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: EC:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cDNA encoding QPc-9.5 kDa (subunit VII) of bovine heart mitochondrial ubiquinol-cytochrome c reductase was cloned and sequenced. This cDNA is 665 base pairs long with an open reading frame of 246 base pairs that encodes an 81-amino acid mature QPc-9.5 kDa. The insert contains 395 base pairs of a 3'-noncoding sequence with a poly(A) tail. The amino acid sequence of QPc-9.5 kDa deduced from this nucleotide sequence is the same as that obtained by protein sequencing except that residue 61 is tryptophan instead of cysteine. The QPc-9.5 kDa was overexpressed in Escherichia coli JM109 cells as a glutathione S-transferase fusion protein (GST-QPc) using the expression vector, pGEX/QPc. The yield of soluble active recombinant GST-QPc fusion protein depends on the induction growth time, temperature, and medium. Maximum yield of recombinant fusion protein was obtained from cells harvested 3 h postinduction of growth at 27 degrees C on LB medium containing betaine and sorbitol. QPc-9.5 kDa was released from the fusion protein by proteolytic cleavage with thrombin. Isolated recombinant QPc-9.5 kDa showed one protein band in SDS-polyacrylamide gel electrophroesis corresponding to subunit VII of mitochondrial ubiquinol-cytochrome c reductase. Although the isolated recombinant QPc-9.5 kDa is soluble in aqueous solution, it is in a highly aggregated form, with an apparent molecular mass of over 1 million. Addition of detergent deaggreates the isolated protein to the monomeric state, suggesting that the recombinant protein exists as a hydrophobic aggregation in aqueous solution. The recombinant QPc-9.5 kDa binds ubiquinone and shows a spectral blue shift. Upon titration of the recombinant protein with ubiquinone, a saturation behavior is observed, suggesting that the binding is specific and that the recombinant protein may be in the functionally active state.
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PMID:Cloning, gene sequencing, and expression of the small molecular mass ubiquinone-binding protein of mitochondrial ubiquinol-cytochrome c reductase. 759 38

We isolated and characterized mutants defective in nuo, encoding NADH dehydrogenase I, the multisubunit complex homologous to eucaryotic mitochondrial complex I. By Southern hybridization and/or sequence analysis, we characterized three distinct mutations: a polar insertion designated nuoG::Tn10-1, a nonpolar insertion designated nuoF::Km-1, and a large deletion designated delta(nuoFGHIJKL)-1. Cells carrying any of these three mutations exhibited identical phenotypes. Each mutant exhibited reduced NADH oxidase activity, grew poorly on minimal salts medium containing acetate as the sole carbon source, and failed to produce the inner, L-aspartate chemotactic band on tryptone swarm plates. During exponential growth in tryptone broth, nuo mutants grew as rapidly as wild-type cells and excreted similar amounts of acetate into the medium. As they began the transition to stationary phase, in contrast to wild-type cells, the mutant cells abruptly slowed their growth and continued to excrete acetate. The growth defect was entirely suppressed by L-serine or D-pyruvate, partially suppressed by alpha-ketoglutarate or acetate, and not suppressed by L-aspartate or L-glutamate. We extended these studies, analyzing the sequential consumption of amino acids by both wild-type and nuo mutant cells growing in tryptone broth. During the lag and exponential phases, both wild-type and mutant cells consumed, in order, L-serine and L-aspartate. As they began the transition to stationary phase, both cell types consumed L-tryptophan. Whereas wild-type cells then consumed L-glutamate, glycine, L-threonine, and L-alanine, mutant cells utilized these amino acids poorly. We propose that cells defective for NADH dehydrogenase I exhibit all these phenotypes, because large NADH/NAD+ ratios inhibit certain tricarboxylic acid cycle enzymes, e.g., citrate synthase and malate dehydrogenase.
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PMID:Mutations in NADH:ubiquinone oxidoreductase of Escherichia coli affect growth on mixed amino acids. 815 82

