Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In 63 children with severe meningococcal infection (MI) and meningitides of another origin red cell metabolism was studied: levels of ATP, ADP, AMP, ATP/ADP, ATP/AMP, energetic charge, 2,3-DPG, FAD, piruvate, lactate, activity of lactate dehydrogenase, piruvate kinase, glucose-6-phosphate dehydrogenase, glutatione reductase, Mg2+, Na+, K(+)-dependent ATPase. All the disease periods were characterized by combined pathobiochemical shifts of different degree typical for varying metabolic systems and correlating with the infection severity. The discussion covers pathogenetic and clinical significance of red cell metabolism shifts in patients with MI and purulent meningitides.
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PMID:[The dynamics of erythrocyte metabolism in severe forms of meningococcal infection and suppurative meningitis in children]. 904 77

This work presents the complete sequences of a cDNA and the two allelic genes of dihydrolipoamide dehydrogenase (LipDH) from Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanosomiasis). The full-length cDNA has an ORF of 1431 bp and encodes a protein of 477 amino acid residues. LipDH is a homodimeric protein with FAD as prosthetic group. The calculated molecular mass of the subunit of the mature protein with bound FAD is 50,066. Comparison of the deduced amino acid sequence of LipDH from T. cruzi with that of Trypanosoma brucei and man shows identities of 81% and 50%, respectively. An N-terminal nonapeptide, not present in the mature enzyme, represents a mitochondrial targeting sequence so far found only in trypanosomatids. The gene lpd1 of T. cruzi LipDH was expressed without the targeting sequence in Escherichia coli JRG1342 cells which are deficient for LipDH. For this purpose an ATG codon was introduced directly upstream the codon for Asn10 which represents the N-terminus of the mature protein. This system allowed the synthesis of 1000 U T. cruzi LipDH/1 bacterial cell culture. The recombinant protein was purified to homogeneity by (NH4)2SO4-precipitation and affinity chromatography on 5' AMP-Sepharose. The K(m) values for NAD+, NADH, lipoamide and dihydrolipoamide are identical with those of the enzyme isolated from the parasite. LipDH is present in all major developmental stages of T. cruzi as shown by northern and western blot analyses. This finding is in agreement with the citric acid cycle being active throughout the whole life cycle of the parasite. In vitro studies on a mammalian LipDH revealed the ability of the flavoenzyme to catalyze the redoxcycling and superoxide anion production of nitrofuran derivatives including the antitrypanosomal drug Nifurtimox. For that reason T. cruzi LipDH is regarded as a promising target for the structure-based development of new antiparasitic drugs. The bacterial expression system for the parasite enzyme will now allow the study of the role of T. cruzi LipDH in drug activation and the crystallization of the protein.
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PMID:Cloning, sequencing and functional expression of dihydrolipoamide dehydrogenase from the human pathogen Trypanosoma cruzi. 905 40

Threonine 244 in the alpha subunit of Paracoccus denitrificans transfer flavoprotein (ETF) lies seven residues to the amino terminus of a proposed dinucleotide binding motif for the ADP moiety of the FAD prosthetic group. This residue is highly conserved in the alpha subunits of all known ETFs, and the most frequent pathogenic mutation in human ETF encodes a methionine substitution at the corresponding position, alphaT266. The X-ray crystal structures of human and P. denitrificans ETFs are very similar. The hydroxyl hydrogen and a backbone amide hydrogen of alphaT266 are hydrogen bonded to N(5) and C(4)O of the flavin, respectively, and the corresponding alphaT244 has the same structural role in P. denitrificans ETF. We substituted a methionine for T244 in the alpha subunit of P. denitrificans ETF and expressed the mutant ETF in Escherichia coli. The mutant protein was purified, characterized, and compared with wild type P. denitrificans ETF. The mutation has no significant effect on the global structure of the protein as inferred from visible and near-ultraviolet absorption and circular dichroism spectra, far-ultraviolet circular dichroism spectra, and infrared spectra in 1H2O and 2H2O. Intrinsic fluorescence due to tryptophan of the mutant protein is 60% greater than that of the wild type ETF. This increased tryptophan fluorescence is probably due to a change in the environment of the nearby W239. Tyrosine fluorescence is unchanged in the mutant protein, although two tyrosine residues are close to the site of the mutation. These results indicate that a change in structure is minor and localized. Kinetic constants of the reductive half-reaction of ETF with porcine medium chain acyl-CoA dehydrogenase are unaltered when alphaT244M ETF serves as the substrate; however, the mutant ETF fails to exhibit saturation kinetics when the semiquinone form of the protein is used as the substrate in the disproportionation reaction catalyzed by P. denitrificans electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). The redox behavior of the mutant ETF was also altered as determined from the equilibrium constant of the disproportionation reaction. The separation of flavin redox potentials between the oxidized/semiquinone couple and semiquinone/hydroquinone couple are -6 mV in the wild type ETF and -27 mV in the mutant ETF. The mutation does not alter the AMP content of the protein, although the extent and fidelity of AMP-dependent, in vitro renaturation of the mutant AMP-free apoETF is reduced by 57% compared to renaturation of wild type apoETF, likely due to the absence of the potential hydrogen bond donor T244.
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PMID:alphaT244M mutation affects the redox, kinetic, and in vitro folding properties of Paracoccus denitrificans electron transfer flavoprotein. 910 14

