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:1.10.3.3 (ascorbate oxidase)
778 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Apoascorbate oxidase has been shown to have a molecular weight of 137,000 +/- 3,000 and essentially the same gross quaternary conformation as native ascorbate oxidase. The apoenzyme, however, lacks much of the conformational stability of the native enzyme. The removal of the copper from the oxidase protein, and the simultaneous reduction of the disulfide bonds results in an apoenzyme of lower structural stability than the native oxidase. The aging of apoascorbate oxidase has been found to involve a loss of ionizable tyrosine residues and a dissociation to subunits and component polypeptide chains, which was not observed with the more stable native and holo enzymes. The molecular weight of holoascorbate oxidase has been determined to be 285,000. An s020, w of 9.79 has been determined for the holoenzyme. Holoascorbate oxidase has been shown to have an electrophoretic mobility on polyacrylamide gels that is 23% lower than either the native or apoenzyme. Furthermore, electrophoresis of the holoenzyme, in buffers containing dodecyl sulfate, and also isoelectric focusing of the holenzyme, produce patterns of greater similarity to those of apoascorbate oxidase than the native enzyme.
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PMID:Concerning the metalloenzyme ascorbate oxidase. 85 93

The genomic clone named Bp10 contains a member of a small pollen-specific gene family of B. napus. The expression of the Bp10 gene family is maximal in early binucleate microspores and declines considerably in mature trinucleate pollen. Homologues of the Bp10 genes are expressed in the pollen of other plant species. The pollen-specific expression of the gene contained in the genomic clone was confirmed in tobacco plants transformed with a chimeric Bp10 promoter/GUS construct. A promoter fragment of 396 bp is sufficient to direct a strong and correct spatial and temporal expression in transgenic plants. The Bp10 gene family codes for proteins of 62 kDa showing approximately 30% sequence identify to cucumber and pumpkin ascorbate oxidases (AAOs). However, the AAO active centres are not conserved in the Bp10 products, suggesting an evolutionary relationship but a different enzymatic activity for these proteins. Expression of a recombinant Bp10 protein in E. coli inhibits bacterial growth on minimal medium, suggesting the production of an enzymatically active polypeptide in bacteria. No AAO activity could be correlated with the expression of the recombinant protein. Moreover, substances affecting AAO activity do not appear to influence the inhibitory activity of the protein produced in bacteria. However, as indicated by the rescue of bacterial growth in the presence of sodium bicarbonate or gaseous CO2, the Bp10 protein activity could be modulated by CO2 levels.
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PMID:A Brassica napus gene family which shows sequence similarity to ascorbate oxidase is expressed in developing pollen. Molecular characterization and analysis of promoter activity in transgenic tobacco plants. 130 99

The crystal structure of the fully oxidized form of ascorbate oxidase (EC 1.10.3.3) from Zucchini has been refined at 1.90 A (1 A = 0.1 nm) resolution, using an energy-restrained least-squares refinement procedure. The refined model, which includes 8764 protein atoms, 9 copper atoms and 970 solvent molecules, has a crystallographic R-factor of 20.3% for 85,252 reflections between 8 and 1.90 A resolution. The root-mean-square deviation in bond lengths and bond angles from ideal values is 0.011 A and 2.99 degrees, respectively. The subunits of 552 residues (70,000 Mr) are arranged as tetramers with D2 symmetry. One of the dyads is realized by the crystallographic axis parallel to the c-axis giving one dimer in the asymmetric unit. The dimer related about this crystallographic axis is suggested as the dimer present in solution. Asn92 is the attachment site for one of the two N-linked sugar moieties, which has defined electron density for the N-linked N-acetyl-glucosamine ring. Each subunit is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type and related to plastocyanin and azurin. An analysis of intra- and intertetramer hydrogen bond and van der Waals interactions is presented. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine and a methionine ligand and represents the type-1 copper. It is located in domain 3. The bond lengths of the type-1 copper centre are comparable to the values for oxidized plastocyanin. The trinuclear cluster has eight histidine ligands symmetrically supplied from domain 1 and 3. It may be subdivided into a pair of copper atoms with histidine ligands whose ligating N-atoms (5 NE2 atoms and one ND1 atom) are arranged trigonal prismatic. The pair is the putative type-3 copper. The remaining copper has two histidine ligands and is the putative spectroscopic type-2 copper. Two oxygen atoms are bound to the trinuclear species as OH- or O2- and bridging the putative type-3 copper pair and as OH- or H2O bound to the putative type-2 copper trans to the copper pair. The bond lengths within the trinuclear copper site are similar to comparable binuclear model compounds. The putative binding site for the reducing substrate is close to the type-1 copper.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Refined crystal structure of ascorbate oxidase at 1.9 A resolution. 154 98