The carboxyl-terminal region of plant ferredoxin-NADP+ reductases is formed by an invariant alpha-helix/loop/beta-strand, culminating in a conserved tyrosine that displays extensive interaction with the prosthetic group FAD. We have investigated the potential role of the terminal region in reductase function, by introducing mutations and deletions on pea ferredoxin-NADP+ reductase overexpressed in Escherichia coli. Replacement of the terminal tyrosine by tryptophan, phenylalanine, serine, and glycine resulted in a 2.2-, 2.0-, 22-, and 302-fold reduction, respectively, in kcat for the diaphorase reaction, whereas elimination of the tyrosine caused a 846-fold decrease in kcat. Km values were largely unaffected by the substitutions. Similar results were obtained when the mutants were assayed for cytochrome c reduction, indicating that aromaticity is the most important factor to the function of the tyrosine in catalysis. The presence of the phenol ring at the carboxyl-terminal position of wild-type reductase is important, but not an absolute requirement for enzyme function or FAD assembly. Deletion of the alpha-helix/beta-strand region prevented reductase proper folding in the bacterial host, while shortening of the terminal region by splicing 3 amino acids at the beginning of the alpha-helix produced a moderately soluble reductase, devoid of FAD and enzymatic activity.
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PMID:Probing the role of the carboxyl-terminal region of ferredoxin-NADP+ reductase by site-directed mutagenesis and deletion analysis. 836 77

Cytochrome P450BM-3 from Bacillus megaterium is a soluble, catalytically self-sufficient fatty acid mono-oxygenase that, in structural organization and amino acid sequence, resembles the Class II (microsomal) P450 systems. Its single polypeptide chain contains both a P450 heme domain and an NADPH:P450 reductase domain, each of which bears significant homology with its microsomal counterparts. We report here the critical nature of three amino acids in the reductase domain of this enzyme with respect to FMN binding and catalytic activity. We used site-directed mutagenesis to change glycine 570 to bulkier amino acids; none of these mutant enzymes contained FMN after purification. We also made substitutions for tryptophan 574 and tyrosine 536, which by sequence analogy (Porter, T. D. (1991) Trends Biochem. Sci. 16, 154-158) were proposed to bind FMN through stacking of the aromatic rings with the isoalloxazine ring of the flavin. Mutants of tryptophan 574 which retained the aromatic side chain contained no less than 0.85 mol of FMN per mol of enzyme, while aspartate and glycine substitutions yielded enzymes which did not incorporate FMN. Substitution of tyrosine 536 with aspartate gave an enzyme which contained 0.44 mol of FMN per mol of enzyme but was inactive as a fatty acid hydroxylase and had only 2% of wild-type cytochrome c reductase activity, while the glycine mutant at this position bound no FMN. Furthermore, although all of the mutant enzymes contained 1 mol of FAD per mol of enzyme, the Y536D mutant and those entirely lacking FMN retained no more than 40% of wild-type ferricyanide reductase activity. By assaying these enzymes in the presence of added FMN, we were able to assess the relative importance of the residues in the wild-type sequence with respect to their contribution to FMN binding. In addition, the aromatic mutants of tryptophan 574, which were nearly as active in cytochrome c reduction as wild-type P450BM-3, were only 20% as active in myristate hydroxylation as the wild-type enzyme, suggesting that this amino acid plays an important role in the flow of electrons between the P450 heme and reductase domains.
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PMID:Critical residues involved in FMN binding and catalytic activity in cytochrome P450BM-3. 846 85

