results in a 70-fold purification. Molecular weights of 30 000 and 40 000 and Km's of 4.75 X 10(-5) M NADH and 4.0 X 10(-5) M NADPH have been determined for the pure NADH and NADPH specific FMN reductases, respectively. The NADPH specific FMN reductase does not utilize NADH, while the NADH specific enzyme does dehydrogenate NADPH with a maximal velocity one-tenth of that for NADH. Separate NADH and NADPH specific FMN reductases from Photobacterium fischeri could not be demonstrated.

Chromatography, Affinity↗

PubMed1997

1H, 15N and 13C NMR resonance assignment, secondary structure and global fold of the FMN-binding domain of human cytochrome P450 reductase.

The FMN-binding domain of human NADPH-cytochrome P450 reductase, corresponding to exons 3-7, has been expressed at high level in an active form and labelled with 13C and 15N. Most of the backbone and aliphatic side-chain 1H, 15N and 13C resonances have been assigned using heteronuclear double- and triple-resonance methods, together with a semiautomatic assignment strategy. The secondary structure as estimated from the chemical shift index and NOE connectivities consists of six alpha-helices and five beta-strands. The global fold was deduced from the long-range NOEs unambiguously assigned in a 4D 13C-resolved HMQC-NOESY-HMQC spectrum. The fold is of the alternating alpha/beta type, with the five beta-strands arranged into a parallel beta-sheet. The secondary structure and global fold are very similar to those of the bacterial flavodoxins, but the FMN-binding domain has an extra short helix in place of a loop, and an extra helix at the N-terminus (leading to the membrane anchor domain in the intact P450 reductase). The experimental constraints were combined with homology modelling to obtain a structure of the FMN-binding domain satisfying the observed NOE constraints. Chemical shift comparisons showed that the effects of FMN binding and of FMN reduction are largely localised at the binding site.

Amino Acid Sequence↗

PubMed1990

Conformational changes in Chondrus crispus flavodoxin on dissociation of FMN and reconstitution with flavin analogues.

The apoflavodoxin produced by precipitation of Chondrus crispus flavodoxin with trichloroacetic acid migrates as a single molecular species on non-denaturing PAGE, but at a much lower Rm than the flavoprotein. Values of s and D were significantly lower than for the flavodoxin, but their substitution in the Svedberg equation indicated the molecular mass was closely similar to that of the flavodoxin. This was confirmed by meniscus-depletion sedimentation-equilibrium studies. The Stokes radius of the apoflavodoxin was 3.65 nm, compared with 2.33 nm for the flavodoxin, and estimates of frictional coefficient ratio suggested the apoprotein was in extended conformation compared with the roughly globular shape of the flavodoxin. The Ka for FMN binding was 2.8 x 10(8)M, and the electrophoretic and physicochemical properties of the reconstituted flavoprotein were closely similar to those of the native flavodoxin. FAD, iso-FMN and thio-FMN were also bound effectively, but methyl-FMN and riboflavin were bound only weakly, if at all. The reconstituted flavoproteins were active to various extents in mediating electron transfer from NADPH to cytochrome c catalysed by flavodoxin-NADP+ oxidoreductase, the highest activity being with the thio-FMN flavodoxin.

Apoproteins↗