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At least 379 records · Page 21Linked to original sources
PubMed1999

Alteration in FAD-glucose dehydrogenase activity and hemocyte behavior contribute to initial disruption of Manduca sexta immune response to Cotesia congregata parasitoids.

Cotesia congregata and Manduca sexta were used as a model system to study the mechanism and effect of a polydnavirus (PDV). We hypothesized that (1) FAD-glucose dehydrogenase (GLD) (EC 1.1.99.10) hemolymph titer would increase in response to parasitism, (2) hemocyte targeting behavior would be altered by parasitism, and (3) changes observed in GLD activity and hemocyte behavior immediately post-parasitization would be due to the presence of PDV. GLD specific activity was measured at four time points early during parasitism using a spectrophotometric enzyme assay. Hemocyte behavior was measured using direct observations of hemocyte response to a foreign target in vitro. Results demonstrate that GLD increases immediately post-oviposition and post-injection of purified PDV, indicating that virions elicit nonself recognition. This increase relative to unparasitized controls also occurs in response to trioxsalen-UV inactivated virus, indicating that the initial disruption of the host immune response is not dependent upon viral transcription. Further, we demonstrate that plasmatocytes are actively spreading and aggregating but are not targeting nonself material in both parasitized and polydnavirus treatments. These results indicate that purified PDV is recognized as nonself and is triggering an immediate cellular immune response prior to viral transcription.

Journal Article↗
PubMed2002

Conserved positions for ribose recognition: importance of water bridging interactions among ATP, ADP and FAD-protein complexes.

Analysis of the spatial arrangement of protein and water atoms that form polar interactions with ribose has been performed for a structurally non-redundant dataset of ATP, ADP and FAD-protein complexes. The 26 ligand-protein structures were separated into two groups corresponding to the most populated furanose ring conformations (N and S-domains). Four conserved positions were found for S-domain protein-ligand complexes and five for N-domain complexes. Multiple protein folds and secondary structural elements were represented at a single conserved position. The following novel points were revealed: (i) Two complementary positions sometimes combine to describe a putative atomic spatial location for a specific conserved binding spot. (ii) More than one third of the interactions scored were water-mediated. Thus, conserved spatial positions rich in water atoms are a significant feature of ribose-protein complexes.

Adenosine Diphosphate↗
PubMed2003

Sub-atomic resolution crystal structure of cholesterol oxidase: what atomic resolution crystallography reveals about enzyme mechanism and the role of the FAD cofactor in redox activity.

The crystal structure of cholesterol oxidase, a 56kDa flavoenzyme was anisotropically refined to 0.95A resolution. The final crystallographic R-factor and R(free) value is 11.0% and 13.2%, respectively. The quality of the electron density maps has enabled modeling of alternate conformations for 83 residues in the enzyme, many of which are located in the active site. The additional observed structural features were not apparent in the previous high-resolution structure (1.5A resolution) and have enabled the identification of a narrow tunnel leading directly to the isoalloxazine portion of the FAD prosthetic group. The hydrophobic nature of this narrow tunnel suggests it is the pathway for molecular oxygen to access the isoalloxazine group for the oxidative half reaction. Resolving the alternate conformations in the active site residues provides a model for the dynamics of substrate binding and a potential oxidation triggered gating mechanism involving access to the hydrophobic tunnel. This structure reveals that the NE2 atom of the active site histidine residue, H447, critical to the redox activity of this flavin oxidase, acts as a hydrogen bond donor rather than as hydrogen acceptor. The atomic resolution structure of cholesterol oxidase has revealed the presence of hydrogen atoms, dynamic aspects of the protein and how side-chain conformations are correlated with novel structural features such as the oxygen tunnel. This new structural information has provided us with the opportunity to re-analyze the roles played by specific residues in the mechanism of the enzyme.

Binding Sites↗
PubMedDate not supplied

FAD mutant PS-1 gene-targeted mice: increased A beta 42 and A beta deposition without APP overproduction.

