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PubMed1981

Nitrate reductase from Penicillium chrysogenum. Purification and kinetic mechanism.

Nitrate reductase (NADPH:nitrate oxidoreductase; EC 1.6.6.1-3) was purified to apparent homogeneity from mycelium of Penicillium chrysogenum. The final preparation catalyzed the NADPH-dependent, FAD-mediated reduction of nitrate with a specific activity of 170-225 units X mg of protein-1. Gel filtration and glycerol density centrifugation yielded, respectively, a Stokes radius of 6.3 nm and an s20,w of 7.4. The molecular weight was calculated to be 199,000. On sodium dodecyl sulfate gels, the enzyme displayed two almost contiguous dye-staining bands corresponding to molecular weights of about 97,000 and 98,000. The enzyme prefers NADPH to NADH (kspec ratio = 2813), FAD to FMN (kspec ratio = 141), FAD (+ NADPH) to FADH2 (kspec ratio = 12,000), and nitrate to chlorate (kspec ratio = 4.33), where the kspec (the specificity constant for a given substrate) represents Vmax/Km. The Penicillium enzyme will also catalyze te NADPH-dependent, FAD-mediated reduction of cytochrome c with a specific activity of 647 units X mg of protein-1 (Kmcyt = 1.25 X 10(-5) M), and the reduced methyl viologen (MVH2, i.e. methyl viologen + dithionite)-dependent, NADPH and FAD-independent reduction of nitrate with a specific activity of 250 units X mg of protein-1 kmMVH2 = 3.5 X 10(-6) M). Initial velocity studies showed intersecting NADPH-FAD and nitrate-FAD reciprocal plot patterns. The NADPH-nitrate pattern was a series of parallel lines at saturating and unsaturating FAD levels. NADP+ was competitive with NADPH, uncompetitive with nitrate (at saturating and unsaturating FAD levels), and a mixed-type inhibitor with respect to FAD. Nitrite was competitive with nitrate, uncompetitive with NADPH (at saturating and unsaturating FAD levels), and a mixed-type inhibitor with respect to FAD. At unsaturating nitrate and FAD, NADPH exhibited substrate inhibition, perhaps as a result of binding to the FAD site(s). At very low FAD concentrations, low concentrations of NADP+ activated the reaction slightly. The initial velocity and product inhibition patterns are consistent with either of the two kinetic mechanisms. One (rather unlikely) mechanism involves the rapid equilibrium random binding of all ligands with (a) NADP+ and NADPH mutually exclusive, (b) nitrate and nitrite mutually exclusive, (c) the binding of NADPH strongly inhibiting the binding of nitrate and vice versa, (d) the binding of NADPH strongly promoting the binding of nitrite and vice versa, and (e) the binding of nitrate strongly promoting the binding of NADP+ and vice versa...

Binding Sites↗
PubMed1999

Mechanism of activation of acyl-CoA substrates by medium chain acyl-CoA dehydrogenase: interaction of the thioester carbonyl with the flavin adenine dinucleotide ribityl side chain.