New resonance Raman (RR) spectra at 15 K are reported for poplar (Populus nigra) and oleander (Oleander nerium) plastocyanins and for Alcaligenes faecalis pseudoazurin. The spectra are compared with those of other blue copper proteins (cupredoxins). In all cases, nine or more vibrational modes between 330 and 460 cm-1 can be assigned to a coupling of the Cu-S(Cys) stretch with Cys ligand deformations. The fact that these vibrations occur at a relatively constant set of frequencies is testimony to the highly conserved ground-state structure of the Cu-Cys moiety. Shifts of the vibrational modes by 1-3 cm-1 upon deuterium exchange can be correlated with N-H...S hydrogen bonds from the protein backbone to the sulfur of the Cys ligand. There is marked variability in the intensities of these Cys-related vibrations, such that each class of cupredoxin has its own pattern of RR intensities. For example, plastocyanins from poplar, oleander, French bean, and spinach have their most intense feature at approximately 425 cm-1; azurins show greatest intensity at approximately 410 cm-1, stellacyanin and ascorbate oxidase at approximately 385 cm-1, and nitrite reductase at approximately 360 cm-1. These variable intensity patterns are related to differences in the electronic excited-state structures. We propose that they have a basis in the protein environment of the copper-cysteinate chromophore. A further insight into the vibrational spectra is provided by the structures of the six cupredoxins for which crystallographic refinements at high resolution are available (plastocyanins from P. nigra, O. nerium, and Enteromorpha prolifera, pseudoazurin from A. faecalis, azurin from Alcaligenes denitrificans, and cucumber basic blue protein). The average of the Cu-S(Cys) bond lengths is 2.12 +/- 0.05 A. Since the observed range of bond lengths falls within the precision of the determinations, this variation is considered insignificant. The Cys ligand dihedral angles are also highly conserved. Cu-S gamma-C beta-C alpha is always near -170 degrees and S gamma-C beta-C alpha-N near 170 degrees. As a result, the Cu-S gamma bond is coplanar with the Cys side-chain atoms and part of the polypeptide backbone. The coplanarity accounts for the extensive coupling of Cu-S stretching and Cys deformation modes as seen in the RR spectrum. The conservation of this copper-cysteinate conformation in cupredoxins may indicate a favored pathway for electron transfer.
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PMID:Resonance Raman spectra of plastocyanin and pseudoazurin: evidence for conserved cysteine ligand conformations in cupredoxins (blue copper proteins). 193 14

Two crystal forms of the multi-copper protein ascorbate oxidase from Zucchini have been analysed at 2.5 A (1 A = 0.1 nm) resolution and a model of the polypeptide chain and the copper ions and their ligands has been built. Crystal forms M2 and M1 contain a dimer of 140,000 Mr and a tetramer of 280,000 Mr, respectively, in the asymmetric unit. The crystallographic analysis proceeded by multiple isomorphous replacement in M2 followed by solvent flattening and averaging about the local dyad axis. M1 was solved by Patterson search techniques using the M2 electron density. M1 was fourfold averaged. M1 and M2 were combined and the process of averaging repeated in cycles. An atomic model was built into the resulting electron density map and refinement initiated. The current R values of M2 and M1 are 24.5% and 32.6%, respectively. Excellent stereo chemistry was maintained, with root-mean-square deviations of bond lengths and bond angles from average values of 0.02 A and 3.1 degrees, respectively. Each subunit of about 550 amino acid residues has a globular shape with dimensions of 49 A x 53 A x 65 A. It is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type. It is distantly related to plastocyanin. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine, and a methionine ligand and represents the type-1 copper. It is located in the third domain. The trinuclear cluster has eight histidine ligands. It may be subdivided into a pair of copper atoms with six histidine ligands arranged trigonal prismatic. The pair probably represents the type-3 copper. The remaining copper has two histidine ligands. Its third site of co-ordination is formed by the pair of copper atoms. The fourth ligand may be OH- represented by a small protrusion of electron density. This copper probably is the type-2 copper. The symmetry of the trinuclear cluster is C2 and the ligands are supplied symmetrically by domains 1 and 3. However, domain 1 does not contain a type-1 copper and lacks the characteristic ligands. The unprecedented trinuclear cluster probably represents the oxygen binding and electron storage site.
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PMID:X-ray crystal structure of the blue oxidase ascorbate oxidase from zucchini. Analysis of the polypeptide fold and a model of the copper sites and ligands. 271 59

