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PubMed2006

Transcriptional regulation of the fad regulon genes of Escherichia coli by ArcA.

ArcA is a global transcription factor required for optimal growth of Escherichia coli during anaerobic growth. In this study, the role of ArcA on the transcriptional regulatory subnetwork of the fad regulon was investigated. Gene expression profiles of deletion mutants (Delta arcA, Delta fadR and Delta arcA/Delta fadR) indicated that (i) ArcA is a major transcription factor for the transcriptional regulation of fatty acid metabolism in the absence of oxygen, and (ii) ArcA and FadR cooperatively regulate the fad regulon under anaerobic conditions. To determine the direct interaction between ArcA and the promoters of the fad regulon genes, chromatin immunoprecipitation (ChIP) analysis was performed. ChIP analysis suggested that ArcA directly binds to the promoter regions of the fad regulon genes in vivo. An ArcA-binding motif was identified from known binding sequences and predicted putative binding sites in the promoter regions of the fad regulon genes. These results indicate that ArcA directly represses the expression of fad regulon genes during anaerobic growth.

Anaerobiosis↗
PubMed1978

FAD is covalently attached to peptidyl-tRNA during cell-free synthesis of 6-hydroxy-D-nicotine oxidase.

The process, by which FAD is attached covalently to the 6-hydroxy-D-nicotine oxidase apoprotein in D-nicotine-induced cells of Arthrobacter oxidans was studied in vitro. [3H]Adenine-labelled FAD prepared biosynthetically in Clostridium kluyveri was incorporated into the 6-hydroxy-D-nicotine oxidase molecule during cell-free translation. FAD rather than FMN or riboflavin was thus shown to be the flavin derivative transferred to the polypeptide chain. After short-term protein synthesis on ribosomes from induced A. oxidans cells in the presence of an Escherichia coli MRE 600 supernatant fraction and [adenine-2-3H]FAD, THE PEPTIDYL-TRNA fraction was separated from completed polypeptides. Labelled FAD was found to be covalently attached to the tRNA-bound polypeptides. Cleavage of the tRNA-peptide bond released labelled polypeptides the largest of which migrated as authentic 6-hydroxy-D-nicotine oxidase during dodecylsulfate/polyacrylamide gel electrophoresis. These results strongly suggest that FAD is incorporated into the nascent polypeptide chains of 6-hydroxy-D-nicotine oxidase during ribosomal translation.

Adenine↗
PubMed1983

FAD analogues as mechanistic and 'binding-domain' probes of spinach ferredoxin-NADP+ reductase.

The native flavin, FAD, of spinach ferredoxin--NADP+ reductase, has been replaced by a number of FAD analogues with modifications of the isoalloxazine ring system. The apoenzyme binds 8-mercapto-FAD in its thiolate anion form and 6-hydroxy-FAD in its neutral form. These results are consistent with classification of this enzyme as a dehydrogenase/electron transferase, an ascription originally made on the basis of its physiological function and in common with other properties of this class, e.g. stabilization of the neutral flavin semiquinone. The chemical reactivity toward methylmethanethiolsulfonate of the 8-mercapto-FAD . enzyme clearly shows that the flavin 8-position is exposed to solvent. On the other hand, the lack of reactivity with the 2-thio-FAD . enzyme indicates that the pyrimidine subnucleus of the flavin is buried within the protein molecule. The seven modified flavins examined all support NADPH--ferricyanide reductase activity, the catalytic velocity being directly proportional to the redox potential of the flavin. No such linear free energy relationship was found between redox potential and activity with ferredoxin or iodonitrotetrazolium as acceptor.

Catalysis↗
PubMed1994

The covalent attachment of FAD to the flavoprotein of Saccharomyces cerevisiae succinate dehydrogenase is not necessary for import and assembly into mitochondria.