The flavin adenine dinucleotide (FAD) cofactor of pig kidney medium-chain specific acyl-coenzyme A (CoA) dehydrogenase (MCADH) has been replaced by ribityl-3'-deoxy-FAD and ribityl-2'-deoxy-FAD. 3'-Deoxy-FAD-MCADH has properties very similar to those of native MCADH, indicating that the FAD-ribityl side-chain 3'-OH group does not play any particular role in cofactor binding or catalysis. 2'-Deoxy-FAD-MCADH was characterized using the natural substrate C8CoA as well as various substrate and transition-state analogues. Substrate dehydrogenation in 2'-deoxy-FAD-MCADH is approximately 1.5 x 10(7)-fold slower than that of native MCADH, indicating that disruption of the hydrogen bond between 2'-OH and substrate thioester carbonyl leads to a substantial transition-state destabilization equivalent to approximately 38 kJ mol-1. The alphaC-H microscopic pKa of the substrate analogue 3S-C8CoA, which undergoes alpha-deprotonation on binding to MCADH, is lowered from approximately 16 in the free state to approximately 11 (+/-0.5) when bound to 2'-deoxy-FAD-MCADH. This compares with a decrease of the same pKa to approximately 5 in the complex with unmodified hwtMCADH, which corresponds to a pK shift of approximately 11 pK units, i.e., approximately 65 kJ mol-1 [Vock, P., Engst, S., Eder, M., and Ghisla, S. (1998) Biochemistry 37, 1848-1860]. The difference of this effect of approximately 6 pK units ( approximately 35 kJ mol-1) between MCADH and 2'-deoxy-FAD-MCADH is taken as the level of stabilization of the substrate carbanionic species caused by the interaction with the FAD-2'-OH. This energetic parameter derived from the kinetic experiments (stabilization of transition state) is in agreement with those obtained from static experiments (lowering of alphaC-H microscopic pKa of analogue, i.e., stabilization of anionic transition-state analogue). The contributions of the two single H-bonds involved in substrate activation (Glu376amide-N-H and ribityl-2'-OH) thus appear to behave additively toward the total effect. The crystal structures of native pMCADH and of 2'-deoxy-FAD-MCADH complexed with octanoyl-CoA/octenoyl-CoA show unambiguously that the FAD cofactor and the substrate/product bind in an identical fashion, implying that the observed effects are mainly due to (the absence of) the FAD-ribityl-2'-OH hydrogen bond. The large energy associated with the 2'-OH hydrogen bond interaction is interpreted as resulting from the changes in charge and the increased hydrophobicity induced by binding of lipophilic substrate. This is the first example demonstrating the direct involvement of a flavin cofactor side chain in catalysis.

Acyl-CoA Dehydrogenase↗
PubMed1991

Autoflavinylation of apo6-hydroxy-D-nicotine oxidase.

6-Hydroxy-D-nicotine oxidase (6-HDNO) was expressed in Escherichia coli JM109 cells from the recombinant plasmid pAX-6-HDNO as a beta-galactosidase-6-HDNO fusion protein. Affinity chromatography of the fusion protein on p-aminobenzyl-1-thio-beta-galactopyranoside-agarose and subsequent digestion with protease Xa yielded highly purified apo6-HDNO. Incubation of the purified protein with [14C]FAD demonstrated that flavinylation of apo6-HDNO proceeds autocatalytically. Phosphorylated three-carbon compounds such as glycerol-3-P, which are known to stimulate the formation of the histidyl (N3)-(8 alpha) FAD between apo6-HDNO and FAD (Brandsch, R., and Bichler, V. (1989) Eur. J. Biochem. 182, 125-128), could be replaced in their action by high concentrations of glycerol (45%) or sucrose (20%). These substances apparently induced and stabilized a conformational state of the apoenzyme compatible with covalent attachment of FAD. In the absence of glycerol the apoenzyme readily lost the ability to form holoenzyme at temperatures above 30 degrees C. Holoenzyme formation protected the 6-HDNO polypeptide from this thermal denaturation. Autoflavinylation of 6-HDNO was inhibited by the sulfhydryl reagents dithionitrobenzoate or N-ethylmaleimide. Inhibition was prevented by mercaptoethanol or FAD, but not 6-hydroxy-D-nicotine, the substrate of the holoenzyme. A cysteine-thiol group may therefore be involved in reactions leading to the covalent attachment of FAD to apo6-HDNO. When flavinylation of apo6-HDNO proceeded under anaerobic conditions, the amount of incorporation of [14C]FAD into the polypeptide was indistinguishable from reactions performed in the presence of O2. None of the FAD-derivatives (8-demethyl-FAD, 8-chloro-FAD, and 5-deaza-FAD) could replace FAD in holoenzyme formation. The failure of covalent attachment of 5-deaza-FAD to apo6-HDNO is in agreement with the assumption that the quinone methide form of the isolloxazine ring is an intermediate in the flavinylation reaction.

Amino Acid Sequence↗
PubMed1985

Inhibition by chlorpromazine of thyroxine modulation of flavin metabolism in liver, cerebrum and cerebellum.