Adenylylsulfate reductase (EC 1.8.99.2) isolated from Desulfovibrio vulgaris Miyazaki catalyzes electron transfer from dihydroflavin coenzyme (FADH2, FMNH2, or dihydroriboflavin) to adenylyl sulfate (APS), and catalyzes flavin-mediated oxidation of ferrocytochrome c3 with APS. The reaction with FAD as an electron mediator was markedly stimulated in the presence of menadione. Km of the enzyme was about 0.015 mM for riboflavin and FAD in the presence of menadione. Free flavin coenzyme was found to be the normal cellular constituent. These observations suggested that free flavin coenzyme may be a physiological electron carrier for APS reductase, and the enzyme may be called AMP, sulfite:flavin oxidoreductase. Km (APS) of this enzyme is lower than 1 microM. The enzyme is not inhibited by ATP and GTP, but was inhibited by AMP and sulfite. Its extremely low Km (APS) enables this enzyme to reduce any traces of cytosolic APS which is present only at micromolar concentration, and inhibition by sulfite makes this organism utilize an energetically favorable electron acceptor, sulfite, preferentially over APS which is produced from sulfate at the cost of ATP.
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PMID:Catalytic properties of adenylylsulfate reductase from Desulfovibrio vulgaris Miyazaki. 911 53

Direct electrochemical studies, utilizing two voltammetric methods-square-wave voltammetry (SWV) and cyclic voltammetry (CV)-have been performed on recombinant forms of the flavin domain of spinach assimilatory nitrate reductase in the presence of NAD+ analogs. The reduction potentials (E degrees ') of the flavin domains have been determined at an edge pyrolytic graphite electrode utilizing MgCl2 as a redox-inactive promoter. Under identical experimental conditions (pH 7.0, 25 degrees C), the two-electron reduction potential for the FAD/FADH2 couple has been determined to be -274 and -257 mV by SWV and CV, respectively. In contrast, the reduction potentials of free FAD have been determined to be -234 and -227 mV by SWV and CV, respectively. The reduction potentials of the complex formed between the FAD prosthetic group in the recombinant flavin domain and various NAD+ analogs have been determined to be as follows: NAD+ (E degrees ' = -192 mV), 5'-ADP ribose (E degrees ' = -199 mV), ADP (E degrees ' = -154 mV), AMP (E degrees ' = -196 mV), adenosine (E degrees ' = -192 mV), adenine (E degrees ' = -220 mV), and NMN (E degrees ' = -208 mV). In contrast to these positive shifts in reduction potential, nicotinamide (E degrees ' = -268 mV) had very little effect on the reduction potential of this flavin complex. Moreover, addition of NAD+ to the FAD prosthetic group in a variety of mutant forms of the recombinant flavin domain resulted in positive shifts in the reduction potential of the complex, although the magnitude of the shifts varied from a minimum of 6 mV obtained for the C240A mutant to a maximum of 79 mV obtained for the C62S mutant. These results represent the first extensive application of direct electrochemistry to examine the redox properties of assimilatory nitrate reductase and indicate that complex formation with NAD+, or various NAD+ analogs, results in a positive shift in the flavin reduction potential, with the magnitude of the shift correlating well with the efficiency of the inhibitor.
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PMID:Direct electrochemistry of the flavin domain of assimilatory nitrate reductase: effects of NAD+ and NAD+ analogs. 928 15