Succinate dehydrogenase of the bacterial or inner mitochondrial membrane catalyses the oxidation of succinate to fumarate and directs reducing equivalents into the electron-transport chain. The enzyme is also able to catalyse the reverse reaction, the reduction of fumarate to succinate. The enzyme is composed of four subunits. These subunits include a catalytic dimer composed of a flavoprotein subunit with a covalently bound FAD, and an iron-sulfur protein subunit with three different iron-sulfur centres, which is anchored to the membrane by two smaller integral membrane proteins. The FAD moiety is attached to the flavoprotein subunit by an 8 alpha-[N(3)-histidyl]FAD linkage at a conserved histidine residue, His90 of the Saccharomyces cerevisiae succinate dehydrogenase. By mutating His90 to a serine residue, we have constructed a flavoprotein subunit that is unable to covalently bind FAD. The mutant flavoprotein is targeted to mitochondria, translocated across the mitochondrial membranes, and is assembled with the other subunits where it binds FAD non-covalently. The resulting holoenzyme has no succinate-dehydrogenase activity but retains fumarate reductase activity. The covalent attachment of FAD is therefore necessary for succinate oxidation but is dispensable for both fumarate reduction and for the import and assembly of the flavoprotein subunit.

Binding Sites↗
PubMed1997

NifL of Klebsiella pneumoniae carries an N-terminally bound FAD cofactor, which is not directly required for the inhibitory function of NifL.

In Klebsiella pneumoniae NifL antagonizes the action of the transcriptional activator NifA in the presence of molecular oxygen or combined nitrogen. To determine what cofactors might be involved in the oxygen sensing mechanism, we purified and analyzed fusion proteins made between the Escherichia coli maltose binding protein, MalE, and NifL. NifL synthesized and purified under strictly anaerobic conditions did not contain significant amounts of iron or acid-labile sulfur indicating the absence of an oxygen sensing iron-sulfur cluster. However, NifL protein purified in its inhibitory form contained 0.3 +/- 0.01 mol FAD and less than 0.01 mol FMN per mol NifL suggesting the presence of FAD as a cofactor. Characterization of NifL synthesized in the absence of oxygen and combined nitrogen showed that the non-inhibitory form of NifL also contained FAD (0.54 mol FAD per mol NifL). Using fusions between MalE and different portions of NifL we localized the binding site of FAD to the N-terminal domain of NifL. These results and our previous observation that the C-terminal domain of NifL is sufficient to inhibit NifA activity indicate that the N-terminally bound FAD is not directly required for the inhibitory activity of NifL. This observation is supported by the finding that purified apoprotein of NifL was still able to inhibit transcriptional activation by NifA in vitro.

Bacterial Proteins↗
PubMed1991

Utilization of red-cell FAD by methaemoglobin reductases at the expense of glutathione reductase in heterozygous beta-thalassaemia.

FAD-dependent methaemoglobin reductases (MHR) were studied in red cells in heterozygous beta-thalassaemia to investigate how they related to low FAD-dependent glutathione reductase (GR). In contrast to GR, MHR activities were usually normal or increased. In particular, whether expressed in relation to haemoglobin or number of red cells, NADPH-MHR activity was markedly increased in most subjects, probably being a response to increased oxidative stress. Oral riboflavin had no effect on MHR activities, indicating saturation with FAD even though GR was deficient. A strong correlation between percent stimulation of GR by FAD and NADPH-MHR activity indicates that FAD is utilized by MHR at the expense of GR. This could be an important influence on GR in heterozygous beta-thalassaemia. Thus, the low activity resulting from an inherited deficiency of FAD is decreased further.

Cytochrome-B(5) Reductase↗
PubMed1990

NADPH-cytochrome P-450 reductase. Physical properties and redox behavior in the absence of the FAD moiety.

NADPH-cytochrome P-450 reductase contains one molecule each of FMN and FAD. The FAD moiety has been selectively removed, producing the FMN reductase. The FMN reductase is stable and enzymatic activity is reconstituted with either FAD or FMN. FMN remains tightly bound, but can both dissociate from the FMN site and bind to the vacant FAD site. The amount of FMN bound in the FAD site is minimal under specific experimental conditions. There are at least two conformational subpopulations of the FMN reductase; NADP dissociates readily from one but extremely slowly from the other. Rapid dissociation of NADP is regained upon reconstitution with FAD. The one-electron redox state of the FMN reductase is thermodynamically stabilized, though to a lesser degree than in the holoreductase. When two-electron reduced FMN reductase is exposed to oxygen, a stable species with an absorbance peak at 580 nm forms rapidly and quantitatively. This species has been identified by electron paramagnetic resonance spectroscopy as the neutral radical of FMN and is indistinguishable from the air-stable radical of the holoreductase. The redox behavior of the FMN reductase is in agreement with properties proposed previously for the FMN site.