In livers of adult rats that have been treated with thyroxine, the rate of incorporation of radiolabeled riboflavin into both flavin adenine dinucleotide (FAD) and FAD covalently attached to specific apoflavoenzymes was enhanced markedly. By contrast, thyroxine diminished riboflavin incorporation into FAD in cerebrum and cerebellum but continued to enhance incorporation into the covalently bound fraction of FAD. Diminished net incorporation of riboflavin into FAD in brains of adult rats may reflect increased utilization of this fraction for covalent attachment into specific apoflavoenzymes rather than down regulation of the FAD biosynthetic enzymes, flavokinase and FAD pyrophosphorylase, inasmuch as covalent attachment of FAD occurs subsequent to the formation of FAD. The psychotropic drug, chlorpromazine, over a wide dose range, exerted an inhibitory effect on both incorporation of riboflavin into FAD and the utilization of FAD for incorporation into covalently bound flavoenzymes in liver, cerebrum, and cerebellum. Thus, chlorpromazine inhibition of FAD metabolism occurred regardless of the direction of the thyroxine effect and was compatible with an observed inhibitory effect by this drug upon the flavin biosynthetic enzymes.

Animals↗
PubMed1997

Stimulation of antiviral activities of interferon by a liver extract preparation.

The liver extract preparation Adelavin9 (referred to as EXT-FAD hereinafter) is a combination product that consists of hog liver extract and flavin-adenine dinucleotide (FAD). EXT-FAD has been reported to improve the histology of chronic liver injury and is widely used in Japan. Interferon (IFN) has been used to treat viral hepatitis. In this study, the action of EXT-FAD on the antiviral activity of IFN was examined using experimental models of viral infection in vitro and in vivo. EXT-FAD significantly enhanced the 2',5'-oligoadenylate synthetase (2-5AS) activity in human peripheral blood mononuclear cells (PBMC) treated with human IFN-alpha-2a. EXT-FAD is likely to affect the late phase of IFN action to produce the antiviral activity. Combination of IFN-alpha-2a with EXT-FAD produced a synergistic inhibitory effect against herpes simplex virus type-1 (HSV-1) in vitro. The effect of EXT-FAD in combination with IFN-alpha-2a against influenza virus type A (Fluv A) was also examined and a similar effect to that found in the anti-HSV-1 assay was observed. In addition, administration of EXT-FAD to mice infected with HSV-1 significantly prolonged the mean survival periods. The intracellular redox state plays a role in inhibiting virus replication. EXT-FAD was found to increase the intracellular glutathione (GSH) level. EXT-FAD also enhanced the nitric oxide synthase (NOS) activity, which plays a role in antiviral effects. This result indicate that the effect of EXT-FAD on the antiviral activity of IFN is partially related to increased intracellular GSH levels and NOS activation.

Animals↗
PubMed1993

Role of flavin in acetoin production by two bacterial pyruvate oxidases.

Escherichia coli pyruvate oxidase (POXEC) requires FAD both for the oxidative decarboxylation of pyruvate to acetate and CO2 and for the formation of acetoin from pyruvate and acetaldehyde. Prior work has shown that the catalytic activity (kcat/Km) for POXEC in the oxidative reaction is stimulated approximately 450-fold by amphiphilic activators. This paper shows that the acetoin reaction does not respond to activation. The FAD requirement for acetoin formation can be replaced by 5-deaza-FAD and 6-hydroxy-FAD, FAD analogs which form kinetically stable oxidized and reduced enzyme species, respectively. As would be expected, the 5-deaza- and 6-hydroxy-FAD enzymes are not active in the oxidative reaction. A second flavin pyruvate oxidase from Pediococcus pseudomonas (POXPP), which catalyzes the oxidative decarboxylation of pyruvate to CO2 and acetyl phosphate, also requires FAD for acetoin formation. POXPP has an oxidative rate comparable to that of POXEC, but in comparison to POXEC, POXPP catalyzes acetoin formation at a much reduced rate. Again, as was found with the POXEC, an FAD analog incapable of undergoing facile oxidation-reduction reactions also could replace the FAD requirement in the POXPP acetoin reaction. The results indicate that the role for FAD in acetoin formation with both enzymes is based on a structural requirement and that FAD does not participate in a redox function in the acetoin reaction.

Acetoin↗
PubMed1987

Kinetic studies on the reaction mechanism of sarcosine oxidase.