An enzyme activity splitting FAD to AMP and riboflavin 4',5'-cyclic phosphate (4',5'-cFMN), with a Km of 6-8 microM, was partially purified from the cytosolic fraction of rat liver homogenates. 4', 5'-cFMN was characterized by enzyme, HPLC, UV-visible and NMR spectroscopic analyses. The data suggest that a novel enzyme, tentatively named FAD-AMP lyase (cyclizing) or FMN cyclase, is involved. Also, 4',5'-cFMN was hydrolysed to 5'-FMN by a rat liver cyclic phosphodiesterase. The results indicate a novel enzymic pathway for flavins in mammals, and support the biological relevance of 4',5'-cFMN, perhaps as a flavocoenzyme or a regulatory signal.
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PMID:Enzymic formation of riboflavin 4',5'-cyclic phosphate from FAD: evidence for a specific low-Km FMN cyclase in rat liver1. 948 Sep 5

A thermostable enzyme previously identified as an NADH oxidase has been purified from Thermus aquaticus YT-1 by chromatography on DEAE-cellulose and AMP-Sepharose. The enzyme is dimeric with subunits of 54 kDa and one molecule FAD/subunit. The FAD is tightly bound, but it can be removed reversibly by hydrophobic chromatography at low pH. The blue flavin semiquinone is stabilised during photo-chemical reduction of the enzyme. Chemical reduction by static titration with dithionite ion showed that the enzyme requires about 5 mol dithionite/mol FAD for full reduction, and that reduction occurs in four phases. Reduction by the substrate NADH is incomplete, with the formation of a new long-wavelength absorption underlying the semiquinone absorption. Amino acid sequencing showed that the T aquaticus enzyme resembles other microbial flavoenzymes that function in two-enzyme systems for the reduction of peroxides, and which contain two redox-active disulphide groups in addition to the flavin. The enzyme catalyses the reduction of O2, ferricyanide ion, 2,6-dichloroindophenol, and 5,5'dithiobis(2,2'-dinitrobenzoate), and of cumene hydroperoxide in the presence of the small protein component (AhpC) of the peroxide-reducing system of Salmonella typhimurium. The reduction of O2 is slow in the absence of exogenous flavin while dye reduction is fast, suggesting that the free flavin that is added to the usual assay for T. aquaticus NADH oxidase functions by mediating electron transfer from enzyme-bound reduced flavin to O2. The physiological function of the enzyme is probably in peroxide reduction with a small protein analogous to AhpC as the natural electron acceptor.
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PMID:Purification and characterisation of NADH oxidase from Thermus aquaticus YT-1 and evidence that it functions in a peroxide-reduction system. 949 70

NADPH:ferredoxin reductase (AvFPR) is involved in the response to oxidative stress in Azotobacter vinelandii. The crystal structure of AvFPR has been determined at 2.0 A resolution. The polypeptide fold is homologous with six other oxidoreductases whose structures have been solved including Escherichia coli flavodoxin reductase (EcFldR) and spinach, and Anabaena ferredoxin:NADP+ reductases (FNR). AvFPR is overall most homologous to EcFldR. The structure is comprised of a N-terminal six-stranded antiparallel beta-barrel domain, which binds FAD, and a C-terminal five-stranded parallel beta-sheet domain, which binds NADPH/NADP+ and has a classical nucleotide binding fold. The two domains associate to form a deep cleft where the NADPH and FAD binding sites are juxtaposed. The structure displays sequence conserved motifs in the region surrounding the two dinucleotide binding sites, which are characteristic of the homologous enzymes. The folded over conformation of FAD in AvFPR is similar to that in EcFldR due to stacking of Phe255 on the adenine ring of FAD, but it differs from that in the FNR enzymes, which lack a homologous aromatic residue. The structure of AvFPR displays three unique features in the environment of the bound FAD. Two features may affect the rate of reduction of FAD: the absence of an aromatic residue stacked on the isoalloxazine ring in the NADPH binding site; and the interaction of a carbonyl group with N10 of the flavin. Both of these features are due to the substitution of a conserved C-terminal tyrosine residue with alanine (Ala254) in AvFPR. An additional unique feature may affect the interaction of AvFPR with its redox partner ferredoxin I (FdI). This is the extension of the C-terminus by three residues relative to EcFldR and by four residues relative to FNR. The C-terminal residue, Lys258, interacts with the AMP phosphate of FAD. Consequently, both phosphate groups are paired with a basic group due to the simultaneous interaction of the FMN phosphate with Arg51 in a conserved FAD binding motif. The fourth feature, common to homologous oxidoreductases, is a concentration of 10 basic residues on the face of the protein surrounding the active site, in addition to Arg51 and Lys258.
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PMID:The crystal structure of NADPH:ferredoxin reductase from Azotobacter vinelandii. 986 48