Animals↗
PubMed1979

Induction of the FAD synthetase system in rat liver by phenobarbital administration.

FMN and FAD synthesizing activities in rat liver were investigated when drug metabolizing enzymes were induced by phenobarbital. The FAD content of the liver mitochondrial and microsomal fractions were increased by either one shot administration or three successive daily administrations of phenobarbital. Similarly the flavokinase and FAD pyrophosphorylase activities also increased in the mitochondrial, microsomal and cytosol fractions. The authors have verified that activation of the FAD synthesizing system is induced by phenobarbital and suggest that the FMN and FAD contents are regulated not only by variations of flavin enzyme apoprotein, but also by changes in flavokinase and FAD pyrophosphorylase activities.

Adenosine Triphosphate↗
PubMed1987

para-Hydroxybenzoate hydroxylase containing 6-hydroxy-FAD is an effective enzyme with modified reaction mechanisms.

The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens, was replaced by 6-hydroxy-FAD (an extra hydroxyl group on the carbon at position 6 of the isoalloxazine ring of FAD). The catalytic cycle of this modified enzyme was analyzed and compared to the function of native (FAD) enzyme. Transient state kinetic analyses of the multiple changes in the chemical state of the flavin were the principal methods used to probe the mechanism. Four known substrates of the native enzyme were used to probe the reaction. With the natural substrate, p-hydroxybenzoate, the 6-hydroxy-FAD enzyme activity was 12-15% of native enzyme, due to a slower release of product from the enzyme, and less than one product molecule was formed per NADPH oxidized, due to an increased rate of nonproductive decomposition of the transient peroxyflavin essential to the catalytic pathway. More extensive changes in mechanism were observed with the substrates, 2,4-dihydroxybenzoate and p-aminobenzoate. The results suggest that, during catalysis, when the reduced state of FAD is ready for oxygen reaction, the substrate is located below and close to the C-4a/N-5 edge of the isoalloxazine ring. The nature of the high extinction, transient state of flavin, formed upon transfer of oxygen to substrate is discussed. It is not a flavin cation, and is unlikely to be an oxygen-substituted analogue of N-3/C-4 dihydroflavin.

4-Hydroxybenzoate-3-Monooxygenase↗
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↗
PubMed1997

Determination of FAD-binding domain in flavin-containing monooxygenase 1 (FMO1).

The flavin-containing monooxygenases (FMOs) are a family of flavoenzymes and contain one molecule of FAD per monomer. In order to demonstrate where FMO interacts with FAD, four mutants for the rat liver FMO1 protein were expressed in yeast and characterized. All four mutants were immunochemically similar to the unmodified form, although the contents of FAD in all four mutants were much lower than that in the unmodified form. Interestingly, the mutant generated by changing the first glycine of the proposed FAD-binding domain (GxGxxG) to alanine revealed catalytic activities, but was lower than those seen with the unmodified form. The conversion of the first glycine to alanine markedly increased and decreased the Km and Vmax values for imipramine N-oxidation, respectively. The other three mutants (RFMOm2, RFMOm3, and RFMOm4) were catalytically inactive. Our results suggest that three glycines, especially the second and third glycines, in the proposed FAD-binding domain were necessary for FMO to show catalytic activities. Using RFMOm1 and the unmodified form, the effects of n-octylamine on the activity of FMO1 were investigated. The activities of both wild-type and RFMOm1 enzymes for all of the compounds examined were enhanced by n-octylamine. The Km and Vmax values of both RFMOm1 and the unmodified form for imipramine N-oxidation were lowered and raised by n-octylamine, respectively.

Animals↗
PubMed1983

Luminometric determination of FAD in subpicomole quantities.

Very small quantities of FAD were able to reactivate apo-D-amino acid oxidase. In the presence of D-alanine, luminol, horseradish peroxidase, and an excess of the apoenzyme, a quantitative luminometric determination of FAD was possible. The maximal photon emission measured in a bicarbonate buffer, pH 9.2, at 37 degrees C was proportional to the amount of FAD added. FMN, riboflavin, or 5-deazaflavin produced no chemiluminescence and had no inhibitory effect in the assay when added together with FAD. With this method, FAD could be quantitatively determined with high accuracy in perchloric acid extracts of animal tissue and bacteria.