A sarcosine oxidase (sarcosine: oxygen oxidoreductase (demethylating), EC 1.5.3.1) isolated from Corynebacterium sp. U-96 contains both covalently bound FAD and noncovalently bound FAD. The noncovalent FAD reacts with sarcosine, the covalent FAD with molecular oxygen (Jorns, M.S. (1985) Biochemistry 24, 3189-3194). To clarify the reaction mechanism of the enzyme, kinetic investigations were performed by the stopped-flow method as well as by analysis of the overall reaction. The absorption spectrum of the enzyme in the steady state was very similar to that of the oxidized enzyme, and no intermediate enzyme species, such as a semiquinoid flavin, was detected. The rate for anaerobic reduction of the noncovalently bound FAD and the covalently bound FAD by sarcosine were 31 and 6.7 s-1, respectively. The latter value was smaller than the value of respective Vmax/e0 obtained by the overall reaction kinetics (Vmax/e0: the maximum velocity per enzyme concentration). Both rate constants for oxidation of the two FADs by molecular oxygen were 100 s-1. A reaction scheme of sarcosine oxidase is proposed to account for the data obtained; 70% of the enzyme functions via a fully reduced enzyme, and 30% of the enzyme goes along a side-path, without forming the fully reduced enzyme. In addition, it is suggested that the reactivity of noncovalently bound FAD with sarcosine is affected by the oxidation-reduction state of the covalently bound FAD, in contrast to the reactivity of the covalently bound FAD with molecular oxygen, which is independent of the oxidation-reduction state of the noncovalently bound FAD.

Anaerobiosis↗
PubMed2004

The presenilin-1 familial Alzheimer disease mutant P117L impairs neurogenesis in the hippocampus of adult mice.

The functions of presenilin 1 (PS1) and how PS1 mutations cause familial Alzheimer's disease (FAD) are incompletely understood. PS1 expression is essential for neurogenesis during embryonic development and may also influence neurogenesis in adult brain. We examined how increasing PS1 expression or expressing an FAD mutant would affect neurogenesis in the adult hippocampus. A neuron-specific enolase (NSE) promoter was used to drive neuronal overexpression of either wild-type human PS1 or the FAD mutant P117L in transgenic mice, and the animals were studied under standard-housing conditions or after environmental enrichment. As judged by bromodeoxyuridine (BrdU) labeling, neural progenitor proliferation rate was mostly unaffected by increasing expression of either wild-type or FAD mutant PS1. However, in both housing conditions, the FAD mutant impaired the survival of BrdU-labeled neural progenitor cells leading to fewer new beta-III-tubulin-immunoreactive neurons being generated in FAD mutant animals during the 4-week postlabeling period. The effect was FAD mutant specific in that neural progenitor survival and differentiation in mice overexpressing wild-type human PS1 were similar to nontransgenic controls. Two additional lines of PS1 wild-type and FAD mutant transgenic mice showed similar changes indicating that the effects were not integration site-dependent. These studies demonstrate that a PS1 FAD mutant impairs new neuron production in adult hippocampus by decreasing neural progenitor survival. They also identify a new mechanism whereby PS1 FAD mutants may impair normal neuronal function and may have implications for the physiological functioning of the hippocampus in FAD.

Alzheimer Disease↗
PubMed1994

Affinity probing of flavin binding sites. 2. Identification of a reactive cysteine in the flavin domain of Escherichia coli DNA photolyase.

8-(Methylsulfonyl)FAD reacts with a single cysteine residue (Cys293) in the flavin domain of Escherichia coli DNA photolyase to form an 8-(cysteinyl)FAD derivative covalently bound to the protein. About 80% protection against covalent attachment with 8-(methylsulfonyl)FAD was observed in the presence of an equimolar amount of FAD. Flavinylated photolyase retains the ability to repair pyrimidine dimers (15% of native activity) and to bind its antenna chromophore, 5,10-methenyltetrahydrofolate. Comparison of the properties of flavinylated enzyme with photolyase containing noncovalently bound 8-(methylthio)-FAD indicate that a perturbation is necessary to accommodate covalent bond formation. 8-(Methylthio)-FAD-reconstituted enzyme exhibits 95% of native activity. The aerobic stability of fully reduced and radical forms of 8-(methylthio)FAD enzyme is similar to that of native enzyme, whereas a radical form is not detected with flavinylated enzyme and the fully reduced enzyme is more easily oxidized by oxygen. The flavin in 8-(methylthio)FAD enzyme or flavinylated photolyase is shielded from solvent. However, the flavin environment in flavinylated enzyme is less hydrophobic as judged by spectral comparison with model 8-(alkylthio)flavins in various solvents. Enzyme containing noncovalently bound 8-(methylsulfonyl)-FAD was prepared by reconstitution with the fully reduced flavin which does not undergo covalent attachment. Covalent attachment was observed after reoxidation but probably involved dissociation and rebinding of oxidized 8-(methylsulfonyl)FAD. The results show that 8-(cysteinyl)FAD in flavinylated photolyase is at or near the normal flavin binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1999