Embryonic rat-brain extract contains a collagen-stimulating factor which enhances the production of collagen types I, III, IV and V by cultured rat muscle cells. Here we report on the partial characterization and possible mechanism of action of a low-molecular-mass fraction with ascorbate-like activity isolated from embryonic rat brain extracts. This activity eluted very close to ascorbate when filtered through Bio-Gel P-2 and Sephadex G-10. The peak of biological activity showed properties of a reducing agent. Both the biological and reducing activities were lost when the fraction was treated with the enzyme ascorbate oxidase. This factor enhanced in a time-dependent manner, the secretion of procollagen, pulse-labeled with [3H] proline. Incubation of the muscle cultures with the factor increased by 15-fold the ratio of hydroxyproline to proline residues in secreted macromolecules over controls. A fourfold increase in the above ratio was obtained for the cellular proteins. Crude homogenates from control and factor-stimulated cultures were tested for prolyl hydroxylase activity using [3H](Pro-Gly-Pro)n as a substrate. Cultures treated with the collagen-stimulating factor showed a 5-50-fold increase in prolyl hydroxylation activity compared to controls. No effect on prolyl hydroxylation was found when the factor was added in vitro to either control or stimulated enzyme preparations. Our results suggest that the collagen-stimulating factor contains ascorbate-like activity which promotes the secretion of collagenous proteins by increasing hydroxylation of proline residues in their polypeptide backbone.
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PMID:Collagen-stimulating factor from embryonic brain has ascorbate-like activity and stimulates prolyl hydroxylation in cultured muscle cells. 396 53

Aspergillus terreus dihydrogeodin oxidase (DHGO) is an enzyme catalyzing the stereospecific phenol oxidative coupling reaction converting dihydrogeodin to (+)- geodin. We previously reported the purification of DHGO from A. terreus and raised polyclonal antibody against DHGO. From the first cDNA library constructed in lambda gt11 using mRNA from 3-day-old mycelium of A. terreus, four clones were identified using anti-DHGO antibody, but all contained partial cDNA inserts around 280 base pairs. This cDNA fragment was used as a probe to clone the genomic DNA and cDNA for dihydrogeodin oxidase from A. terreus. The sequence of the cloned DHGO genomic DNA and cDNA predicted that the DHGO polypeptide consists of 605 amino acids showing significant homology with multicopper blue proteins such as laccase and ascorbate oxidase. Four potential copper binding domains exist in DHGO polypeptide. The DHGO gene consists of seven exons separated by six short introns. Expression of the DHGO gene in Aspergillus nidulans under the starch or maltose-inducible Taka-amylase A promoter as an active enzyme established the functional identity of the gene. Also, introduction of the genomic DNA for DHGO into Penicillium frequentans led to the production of DHGO polypeptide as judged by Western blot analysis.
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PMID:Molecular cloning and heterologous expression of the gene encoding dihydrogeodin oxidase, a multicopper blue enzyme from Aspergillus terreus. 766 60