Alanine↗
PubMed1990

FAD and GSH participate in macrophage synthesis of nitric oxide.

Following partial purification of macrophage nitric oxide (NO) synthase, enzyme activity requires L-arginine, NADPH, and constitutive cytosolic factors, one of which is tetrahydrobiopterin (BH4) (Kwon, N.S., Nathan, C.F. and Stuehr, D.J. [1989] J. Biol. Chem. 264, 20496). Here we identify FAD and GSH as two additional cofactors needed for full enzyme activity. With all defined cytosolic cofactors in excess, NO synthesis was linear over 3 h and was approximately 50% dependent on exogenous FAD, approximately 50% on glutathione (GSH), 84% on tetrahydrobiopterin (BH4), 95% on NADPH, and 98% on L-arginine. The concentrations of added FAD, GSH, and BH4 required for optimal activity were consistent with their levels in macrophage cytosol. Kinetic studies showed that GSH (or DTT) had little or no effect on the rate of NO generation over the first 20-30 min of the reaction, but prevented a subsequent dropoff in rate. This effect was distinct from thiol participation in BH4 regeneration. In contrast, exogenous FAD doubled the rate of NO synthesis throughout the assay period, consistent with a cofactor role. The role of NADPH was not to regenerate BH4, furnish NADP+, nor form reactive oxygen intermediates. These findings demonstrate NO synthesis by a partially purified enzyme in an otherwise defined system, and suggest that an NADPH-utilizing FAD flavoprotein may participate in the reaction.

Amino Acid Oxidoreductases↗
PubMed1995

The Na(+)-translocating NADH:ubiquinone oxidoreductase from the marine bacterium Vibrio alginolyticus contains FAD but not FMN.

The Na(+)-translocating NADH:ubiquinone oxidoreductase from Vibrio alginolyticus was extracted from the bacterial membranes and purified by ion exchange chromatographic procedures. The enzyme catalyzed NADH oxidation by suitable electron acceptors, e.g. menadione, and the Na+ and NADH-dependent reduction of ubiquinone-1. Four dominant bands and a number of minor bands were visible on SDS-PAGE that could be part of the enzyme complex. Flavin analyses indicated the presence of FAD but no FMN in the purified enzyme. FAD but no FMN were also present in V. alginolyticus membranes. FAD is therefore a prosthetic group of the Na(+)-translocating NADH:ubiquinone oxidoreductase and FMN is not present in the enzyme. The FAD was copurified with the NADH dehydrogenase. The purified enzyme exhibited an absorption spectrum with a maximum at 450 nm that is typical for a flavoprotein. Upon incubation with NADH this absorption disappeared indicating reduction of the enzyme-bound FAD.

Cell Membrane↗
PubMed1982

Androgenic suppression of mouse hepatic FAD-containing monooxygenase activity.

Sex-related differences in the activity of hepatic FAD-containing monooxygenase (FAD-M) were found in C3H/St mice. Adult female mice had enzyme activities nearly two-fold greater than male mice and these differences, which were absent in sexually immature mice, became apparent at the onset of puberty. The sex differences in hepatic FAD-M appeared to be mediated through the suppressive effect of testosterone; castration of male mice enhanced enzyme activity, while androgenic replacement returned activities to control levels. Testosterone's suppressive effect was found to be relatively specific for hepatic FAD-M. Treatment of castrated male mice with both the anti-androgen flutamide and testosterone returned enzyme activity to control levels, suggesting that testosterone's regulation of hepatic microsomal FAD-M is receptor-mediated. Female gonadectomy had no effect on this enzyme's activity.

Aging↗
PubMed2006

Crystal structure of archaeal photolyase from Sulfolobus tokodaii with two FAD molecules: implication of a novel light-harvesting cofactor.