Treatment of red cell ghosts with ozone inhibited both AChE (marking the outside of the membrane) and G3PDH (marking the inside of the membrane). There was no change in tryptophan fluorescence of the ghosts after the ozone treatment. Band 3 protein was isolated from the ozone-treated ghosts. The protein was digested with trypsin to obtain water soluble peptides from the cytoplasmic N-terminal tail and the interhelical loops. Fluorescent peptides included GWVIHPLGLR from the outer loop between helices 7 and 8, and peptide WMEAAR from the N-terminal cytoplasmic tail. Neither one of these peptides was oxidized by ozone. This was true whether or not the ghosts were sealed. We conclude that the position of these tryptophans either in the membrane structure, or because of binding to other proteins in the cytoplasmic tail, protects them from oxidation by ozone. Treatment of horse heart cytochrome c with ozone did not change the absorbance spectrum in the heme region or the tryptophan absorbing region. HPLC of the ozone-treated cytochrome c showed that cytochrome c was being modified, indicated by a change in the elution time. Treatment of cytochrome c with ozone did not change the activity in the NADH-cytochrome c reductase assay. Digestion of the ozone-treated cytochrome c with trypsin gave peptides which demonstrated normal fluorescence. (Cytochrome c has abnormally low fluorescence, which is not changed by ozone exposure.) The peptides were separated by HPLC. The fluorescence of the tryptophan-containing peptide (GITWK) was not decreased by treatment of the cytochrome c by ozone. Amino acid analysis of the ozone-treated cytochrome c indicated that methionine was oxidized. We conclude that tryptophan in cytochrome c is protected from oxidation by ozone because of the interaction with the porphyrin ring. Bovine serum albumin and human serum albumin were treated with ozone. There was a monotonic decrease in tryptophan fluorescence in both cases. Digestion of BSA with trypsin produced two fluorescent peptides. The peptide FWGK was identified by coelution with the authentic peptide. The putative peptide AWSVAR was not the same as the chemically synthesized peptide. The peptide sequences FWGK and "AWSVAR" were both oxidized in ozone-treated bovine serum albumin, with no detectable discrimination. Tryptic digestion of the ozone-treated human serum albumin produced a single fluorescent peptide, which was oxidized by ozone. The putative peptide AWAVAR in the tryptic digest of HSA was distinct from chemically synthesized peptide. The oxidation of tryptophans in proteins by ozone is markedly influenced by position in tertiary structure, position in membrane structure, and by chemical interactions within the protein.
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PMID:Reaction of ozone with protein tryptophans: band III, serum albumin, and cytochrome C. 902 65

The complete nucleotide sequence of the Chlamydomonas eugametos (Chlamydomonadales, Chlorophyceae, sensu Mattox and Stewart) mitochondrial genome has been determined (22,897 bp, 34.6% G + C). The genes identified in this circular-mapping genome include those for apocytochrome b, subunit 1 of the cytochrome oxidase complex, subunits 1, 2, 4, 5, and 6 of the NADH dehydrogenase complex, discontinuous large and small subunit ribosomal rRNAs and three tRNAs whose anticodons CAU, CCA and UUG are specific for methionine, tryptophan and glutamine, respectively. The C. eugametos mitochondrial DNA (mtDNA), therefore, shares almost the same reduced set of coding functions and similar unusual features of rRNA gene organization with the linear 15.8 kb mtDNA of Chlamydomonas reinhardtii, the only other completely sequenced chlamydomonadalean mtDNA. However, sequence analysis of the C. eugametos mtDNA has revealed the following distinguishing features relative to those of C. reinhardtii: (1) the absence of a reverse transcriptase-like gene homologue, (2) the presence of an additional gene for tRNA(met) that may be a pseudogene, (3) a completely different gene order, (4) transcription of all genes from the same mtDNA strand, (5) a lower G + C content, (6) less pronounced bias in codon usage, and (7) nine group I introns, several of which contain open reading frames coding for potential maturases/endonucleases and two have a nucleotide at the 5' or 3' splice site of the deduced precursor RNAs that deviates from highly conserved nucleotides reported in other group I introns. The features of mitochondrial genome organization and gene content shared by C. eugametos and C. reinhardtii contrast with those of other green algal mtDNAs that have been characterized in detail. The deep evolutionary divergence between these two Chlamydomonas taxa within the Chlamydomonadales suggests that their shared features of mitochondrial genome organization evolved prior to the origin of this group.
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PMID:Complete sequence of the mitochondrial DNA of Chlamydomonas eugametos. 948 40