To investigate the consequences of mutant presenilin-1 (PS-1) expression under the control of the normal PS-1 gene, a gene-targeted mouse bearing the FAD mutation P264L was made. Gene-targeted models are distinct from transgenic models because the mutant gene is expressed at normal levels, in the absence of the wild-type protein. PS-1(P264L/P264L) mice had normal expression of PS-1 mRNA, but levels of the N- and C-terminal protein fragments of PS-1 were reduced while levels of the holoprotein were increased. When crossed into Tg(HuAPP695.K670N/M671L)2576 mice, the PS-1(P264L) mutation accelerated the onset of amyloid (Abeta) deposition in a gene-dosage dependent manner. Tg2576/PS-1(P264L/P264L) mice also had Abeta deposition that was widely distributed throughout the brain and spinal cord. APP(NLh/NLh)/PS-1(P264L/P264L) double gene-targeted mice had elevated levels of Abeta42, sufficient to cause Abeta deposition beginning at 6 months of age. Abeta deposition increased linearly over time in APP(NLh/NLh)/PS-1(P264L/P264L) mice, whereas the increase in Tg2576 mice was exponential. The APP(NLh/NLh)/PS-1(P264L/P264L) double gene-targeted mouse represents an animal model that exhibits Abeta deposition without overexpression of APP.

Alzheimer Disease↗
PubMed2000

RNA-Catalyzed CoA, NAD, and FAD synthesis from phosphopantetheine, NMN, and FMN.

A novel in vitro selection method was developed to isolate RNA sequences with coenzyme-synthesizing activities. We used size-heterogeneous libraries containing randomized ribonucleotide sequences of four different lengths (30N, 60N, 100N, and 140N), all with 5'-ATP initiation. Two RNAs, CoES7 (30N) and CoES21 (60N), are able to catalyze the synthesis of three common coenzymes, CoA, NAD, and FAD, from their precursors, 4'-phosphopantetheine, NMN, and FMN, respectively. Both ribozymes require divalent manganese for activities. The results support the availability of these coenzymes in an RNA world, and point to a chemical explanation for the complex bipartite structures of many coenzymes.

Coenzyme A↗
PubMed2002

Ring scission of diastereomeric 4-butylspiropentylcarbinyl radicals as a chemical model for identifying enzyme-catalyzed FAD adducts resulting from spiropentylacetyl-CoA.

Both diastereomeric 4-butylspiropentylcarbinyl bromides (14a and 14b) were synthesized in seven steps starting from 1-heptyne, and the stereochemical assignments based upon NOE experiments were confirmed by converting their immediate alcohol precursors (13a and 13b) to 1,4-dibutylspiropentanes (17a and 17b) with C(1) and C(2) symmetry. Each bromide was used to generate its corresponding spiropentylcarbinyl radical (18a and 18b) via its AIBN-initiated tri-n-butyltin hydride reduction. The radical-trapped products are identified, the preferred ring scission mode is identified (C1[bond] C2 bond cleavage), and the estimated rates for the ring opening of 4-butylspiropentylcarbinyl radical (18, k(25) degrees C > or = approximately 5 x 10(9) s(-1)) and 2-butyl-1-vinylcyclopropylcarbinyl radical (33, k(25) degrees C approximately 5 x 10(8) s(-1)) are reported. High-level ab initio calculations addressing the ring-opening isomerizations of cyclopropylcarbinyl and spiropentylcarbinyl radicals also are presented. These results in conjunction with a previous study enable us to propose two structures for the enzyme-catalyzed FAD adducts resulting from spiropentylacetic acid-CoA, a synthetic byproduct of fatty acid metabolism.

Coenzyme A↗
PubMed2003

Stereospecific biocatalytic epoxidation: the first example of direct regeneration of a FAD-dependent monooxygenase for catalysis.

Catalysis for chemical synthesis by cell-free monooxygenases necessitates an efficient and robust in situ regeneration system to supply the enzyme with reducing equivalents. We report on a novel approach to directly regenerate flavin-dependent monooxygenases. The organometallic complex [CpRh(bpy)(H(2)O)](2+) catalyzes the transhydrogenation reaction between formate and isoalloxazine-based cofactors such as FAD and FMN. Coupling this FADH(2) regeneration reaction to the FADH(2)-dependent styrene monooxygenase (StyA) resulted in a chemoenzymatic epoxidation reaction where the organometallic compound substitutes for the native reductase (StyB), the nicotinamide coenzyme (NAD), and an artificial NADH regeneration system such as formate dehydrogenase. Various styrene derivatives were converted into the essentially optically pure (S)-epoxides (ee > 98%). In addition, StyA was shown to be capable of performing sulfoxidation reactions. The productivity of the chemoenzymatic epoxidation reaction using 6.5 microM StyA reached up to 6.4 mM/h, corresponding to approximately 70% of a comparable fully enzymatic reaction using StyB, NADH, and formate dehydrogenase for regeneration. The coupling efficiency of the nonenzymatic regeneration reaction to enzymatic epoxidation was examined in detail, leading to an optimized reaction setup with minimized quenching of the electron supply for the epoxidation reaction. Thus, up to 60% of the reducing equivalents provided via [CpRh(bpy)(H(2)O)](2+) could be channeled into epoxide rather than hydrogen peroxide formation, allowing selective synthesis with high yields.