The intraflavin hydrogen bond in human electron transfer flavoprotein modulates redox potentials and may participate in electron transfer.

Electron-transfer flavoprotein (ETF) serves as an intermediate electron carrier between primary flavoprotein dehydrogenases and terminal respiratory chains in mitochondria and prokaryotic cells. The three-dimensional structures of human and Paracoccus denitrificans ETFs determined by X-ray crystallography indicate that the 4'-hydroxyl of the ribityl side chain of FAD is hydrogen bonded to N(1) of the flavin ring. We have substituted 4'-deoxy-FAD for the native FAD and investigated the analog-containing ETF to determine the role of this rare intra-cofactor hydrogen bond. The binding constants for 4'-deoxy-FAD and FAD with the apoprotein are very similar, and the energy of binding differs by only 2 kJ/mol. The overall two-electron oxidation-reduction potential of 4'-deoxy-FAD in solution is identical to that of FAD. However, the potential of the oxidized/semiquinone couple of the ETF containing 4'-deoxy-FAD is 0.116 V less than the oxidized/semiquinone couple of the native protein. These data suggest that the 4'-hydoxyl-N(1) hydrogen bond stabilizes the anionic semiquinone in which negative charge is delocalized over the N(1)-C(2)O region. Transfer of the second electron to 4'-deoxy-FAD reconstituted ETF is extremely slow, and it was very difficult to achieve complete reduction of the flavin semiquinone to the hydroquinone. The turnover of medium chain acyl-CoA dehydrogenase with native ETF and ETF containing the 4'-deoxy analogue was essentially identical when the reduced ETF was recycled by reduction of 2,6-dichlorophenolindophenol. However, the steady-state turnover of the dehydrogenase with 4'-deoxy-FAD was only 23% of the turnover with native ETF when ETF semiquinone formation was assayed directly under anaerobic conditions. This is consistent with the decreased potential of the oxidized semiquinone couple of the analog-containing ETF. ETF containing 4'-deoxy-FAD neither donates to nor accepts electrons from electron-transfer flavoprotein ubiquinone oxidoreductase (ETF-QO) at significant rates (</=0.5% the wild-type rates). These results indicate that the 4'-hydroxyl-N(1) hydrogen bond plays a major role in the stabilization of the anionic semiquinone and anionic hydroquinone oxidation states of ETF and that this hydrogen bond may provide a pathway for electron transfer between the ETF flavin and the flavin of ETF-QO.

Animals↗
PubMed1995

Cloning and characterization of FAD1, the structural gene for flavin adenine dinucleotide synthetase of Saccharomyces cerevisiae.