Intramolecular long-range electron transfer (ET) processes have been investigated in two types of blue copper proteins; the single-copper protein, azurin and the multi-copper oxidase, ascorbate oxidase. These have several advantages for investigating the parameters that control the above reactions: (1) Their sole physiological role is mediating or catalyzing ET processes via the evolutionary optimized copper sites. (2) The three-dimensional structures of a considerable number of blue single copper containing proteins, e.g. azurins, and of ascorbate oxidase, have been determined at high resolution. (3) These proteins have no other cofactors except for the copper ions, thus the role of the polypeptide matrix can be addressed in a more straightforward manner. In azurins, the ET from the cystine (3-26) radical-ion produced by pulse-radiolytic reduction of this single disulfide bridge, to the Cu(II) ion bound at a distance of approximately 2.6 nm has been studied, in naturally occurring and in single-site mutated azurins. The role of changing specific amino acid residues on the internal long-range electron transfer (LRET) process and its potential pathways has been investigated. It is noteworthy that this process is most probably not part of the physiological function of azurin, hence, there has not been any evolutionary selection of structural elements for the reaction. Therefore, this provides a system for an unbiased examination of structural and chemical requirements for control of this process. By contrast, in blue copper oxidases, the internal ET from the electron uptake site from substrate to the O2 reduction site is part of these enzymes catalytic cycle, presumably optimized by selective pressure. We are investigating this internal ET in ascorbate oxidase and try to resolve the relation between this enzyme's distinct functional states and the internal ET rates. We conclude that in both types of proteins, the investigated LRET proceed primarily along covalent pathways, thus providing suitable systems where the parameters controlling the efficiency of these processes can be pursued.
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PMID:Blue copper proteins as a model for investigating electron transfer processes within polypeptide matrices. 801 35

Copper-resistant strains of Xanthomonas campestris pv. juglandis occur in walnut orchards throughout northern California. The copper resistance genes from a copper-resistant strain C5 of X. campestris pv. juglandis were cloned and located on a 4.9-kb ClaI fragment, which hybridized only to DNA of copper-resistant strains of X. campestris pv. juglandis, and was part of an approximately 20-kb region which was conserved among such strains of X. campestris pv. juglandis. Hybridization analysis indicated that the copper resistance genes were located on the chromosome. Plasmids conferring copper resistance were not detected in copper-resistant strains, nor did mating with copper-sensitive strains result in copper-resistant transconjugants. Copper resistance genes from X. campestris pv. juglandis shared nucleotide sequence similarity with copper resistance genes from Pseudomonas syringae pv. tomato, P. syringae, and X. campestris pv. vesicatoria. DNA sequence analysis of the 4.9-kb fragment from strain C5 revealed that the sequence had an overall G+C content of 58.7%, and four open reading frames (ORF1 to ORF4), oriented in the same direction. All four ORFs were required for full expression of copper resistance, on the basis of Tn3-spice insertional inactivation and deletion analysis. The predicted amino acid sequences of ORF1 to ORF4 showed 65, 45, 47, and 40% identity with CopA, CopB, CopC, and CopD, respectively, from P. syringae pv. tomato. The most conserved regions are ORF1 and CopA and the C-terminal region (166 amino acids from the C terminus) of ORF2 and CopB. The hydrophobicity profiles of each pair of predicted polypeptides are similar except for the N terminus of ORF2 and CopB. Four histidine-rich polypeptide regions in ORF1 and CopA strongly resembled the copper-binding motifs of small blue copper proteins and multicopper oxidases, such as fungal laccases, plant ascorbate oxidase, and human ceruloplasmin. Putative copper ligands of the ORF1 polypeptide product are proposed, indicating that the polypeptide of ORF1 might bind four copper ions: one type 1, one type 2, and two type 3.
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PMID:Molecular cloning, chromosomal mapping, and sequence analysis of copper resistance genes from Xanthomonas campestris pv. juglandis: homology with small blue copper proteins and multicopper oxidase. 828 94

Cultured cells of tobacco BY2 secrete more than 100 proteins into culture medium. Six major proteins were purified, and partial protein sequences were determined. Five of them were found to be similar to an ascorbic acid oxidase, three peroxidase isozymes and a beta-1,3-exoglucanase, respectively. A cDNA clone encoding the remaining polypeptide, whose amino acid sequence showed no similarity with earlier reported proteins, was isolated. It encoded a putative 27 kDa protein of 242 amino acids with resemblance to WCI-5, a wheat protein induced by benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) which activates genes involved in systemic acquired resistance. Transcripts of this clone accumulated upon tobacco mosaic virus infection, mechanical wounding and drought treatment, an induction profile that satisfies the definition of pathogenesis-related (PR) proteins by van Loon et al. (Plant Mol. Biol. Rep. 12 (1994) 245). No similar PR proteins have so far been reported, and therefore our newly designated NtPRp27 points to the existence of a novel PR protein family in tobacco plants.
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PMID:Secreted proteins of tobacco cultured BY2 cells: identification of a new member of pathogenesis-related proteins. 1079 17


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