Biomimetic Materials↗
PubMed2005

Dynamics of intramolecular electron transfer reaction of FAD studied by magnetic field effects on transient absorption spectra.

The kinetics of intermediates generated from intramolecular electron-transfer reaction by photo irradiation of the flavin adenine dinucleotide (FAD) molecule was studied by a magnetic field effect (MFE) on transient absorption (TA) spectra. Existence time of MFE and MFE action spectra have a strong dependence on the pH of solutions. The MFE action spectra have indicated the existence of interconversion between the radical pair and the cation form of the triplet excited state of flavin part. All rate constants of the triplet and the radical pair were determined by analysis of the MFE action spectra and decay kinetics of TA. The obtained values for the interconversion indicate that the formation of cation radical promotes the back electron-transfer reaction to the triplet excited state. Further, rate constants of spin relaxation and recombination have been studied by the time profiles of MFE at various pH. The drastic change of those two factors has been obtained and can be explained by SOC (spin-orbit coupling) induced back electron-transfer promoted by the formation of a stacking conformation at pH > 2.5.

Electrochemistry↗
PubMed2004

Fludarabine, adriamycin and dexamethasone (FAD) in newly diagnosed advanced follicular lymphoma: a phase II study by the British National Lymphoma Investigation (BNLI).

The optimal first-line treatment for symptomatic patients with advanced stage follicular lymphoma remains unclear. Fludarabine-based combination regimens have been extensively used in relapsed disease and merit consideration as first-line therapy. We here report the results of a phase II study of FAD (fludarabine, adriamycin, dexamethasone) regimen in 30 patients with advanced stage follicular lymphoma requiring treatment. The response rate was in excess of 90% with 39% achieving a complete remission. The major toxicity was myelosuppression, but only 3% of cycles were associated with grade IV leucopenia. The high response rate has not translated into major improvements in failure-free survival and consideration must be given to alternative treatment modalities to consolidate the high rate of initial responses.

Administration, Oral↗
PubMed1980

Human FAD-dependent NAD(P)H diaphorase.

A newly discovered human diaphorase, designated diaphorase-4, which accounts for a major part of the diaphorase activity of most tissues but does not occur in erythrocytes, is described. In contrast with other human diaphorases, it is dependent on FAD for activity after electrophoresis, inhibited by low concentrations of dicoumarol and shows a marked affinity for Cibacron Blue. The molecular weight was estimated to be 49000 +/- 1800 by gel filtration. Diaphorase-4 appears to show person-to-person quantitative variation, so that about 4% of the population lack appreciable enzyme activity, but it is not yet clear whether this variation is of genetic or non-genetic origin.

Anthracenes↗
PubMed1998

A fully active FAD-containing precursor remains folded up to its translocation across the chloroplast membranes.

The cytosolic and two recombinant precursors, containing 10 and 30 amino acid spacers between the transit peptide and the mature region of the chloroplast flavoprotein ferredoxin-NADP+ reductase (FNR), were expressed in Escherichia coli cells. These proteins were purified rendering fully active precursors that contained bound FAD. Neither the transit peptide nor the spacers affected the formation of the tightly folded enzyme structure. Protease treatment of the folded precursors resulted in a rapid removal of the transit sequence, rendering an enzymatically active resistant core, even at high protease concentration. All three preproteins could be efficiently imported by isolated pea chloroplasts. Addition of the enzyme substrate NADP+ to the import medium slightly decreased the polypeptide translocation. The precursor bound to isolated chloroplasts in the presence or absence of leaf extracts was as resistant to proteolysis as the folded precursor in solution. In contrast, the FNR precursor unfolded by urea was rapidly digested even at the lowest protease concentration. Together, our results indicate that precursor unfolding may take place during translocation but not during binding to chloroplast envelopes or by interaction with leaf extract soluble factors, and that this process is independent of the distance between the transit peptide and the folded mature region of the protein.

Amino Acid Sequence↗
PubMed1999

Class II DNA photolyase from Arabidopsis thaliana contains FAD as a cofactor.