The FAD1 gene of Saccharomyces cerevisiae has been selected from a genomic library on the basis of its ability to partially correct the respiratory defect of pet mutants previously assigned to complementation group G178. Mutants in this group display a reduced level of flavin adenine dinucleotide (FAD) and an increased level of flavin mononucleotide (FMN) in mitochondria. The restoration of respiratory capability by FAD1 is shown to be due to extragenic suppression. FAD1 codes for an essential yeast protein, since disruption of the gene induces a lethal phenotype. The FAD1 product has been inferred to be yeast FAD synthetase, an enzyme that adenylates FMN to FAD. This conclusion is based on the following evidence. S. cerevisiae transformed with FAD1 on a multicopy plasmid displays an increase in FAD synthetase activity. This is also true when the gene is expressed in Escherichia coli. Lastly, the FAD1 product exhibits low but significant primary sequence similarity to sulfate adenyltransferase, which catalyzes a transfer reaction analogous to that of FAD synthetase. The lower mitochondrial concentration of FAD in G178 mutants is proposed to be caused by an inefficient exchange of external FAD for internal FMN. This is supported by the absence of FAD synthetase activity in yeast mitochondria and the presence of both extramitochondrial and mitochondrial riboflavin kinase, the preceding enzyme in the biosynthetic pathway. A lesion in mitochondrial import of FAD would account for the higher concentration of mitochondrial FMN in the mutant if the transport is catalyzed by an exchange carrier. The ability of FAD1 to suppress impaired transport of FAD is explained by mislocalization of the synthetase in cells harboring multiple copies of the gene. This mechanism of suppression is supported by the presence of mitochondrial FAD synthetase activity in S. cerevisiae transformed with FAD1 on a high-copy-number plasmid but not in mitochondrial of a wild-type strain.

Amino Acid Sequence↗
PubMed1997

Cerebellar pathology in sporadic and familial Alzheimer's disease including APP 717 (Val-->Ile) mutation cases: a morphometric investigation.

Familial Alzheimer's disease (FAD) tends to present with more prominent neurological symptoms including cerebellar signs than sporadic Alzheimer's disease (SAD). In order to elucidate the pathological differences in the cerebellum, which may be associated with the cerebellar symptoms, we have investigated morphometrically beta-amyloid deposits, atrocytosis, Purkinje cells and dentate neurons in the cerebellum of 10 FAD patients including two cases with the beta-amyloid precursor protein (APP) gene mutation (APP717 Val-->Ile), 10 SAD patients and 10 non-demented age-matched controls. The regions examined included the molecular, Purkinje cell and granular cell layers, the cerebellar white matter and the dentate nucleus. Purkinje cell density in FAD was significantly lower than in SAD. There were no significant differences in the density of dentate neurons among the three groups. The density of astrocytes in FAD was significantly greater than that in SAD in the granular cell and Purkinje cell layers and in the white matter. There were no significant differences in the amount and subtypes of beta-amyloid deposits (extracellular, vascular and perivascular) between FAD and SAD in all the regions investigated. In two cases with the APP mutation, both Purkinje cell loss and beta-amyloid deposition in the cerebellum were greater than the mean for FAD and SAD cases. Astrocytosis in the mutation cases was not greater than the mean for FAD cases except for the dentate nucleus in one case. Extracellular beta-amyloid deposits were not seen in any of the control cases although amyloid angiopathy was observed in one case. This study demonstrates for the first time that Purkinje cell loss and reactive astrocytosis of the cerebellum in FAD are more severe than in SAD, but that beta-amyloid deposition in the cerebellum in both FAD and SAD are similar. The more prominent neurological signs observed in FAD may be explained by more severe neurodegeneration than are found in sporadic cases.

Aged↗
PubMed2006

Structural perturbations in the Ala --> Val polymorphism of methylenetetrahydrofolate reductase: how binding of folates may protect against inactivation.

In human methylenetetrahydrofolate reductase (MTHFR) the Ala222Val (677C-->T) polymorphism encodes a heat-labile gene product that is associated with elevated levels of homocysteine and possibly with risk for cardiovascular disease. Generation of the equivalent Ala to Val mutation in Escherichia coli MTHFR, which is 30% identical to the catalytic domain of the human enzyme, creates a protein with enhanced thermolability. In both human and E. coli MTHFR, the A --> V mutation increases the rate of dissociation of FAD, and in both enzymes, loss of FAD is linked to changes in quaternary structure [Yamada, K., Chen, Z., Rozen, R., and Matthews, R. G. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 14853-14858; Guenther, B. D., Sheppard, C. A., Tran, P., Rozen, R., Matthews, R. G., and Ludwig, M. L. (1999) Nat. Struct. Biol. 6, 359-365]. Folates have been shown to protect both human and bacterial enzymes from loss of FAD. Despite its effect on affinity for FAD, the A --> V mutation is located at the bottom of the (betaalpha)(8) barrel of the catalytic domain in a position that does not contact the bound FAD prosthetic group. Here we report the structures of the Ala177Val mutant of E. coli MTHFR and of its complex with the 5,10-dideazafolate analogue, LY309887, and suggest mechanisms by which the mutation may perturb FAD binding. Helix alpha5, which immediately precedes the loop bearing the mutation, carries several residues that interact with FAD, including Asn168, Arg171, and Lys172. In the structures of the mutant enzyme this helix is displaced, perturbing protein-FAD interactions. In the complex with LY309887, the pterin-like ring of the analogue stacks against the si face of the flavin and is secured by hydrogen bonds to residues Gln183 and Asp120 that adjoin this face. The direct interactions of bound folate with the cofactor provide one mechanism for linkage between binding of FAD and folate binding that could account in part for the protective action of folates. Conformation changes induced by folate binding may also suppress dissociation of FAD.