The crystal structure of electron transfer flavoprotein (ETF) from Paracoccus denitrificans was determined and refined to an R-factor of 19.3% at 2.6 A resolution. The overall fold is identical to that of the human enzyme, with the exception of a single loop region. Like the human structure, the structure of the P. denitrificans ETF is comprised of three distinct domains, two contributed by the alpha-subunit and the third from the beta-subunit. Close analysis of the structure reveals that the loop containing betaI63 is in part responsible for conferring the high specificity of AMP binding by the ETF protein. Using the sequence and structures of the human and P. denitrificans enzymes as models, a detailed sequence alignment has been constructed for several members of the ETF family, including sequences derived for the putative FixA and FixB proteins. From this alignment, it is evident that in all members of the ETF family the residues located in the immediate vicinity of the FAD cofactor are identical, with the exception of the substitution of serine and leucine residues in the W3A1 ETF protein for the human residues alphaT266 and betaY16, respectively. Mapping of ionic differences between the human and P. denitrificans ETF onto the structure identifies a surface that is electrostatically very similar between the two proteins, thus supporting a previous docking model between human ETF and pig medium-chain acyl-CoA dehydrogenase (MCAD). Analysis of the ionic strength dependence of the electron transfer reaction between either human or P. denitrificans ETF and MCAD demonstrates that the human ETF functions optimally at low ( approximately 10 mequiv) ionic strength, while P. denitrificans ETF is a better electron acceptor at higher (>75 mequiv) ionic strength. This suggests that the electrostatic surface potential of the two proteins is very different and is consistent with the difference in isoelectric points between the proteins. Analysis of the electrostatic potentials of the human and P. denitrificans ETFs reveals that the P. denitrificans ETF is more negatively charged. This excess negative charge may contribute to the difference in redox potentials between the two ETF flavoproteins and suggests an explanation for the opposing ionic strength dependencies for the reaction of MCAD with the two ETFs. Furthermore, by analysis of a model of the previously described human-P. denitrificans chimeric ETF protein, it is possible to identify one region of ETF that participates in docking with ETF-ubiquinone oxidoreductase, the physiological electron acceptor for ETF.
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PMID:Crystal structure of Paracoccus denitrificans electron transfer flavoprotein: structural and electrostatic analysis of a conserved flavin binding domain. 1002 81

Recombinant house fly (Musca domestica) cytochrome P450 reductase has been purified by anion exchange and affinity chromatography. Steady-state kinetics of cytochrome c reductase activity revealed a random Bi-Bi mechanism with formation of a ternary P450 reductase-NADPH-electron acceptor complex as catalytic intermediate. NADP(H) binding is essential for fast hydride ion transfer to FAD, as well as for electron transfer from FMN to cytochrome c. Reduced cytochrome c had no effect on the enzyme activity, while NADP+ and 2'-AMP inhibited P450 reductase competitively with respect to NADPH and noncompetitively with respect to cytochrome c. The affinity of the P450 reductase to NADPH is 10 times higher than to NADP+ (Kd of 0.31 and 3.3 microM, respectively). Such an affinity change during catalysis could account for a +30 mV shift of the redox potential of FAD. Cys560 was substituted for Tyr by site-directed mutagenesis. This mutation decreased enzyme affinity to NADPH 35-fold by decreasing the bimolecular rate constant of nucleotide binding with no detectable effect on the kinetic mechanism. The affinity of the C560Y mutant enzyme to NADP+ decreased 9-fold compared to the wild-type enzyme, while the affinity to 2'-AMP was not significantly affected, suggesting that Cys560 is located in the nicotinamide binding site of the active, full-size enzyme in solution.
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PMID:Kinetic mechanism of cytochrome P450 reductase from the house fly (Musca domestica). 1031 36


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