The major UV-B photoproduct in DNA is the cyclobutane pyrimidine dimer (CPD). CPD-photolyases repair this DNA damage by a light-driven electron transfer. The chromophores of the class II CPD-photolyase from Arabidopsis thaliana, which was cloned recently [Taylor, R., Tobin, A. & Bray, C. (1996) Plant Physiol. 112, 862; Ahmad, M., Jarillo, J.A., Klimczak, L.J., Landry, L.G., Peng, T., Last, R.L. & Cashmore, A.R. (1997) Plant Cell 9, 199-207], have not been characterized so far. Here we report on the overexpression of the Arabidopsis CPD photolyase in Escherichia coli as a 6 x His-tag fusion protein, its purification and the analysis of the chromophore composition and enzymatic activity. Like class I photolyase, the Arabidopsis enzyme contains FAD but a second chromophore was not detectable. Despite the lack of a second chromophore the purified enzyme has photoreactivating activity.

Arabidopsis↗
PubMed2000

The hybrid-cluster protein ('prismane protein') from Escherichia coli. Characterization of the hybrid-cluster protein, redox properties of the [2Fe-2S] and [4Fe-2S-2O] clusters and identification of an associated NADH oxidoreductase containing FAD and [2Fe-2S].

Hybrid-cluster proteins ('prismane proteins') have previously been isolated and characterized from strictly anaerobic sulfate-reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe-4S] cubane cluster with spin-admixed S = 3/2 ground-state paramagnetism and a novel type of hybrid [4Fe-2S-2O] cluster, which can attain four redox states. Genomic sequencing reveals that genes encoding putative hybrid-cluster proteins are present in a range of bacterial and archaeal species. In this paper we describe the isolation and spectroscopic characterization of the hybrid-cluster protein from Escherichia coli. EPR spectroscopy shows the presence of a hybrid cluster in the E. coli protein with characteristics similar to those in the proteins of anaerobic sulfate reducers. EPR spectra of the reduced E. coli hybrid-cluster protein, however, give evidence for the presence of a [2Fe-2S] cluster instead of a [4Fe-4S] cluster. The hcp gene encoding the hybrid-cluster protein in E. coli and other facultative anaerobes occurs, in contrast with hcp genes in obligate anaerobic bacteria and archaea, in a small operon with a gene encoding a putative NADH oxidoreductase. This NADH oxidoreductase was also isolated and shown to contain FAD and a [2Fe-2S] cluster as cofactors. It catalysed the reduction of the hybrid-cluster protein with NADH as an electron donor. Midpoint potentials (25 degrees C, pH 7.5) for the Fe/S clusters in both proteins indicate that electrons derived from the oxidation of NADH (Em NADH/NAD+ couple: -320 mV) are transferred along the [2Fe-2S] cluster of the NADH oxidoreductase (Em = -220 mV) and the [2Fe-2S] cluster of the hybrid-cluster protein (Em = -35 mV) to the hybrid cluster (Em = -50, +85 and +365 mV for the three redox transitions). The physiological function of the hybrid-cluster protein has not yet been elucidated. The protein is only detected in the facultative anaerobes E. coli and Morganella morganii after cultivation under anaerobic conditions in the presence of nitrate or nitrite, suggesting a role in nitrate-and/or nitrite respiration.

Amino Acid Sequence↗
PubMed2001

The NADH-dependent reductase of a putative multicomponent tetrahydrofuran mono-oxygenase contains a covalently bound FAD.

NADH-cytochrome c oxidoreductase activity specifically expressed during growth on tetrahydrofuran was detected in cell extracts of Pseudonocardia sp. strain K1. The enzyme catalyzing this reaction was purified to apparent homogeneity by a three-step purification procedure. It was characterized as a monomer of apparent molecular mass 40 kDa. Spectroscopic studies indicated that it contains an iron-sulfur cluster and a flavin cofactor. An amount of 1 mol of flavin and 1 mol of iron was determined per mol of homogeneous protein. The N-terminal amino-acid sequence exhibited great similarity to the reductase component of various oxygenases. Cloning and sequencing of the corresponding gene designated as thmD revealed an ORF encoding a protein of 360 amino acids. An overall similarity of up to 38% was obtained to the NAD(P)H-acceptor reductase of several binuclear iron-containing mono-oxygenases. Conserved sequence motifs were identified that were similar to the chloroplast-type ferredoxin 2Fe-2S centre and to nucleotide-binding domains. Studies on the flavin cofactor showed that it could not be removed from the protein by denaturation, indicating a covalent attachment. Spectroscopic studies revealed that the flavin is at the FAD level and covalently bound to the protein via the flavin 8alpha-methyl group. Thus, the isolated reductase component is the first enzyme of this type for which a covalent attachment of the flavin has been observed.