The nucleotide sequences of two segments of 6,737 ntp and 258 nto of the 18.4-kb circular mitochondrial (mt) DNA molecule of the soft coral Sarcophyton glaucum (phylum Cnidaria, class Anthozoa, subclass Octocorallia, order Alcyonacea) have been determined. The larger segment contains the 3' 191 ntp of the gene for subunit 1 of the respiratory chain NADH dehydrogenase (ND1), complete genes for cytochrome b (Cyt b), ND6, ND3, ND4L, and a bacterial MutS homologue (MSH), and the 5' terminal 1,124 ntp of the gene for the large subunit rRNA (1-rRNA). These genes are arranged in the order given and all are transcribed from the same strand of the molecule. The smaller segment contains the 3' terminal 134 ntp of the ND4 gene and a complete tRNA(f-Met) gene, and these genes are transcribed in opposite directions. As in the hexacorallian anthozoan, Metridium senile, the mt-genetic code of S. glaucum is near standard: that is, in contrast to the situation in mt-genetic codes of other invertebrate phyla, AGA and AGG specify arginine, and ATA specifies isoleucine. However, as appears to be universal for metazoan mt-genetic codes, TGA specifies tryptophan rather than termination. Also, as in M. senile the mt-tRNA(f-Met) gene has primary and secondary structural features resembling those of Escherichia coli initiator tRNA, including standard dihydrouridine and T psi C loop sequences, and a mismatched nucleotide pair at the top of the amino-acyl stem. The presence of a mutS gene homologue, which has not been reported to occur in any other known mtDNA, suggests that there is mismatch repair activity in S. glaucum mitochondria. In support of this, phylogenetic analysis of MutS family protein sequences indicates that the S. glaucum mtMSH protein is more closely related to the nuclear DNA-encoded mitochondrial mismatch repair protein (MSH1) of the yeast Saccharomyces cerevisiae than to eukaryotic homologues involved in nuclear function, or to bacterial homologues. Regarding the possible origin of the S. glaucum mtMSH gene, the phylogenetic analysis results, together with comparative base composition considerations, and the absence of an MSH gene in any other known mtDNA best support the hypothesis that S. glaucum mtDNA acquired the mtMSH gene from nuclear DNA early in the evolution of octocorals. The presence of mismatch repair activity in S. glaucum mitochondria might be expected to influence the rate of evolution of this organism's mtDNA.
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PMID:Mitochondrial DNA of the coral Sarcophyton glaucum contains a gene for a homologue of bacterial MutS: a possible case of gene transfer from the nucleus to the mitochondrion. 954 36

Dihydrolipoamide dehydrogenase (LADH) lipoamide reductase activity decreased whereas enzyme diaphorase activity increased after LADH treatment with myeloperoxidase (MPO) dependent systems (MPO/H2O2/halide, MPO/NADH/halide and MPO/H2O2/nitrite systems. LADH inactivation was a function of the composition of the inactivating system and the incubation time. Chloride, iodide, bromide, and the thiocyanate anions were effective complements of the MPO/H2O2 system. NaOCl inactivated LADH, thus supporting hypochlorous acid (HOCl) as putative agent of the MPO/H2O2/NaCl system. NaOCl and the MPO/H2O2/NaCl system oxidized LADH thiols and NaOCl also oxidized LADH methionine and tyrosine residues. LADH inactivation by the MPO/NADH/halide systems was prevented by catalase and enhanced by superoxide dismutase, in close agreement with H2O2 production by the LADH/NADH system. Similar effects were obtained with lactoperoxidase and horse-radish peroxidase supplemented systems. L-cysteine, N-acetylcysteine, penicillamine, N-(2-mercaptopropionylglycine), Captopril and taurine protected LADH against MPO systems and NaOCl. The effect of the MPO/H2O2/NaNO2 system was prevented by MPO inhibitors (sodium azide, isoniazid, salicylhydroxamic acid) and also by L-cysteine, L-methionine, L-tryptophan, L-tyrosine, L-histidine and reduced glutathione. The summarized observations support the hypothesis that peroxidase-generated "reactive species" oxidize essential thiol groups at LADH catalytic site.
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PMID:Inactivation of myocardial dihydrolipoamide dehydrogenase by myeloperoxidase systems: effect of halides, nitrite and thiol compounds. 1019 78