Alanine↗
PubMed1995

Flavinylation of monoamine oxidase B.

Monoamine oxidase B (MAO B) catalyzes the oxidative deamination of biogenic and xenobiotic amines. The oxidative step is coupled to the reduction of an obligatory cofactor, FAD, which is covalently linked to the enzyme at Cys397. In this study, we developed a novel riboflavin-depleted (Rib-) COS-7 cell line to study the flavinylation of MAO B. ApoMAO B can be obtained by expressing MAO B cDNA in these cells. We found that MAO B is expressed equally in the presence or absence of FAD and that apoMAO B can be inserted into the outer mitochondrial membrane. Flavinylation of MAO B was achieved by introducing MAO B cDNA and different flavin derivatives simultaneously into Rib- COS-7 cells via electroporation. Since the addition of riboflavin, FMN, or FAD resulted in equal levels of MAO B activity, we conclude that the flavin which initially binds to apoMAO B is FAD. In our previous work, we used site-directed mutagenesis to show that Glu34 in the dinucleotide-binding motif of MAO B is essential for MAO B activity, and we postulated that this residue is involved in FAD binding. In this study, we tested the role of residue 34 in flavin binding by expressing wild-type or mutant MAO B cDNA in Rib- COS-7 cells with the addition of [14C]FAD. We found that Glu34 is essential for both FAD binding and catalytic activity. Thus, FAD binds to MAO B in a dual manner at Glu34 noncovalently and Cys397 covalently. We conclude that Glu34 is critical for the initial non-covalent binding of FAD and is instrumental in delivering FAD to the covalent attachment site at Cys397.

Animals↗
PubMed2003

Structural studies on flavin reductase PheA2 reveal binding of NAD in an unusual folded conformation and support novel mechanism of action.

The catabolism of toxic phenols in the thermophilic organism Bacillus thermoglucosidasius A7 is initiated by a two-component enzyme system. The smaller flavin reductase PheA2 component catalyzes the NADH-dependent reduction of free FAD according to a ping-pong bisubstrate-biproduct mechanism. The reduced FAD is then used by the larger oxygenase component PheA1 to hydroxylate phenols to the corresponding catechols. We have determined the x-ray structure of PheA2 containing a bound FAD cofactor (2.2 A), which is the first structure of a member of this flavin reductase family. We have also determined the x-ray structure of reduced holo-PheA2 in complex with oxidized NAD (2.1 A). PheA2 is a single domain homodimeric protein with each FAD-containing subunit being organized around a six-stranded beta-sheet and a capping alpha-helix. The tightly bound FAD prosthetic group (K(d) = 10 nm) binds near the dimer interface, and the re face of the FAD isoalloxazine ring is fully exposed to solvent. The addition of NADH to crystalline PheA2 reduced the flavin cofactor, and the NAD product was bound in a wide solvent-accessible groove adopting an unusual folded conformation with ring stacking. This is the first observation of an enzyme that is very likely to react with a folded compact pyridine nucleotide. The PheA2 crystallographic models strongly suggest that reactive exogenous FAD substrate binds in the NADH cleft after release of NAD product. Nanoflow electrospray mass spectrometry data indeed showed that PheA2 is able to bind one FAD cofactor and one FAD substrate. In conclusion, the structural data provide evidence that PheA2 contains a dual binding cleft for NADH and FAD substrate, which alternate during catalysis.

Amino Acid Sequence↗
PubMed1992

The binding of adenine nucleotides to apo-electron-transferring flavoprotein.