Actinomycetales↗
PubMed1996

Ontogeny of FAD-linked glycerophosphate dehydrogenase in rat pancreatic islets.

The activities of the mitochondrial FAD-linked glycerophosphate dehydrogenase (m-GDH), glutamate dehydrogenase, alpha-ketoglutarate dehydrogenase, glutamate-pyruvate transaminase (GPT) and glutamate-oxaloacetate transaminase were measured in islet and liver homogenates from fetal, neonatal, adult male, adult female, pregnant and lactating rats. Either parallel or dissociated ontogenic changes were observed in islet and liver homogenates. The activity of islet m-GDH was slightly, albeit not significantly, lower in neonates than in adult rats, comparable in male and female adult animals, unaffected by pregnancy, and increased during lactation. It was much higher in fetal or adult islets cultured for 7 days than in freshly isolated islets from adult rats. In cultured islets from adult rats, the increase in m-GDH activity coincided with a dramatic decrease of GPT activity, a situation the mirror image of that found in several animal models of non-insulin-dependent diabetes mellitus. The intrinsic properties of m-GDH, as judged by comparison of measurements made by either a radioisotopic or a colorimetric procedure, were not identical in islet and liver homogenates and differed between fetal and adult islets, suggesting the existence of distinct iso-enzymes. These findings illustrate adaptive changes of islet enzymes, with exclusive or partial mitochondrial location, in ontogenic situations characterized by a remodelling of fuel homeostasis.

Animals↗
PubMed1994

Cloning of a cDNA for the FAD-linked glycerol-3-phosphate dehydrogenase from rat liver and its regulation by thyroid hormones.

A full-length 2.4-kb cDNA for the FAD-linked glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) was cloned from rat liver using PCR techniques. The cloned gene encodes a protein of 727 amino acids. The calculated molecular mass of 80,898 Da is higher than the apparent molecular mass observed by SDS/PAGE (74,000 Da) of the purified enzyme. This result indicates that the enzyme is synthesized as a precursor with a putative mitochondrial signal sequence. mRNA for this gene was detected in liver, heart, muscle, brain, testes, and pancreas. With the exception of testes, basal expression levels were very low in all tissues examined. However, application of thyroid hormones led to a 10- to 15-fold increase in liver glycerol-3-phosphate dehydrogenase mRNA, whereas hypothyroidism further decreased the mRNA level.

Amino Acid Sequence↗
PubMed1996

The B form of dihydroorotate dehydrogenase from Lactococcus lactis consists of two different subunits, encoded by the pyrDb and pyrK genes, and contains FMN, FAD, and [FeS] redox centers.

The B form of dihydroorotate dehydrogenase from Lactococcus lactis (DHOdehase B) is encoded by the pyrDb gene. However, recent genetic evidence has revealed that a co-transcribed gene, pyrK, is needed to achieve the proper physiological function of the enzyme. We have purified DHOdehase B from two strains of Escherichia coli, which harbored either the pyrDb gene or both the pyrDb and the pyrK genes of L. lactis on multicopy plasmids. The enzyme encoded by pyrDb alone (herein called the delta-enzyme) was a bright yellow, dimeric protein that contained one molecule of tightly bound FMN per subunit. The delta-enzyme exhibited dihydroorotate dehydrogenase activity with dichloroindophenol, potassium hexacyanoferrate(III), and molecular oxygen as electron acceptors but could not use NAD+. The DHOdehase B purified from the E. coli strain that carried both the pyrDb and pyrK genes on a multicopy plasmid (herein called the deltakappa-enzyme) was quite different, since it was formed as a complex of equal amounts of the two polypeptides, i.e. two PyrDB and two PyrK subunits. The deltakappa-enzyme was orange-brown and contained 2 mol of FAD, 2 mol of FMN, and 2 mol of [2Fe-2S] redox clusters per mol of native protein as tightly bound prosthetic groups. The deltakappa-enzyme was able to use NAD+ as well as dichloroindophenol, potassium hexacyanoferrate(III), and to some extent molecular oxygen as electron acceptors for the conversion of dihydroorotate to orotate, and it was a considerably more efficient catalyst than the purified delta-enzyme. Based on these results and on analysis of published sequences, we propose that the architecture of the deltakappa-enzyme is representative for the dihydroorotate dehydrogenases from Gram-positive bacteria.

Amino Acid Sequence↗