Dihydrolipoamide dehydrogenase (LADH) from Trypanosoma cruzi was inactivated by treatment with myeloperoxidase (MPO)-dependent systems. With MPO/H2O2/NaCl, LADH lipoamide reductase and diaphorase activities significantly decreased as a function of incubation time. Iodide, bromide, thiocyanide and chloride effectively supplemented the MPO/H2O2 system, KI and NaCl being the most and the least effective supplements, respectively. LADH inactivation by MPO/H2O2/NaCl and by NaOCl was similarly prevented by thiol compounds such as GSH, L-cysteine, N-acetylcysteine, penicillamine and N-(2-mercaptopropionyl-glycine) in agreement with the role of HOCI in LADH inactivation by MPO/H2O2/NaCl. LADH was also inactivated by MPO/NADH/halide, MPO/H2O2/NaNO2 and MPO/NADH/NaNO2 systems. Catalase prevented the action of the NADH-dependent systems, thus supporting H2O2 production by NADH-supplemented LADH. MPO inhibitors (4-aminobenzoic acid hydrazide, and isoniazid), GSH, L-cysteine, L-methionine and L-tryptophan prevented LADH inactivation by MPO/H2O2/NaNO2. Other MPO systems inactivating LADH were (a) MPO/H2O2/chlorpromazine; (b) MPO/H2O2/monophenolic systems, including L-tyrosine, serotonin and acetaminophen and (c) MPO/H2O2/di- and polyphenolic systems, including norepinephrine, catechol, nordihydroguaiaretic acid, caffeic acid, quercetin and catechin. Comparison of the above effects and those previously reported with pig myocardial LADH indicates that both enzymes were similarly affected by the MPO-dependent systems, allowance being made for T. cruzi LADH diaphorase inactivation and the greater sensitivity of its LADH lipoamide reductase activity towards the MPO/H2O2/NaCl system and NaOCl.
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PMID:Trypanosoma cruzi dihydrolipoamide dehydrogenase is inactivated by myeloperoxidase-generated "reactive species". 1082 17

The globule dimensions and some electron and conformational properties of the flavoprotein (peripheral) fragment of the mitochondrial NADH dehydrogenase were determined by the time-resolved, phase-modulating, and polarization fluorescence spectroscopies, as well as correlated confocal microscopy. The rotational and the diffusion (translocation) diameters of the protein fragment were shown to be no less than 44 A and approximately 72 A, respectively. The diameter of protomitochondrial particles from the bovine heart, which were used for the isolation of the fraction of peripheral fragments, was no less than 2300 A. The fluorescence from tryptophan and flavin fluorophores in the fragment is strongly quenched by iron of the iron-sulfur clusters, which suggests that a strong electron-vibrational interaction of iron with Trp residues and flavin takes place. An overlapping of the electron clouds of iron-sulfur clusters, Trp residues, and flavin is likely to facilitate the electron transfer through the protein. The heat inactivation of the enzyme was accompanied by neither its substantial conformational changes, nor a considerable release of iron ions from the clusters located near the Trp residues.
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PMID:[The globule diameter and various electron and conformational properties of the flavoprotein fragment of mitochondrial NADH dehydrogenase studied by fluorescence spectroscopy]. 1122 Dec 52


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