Apoprotein of electron-transferring flavoprotein (ETF) reacts with FAD as follows: A*<-->A, A+FAD<-->holoETF. Two different forms of apoETF (A* and A) convert into each other and only one of them, A, can associate with FAD [Sato, K. et al. (1991) J. Biochem. 109, 734-740]. In the present study, the reactions between apoETF and ATP, ADP, AMP, riboflavin, or FMN were investigated. It was revealed that all three adenine nucleotides bind with apoETF with the same kinetic reaction scheme as FAD, and compete with FAD. These results suggest that the nucleotides bind to A with the same location as the ADP part of FAD in holoETF and that the ADP-binding site of apoETF is generated upon conversion from A* to A. Neither riboflavin nor FMN bound to apoETF regardless of the presence or absence of the nucleotides, indicating that the ADP part of the FAD molecule is essential to the incorporation of the isoalloxazine ring into ETF. The binding rate constant of FAD to A was 1/20 of that of ADP while the dissociation rate constant was 1/1,000. This indicates that the riboflavin part of FAD inhibits the binding of FAD by steric hindrance, while after the binding, it stabilizes the complex.

Adenine Nucleotides↗
PubMed1986

Comparison of phase analysis with factor analysis in equilibrium gated radionuclide angiography.

This study is the intercomparison of phase analysis (PA), factor analysis of dynamic structures (FADS) and Karhunen-Loeve analysis (KLA) in the diagnosis of regional wall motion abnormalities, RWMA, of the LV. One hundred and twenty eight patients with proven or suspected CAD have been investigated by both X-ray angiography and radionuclide equilibrium angiography performed in the LOA view. FADS and KLA are performed twice, once on the whole-image (WI-FADS, WI-KLA), and once on the LV ROI (LV-FADS, LV-KLA) as suggested by Pavel. Resulting images and factors are interpreted by a well trained observer. In an attempt to quantify LV-FADS images, two numeric parameters, P1 and P2, are defined. They measure the relative weight of the so-called ventricular factor for 2 and 3 factor analysis, respectively. A ROC curve is calculated for each method, taking X-ray angiography as the gold-standard. The areas under the ROC curves are estimated by the maximum likelihood method and are compared using a test described by Hanley which takes into account the correlation between the responses. The areas are: 0.90 for PA, 0.84 for WI-FADS, 0.86 for LV-FADS, 0.83 for WI-KLA, 0.86 for LV-KLA, 0.65 for P1 and 0.72 for P2. The observed differences are significant (at 5% level) between PA and WI-FADS and between FA and WI-KLA; whereas they are not between PA and LV-FADS, between PA and LV-KLA and between LV-FADS and LV-KLA. The diagnostic value of the two numeric parameters is poor.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1996

Intrinsic signaling function of APP as a novel target of three V642 mutations linked to familial Alzheimer's disease.

APP695 is a transmembrane precursor of Abeta amyloid. In familial Alzheimer's disease (FAD), three mutations V642I/F/G were discovered in APP695, which has been suggested by multiple studies to be a cell surface signaling receptor. We previously reported that normal APP695 encodes a potential GO-linked receptor with ligand-regulated function and that expression of the three FAD mutants (FAD-APPs), not normal APP, induces cellular outputs by GO-dependent mechanisms. This suggests that FAD-APPs are constitutively active GO-linked receptors. Here, we provide direct evidence for this notion. Reconstitution of either recombinant FAD-APP with GO vesicles induced activation of GO, which was inhibitable by pertussis toxin, sensitive to Mg2+ and proportional in quantity to the reconstituted amounts of FAD-APP. Consistent with the dominant inheritance of this type of FAD, this function was dominant over normal APP, because little activation was observed in APP695-GO vesicles. Experiments with antibody competition and sequence deletion indicated that His657-Lys676 of FAD-APP, which has been specified as the ligand-dependent GO-coupling domain of normal APP, was responsible for this constitutive activation, confirming that the three FAD-APPs are mutationally activated APP695. This study identifies the intrinsic signaling function of APP to be a novel target of hereditary Alzheimer's disease mutations, providing an in vitro system for the screening of potential FAD inhibitors.

Alzheimer Disease↗