UV exposure of DNA molecules induces serious DNA lesions. The cyclobutane pyrimidine dimer (CPD) photolyase repairs CPD-type - lesions by using the energy of visible light. Two chromophores for different roles have been found in this enzyme family; one catalyzes the CPD repair reaction and the other works as an antenna pigment that harvests photon energy. The catalytic cofactor of all known photolyases is FAD, whereas several light-harvesting cofactors are found. Currently, 5,10-methenyltetrahydrofolate (MTHF), 8-hydroxy-5-deaza-riboflavin (8-HDF) and FMN are the known light-harvesting cofactors, and some photolyases lack the chromophore. Three crystal structures of photolyases from Escherichia coli (Ec-photolyase), Anacystis nidulans (An-photolyase), and Thermus thermophilus (Tt-photolyase) have been determined; however, no archaeal photolyase structure is available. A similarity search of archaeal genomic data indicated the presence of a homologous gene, ST0889, on Sulfolobus tokodaii strain7. An enzymatic assay reveals that ST0889 encodes photolyase from S. tokodaii (St-photolyase). We have determined the crystal structure of the St-photolyase protein to confirm its structural features and to investigate the mechanism of the archaeal DNA repair system with light energy. The crystal structure of the St-photolyase is superimposed very well on the three known photolyases including the catalytic cofactor FAD. Surprisingly, another FAD molecule is found at the position of the light-harvesting cofactor. This second FAD molecule is well accommodated in the crystal structure, suggesting that FAD works as a novel light-harvesting cofactor of photolyase. In addition, two of the four CPD recognition residues in the crystal structure of An-photolyase are not found in St-photolyase, which might utilize a different mechanism to recognize the CPD from that of An-photolyase.

Amino Acid Sequence↗
PubMed2006

Over-expression in Escherichia coli, purification and characterization of isoform 2 of human FAD synthetase.

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. The human isoform 2 of FADS (hFADS2), which is the product of FLAD1 gene, was over-expressed in Escherichia coli as a T7-tagged protein and identified by MALDI-TOF MS analysis. Its molecular mass, calculated by SDS-PAGE, was approx. 55 kDa. The expressed protein accounted for more than 40% of the total protein extracted from the cell culture; 10% of it was recovered in a soluble and nearly pure form by Triton X-100 treatment of the insoluble cell fraction. hFADS2 possesses FADS activity and has a strict requirement for MgCl2, as demonstrated in a spectrophotometric assay. The purified recombinant isoform 2 showed a kcat of 3.6 x 10(-3)s(-1) and exhibited a KM value for FMN of about 0.4 microM. The expression of the hFADS2 isoform opens new perspectives in the structural studies of this enzyme and in the design of antibiotics based on the functional differences between the bacterial and the human enzymes.

Amino Acid Sequence↗
PubMed2005

Identification of the human mitochondrial FAD transporter and its potential role in multiple acyl-CoA dehydrogenase deficiency.

Multiple acyl-CoA dehydrogenase deficiency (MADD) or glutaric aciduria type II (GAII) is most often caused by mutations in the genes encoding the alpha- or beta-subunit of electron transfer flavoprotein (ETF) or electron transfer flavoprotein dehydrogenase (ETF-DH). Since not all patients have mutations in these genes, other as yet unidentified genes are predicted to be involved as well. Because all affected mitochondrial flavoproteins in MADD have FAD as a prosthetic group, the underlying defect in these patients may be due to a thus far undisclosed disturbance in the metabolism of FAD. Since a proper mitochondrial flavin balance is maintained by a mitochondrial FAD transporter, a defect of this transporter could also cause an MADD-like phenotype. In yeast, FAD is transported across the mitochondrial inner membrane by the FLX1 protein. An FLX1-mutated Saccharomyces cerevisiae strain exhibits a decreased activity of several mitochondrial flavoproteins. In the present study, we report the identification of the human mitochondrial FAD transporter. Based on sequence similarity to FLX1, we identified two human candidate genes (MFT and N111), which were cloned and characterized by functional expression in an FLX1-mutated yeast strain. Of the two candidate genes, only the previously described mitochondrial folate transporter (MFT) was able to functionally complement the FLX1 mutant. Candidates for mutations in the MFT gene are patients with a clinical suspicion of MADD but without any mutation in the alpha- or beta-subunit of ETF or ETF-DH.

Acyl-CoA Dehydrogenase↗