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Query: UNIPROT:P06889 (Mol)
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The gene encoding laccase in the chestnut blight fungus, Cryphonectria parasitica, has been cloned and characterized. The predicted C. parasitica laccase amino acid sequence (591 aa) was 57% identical to the Neurospora crassa laccase sequence and contained four potential copper-binding regions that are conserved in a number of copper-binding proteins. Treatment of a virulent C. parasitica strain with 3 microM cycloheximide resulted in a marked increase in laccase mRNA accumulation, whereas identical treatment of an isogenic strain that contained a hypovirulence-associated virus failed to significantly increase laccase mRNA levels. In contrast, the accumulation of mRNAs encoding beta-tubulin, actin, or glyceraldehyde-3-phosphate dehydrogenase was not appreciably altered by either the presence of a hypovirulence-associated virus or treatment with cycloheximide. These results provide evidence that the expression of a specific fungal gene encoding a known protein product is selectively modulated by a hypovirulence-associated virus.
Mol Plant Microbe Interact
PMID:Molecular analysis of the laccase gene from the chestnut blight fungus and selective suppression of its expression in an isogenic hypovirulent strain. 153 23

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)
J Mol Biol 1992 Mar 05
PMID:Refined crystal structure of ascorbate oxidase at 1.9 A resolution. 154 98

A crude protein extract of Bacillus subtilis W23 contains a sequence-specific DNA binding activity for the xyl operator as detected by the gel mobility shift assay. A xylR determinant encoded on a multicopy plasmid leads to increased expression of this binding activity. In situ footprinting analysis of the protein-DNA complex in a polyacrylamide gel shows that the xyl operator is sequence-specifically bound and protected from cleavage by copper-phenanthroline at 26 phosphodiester bonds on each strand. Quantitative competition assays for repressor binding reveal that a 25 bp synthetic xyl operator cloned into a polylinker is bound with the same affinity as the operator in the wild-type xyl regulatory region. This confirms that no additional sites in the wild-type sequence contribute to repressor binding. The xyl operator consists of ten palindromic base pairs flanking five central non-palindromic base pairs. A mutational analysis shows that the sequence of the central base pairs contributes to recognition by the repressor protein and that the spacing of the palindromic elements is crucial for repressor binding. An operator half site is not bound by the repressor. In vivo and in vitro induction studies suggest that, of several structurally similar sugars, xylose is the only molecular inducer of the Xyl repressor.
Mol Gen Genet 1992 Apr
PMID:Regulation of the Bacillus subtilis W23 xylose utilization operon: interaction of the Xyl repressor with the xyl operator and the inducer xylose. 158 10

The effects of oxidized human plasma low density lipoproteins (Ox-LDL) on the proliferation of cultured aortic smooth muscle cells was studied, employing viable cell counting, [3H] thymidine incorporation into DNA, and the release of lactate dehydrogenase (LDH) into the medium. Oxidized LDL (prepared by incubation of LDL with copper sulfate) exerted a concentration-dependent stimulation (2 fold, compared to control) of aortic smooth muscle cell proliferation at low concentrations (0.1 micrograms-10 micrograms/ml medium). On the other hand, at high concentrations (25-200 micrograms/ml), Ox-LDL produced a pronounced decrease in viable cells, a decrease in the incorporation of [3H] thymidine into DNA, and an increase in the release of LDH in the medium. In this report, the previously postulated biological roles of oxidized-LDL in atherosclerosis are discussed in view of these findings.
Mol Cell Biochem 1992 Apr
PMID:Role of oxidized human plasma low density lipoproteins in atherosclerosis: effects on smooth muscle cell proliferation. 158 38

The naturally occurring flavonoid, quercetin, in the presence of Cu(II) and molecular oxygen caused breakage of calf thymus DNA, supercoiled pBR322 plasmid DNA and single stranded M13 phage DNA. In the case of the plasmid, the product(s) were relaxed circles or a mixture of these and linear molecules depending upon the conditions. For the breakage reaction, Cu(II) could be replaced by Fe(III) but not by other ions tested [Fe(II), Co(II), Ni(II), Mn(II) and Ca(II)]. Structurally related flavonoids, rutin, galangin, apigenin and fisetin were effective or less effective than quercetin in causing DNA breakage. In the case of the quercetin-Cu(II) reaction, Cu(I) was shown to be essential intermediate by using the Cu(I)-sequestering reagent, bathocuproine. By using Job plots we established that, in the absence of DNA, five Cu(II) ions were reduced by one quercetin molecule; in contrast two ions were reduced per quercetin molecule in the DNA breakage reaction. Equally neocuproine inhibited the DNA breakage reaction. The involvement of active oxygen in the reaction was established by the inhibition of DNA breakage by superoxide dismutase, iodide, mannitol, formate and catalase (the inhibition was complete in the last case). The strand scission reaction was shown to account for the biological activity of quercetin as assayed by bacteriophage inactivation. From these data we propose a mechanism for the DNA strand scission reaction of quercetin and related flavonoids.
Mol Cell Biochem 1992 Apr
PMID:Strand scission in DNA induced by dietary flavonoids: role of Cu(I) and oxygen free radicals and biological consequences of scission. 158 40

This report describes the isolation and characterization of a cDNA clone representing a gene specifically expressed in pollen. A cDNA library was constructed against mRNA from mature pollen of Nicotiana tabacum. It was screened differentially against cDNA from mRNA of leaf and of pollen. One clone, NTPc303, was further characterized. On northern blot this clone hybridizes to a transcript 2100 nucleotides in length. NTPc303 is abundant in pollen. Expression of the corresponding gene is restricted to pollen, because no other generative or vegetative tissue contains transcripts hybridizing to NTPc303. Expression of NTP303 is evolutionarily conserved: homologous transcripts are present in pollen from various plant species. The first NTP303 transcripts are detectable on northern blot at the early bi-nucleate stage and accumulate until the pollen has reached maturity. During germination and pollen tube growth in vitro new NTP303 transcripts appear. This transcription has been proved by northern blots as well as by pulse labelling experiments. Nucleotide sequence analysis revealed that NTPc303 has an open reading frame coding for a predicted protein of 62 kDa. This protein shares homology to ascorbate oxidase and other members of the blue copper oxidase family. A possible function for this clone during pollen germination is discussed.
Plant Mol Biol 1992 Apr
PMID:Characterization of a pollen-specific cDNA clone from Nicotiana tabacum expressed during microgametogenesis and germination. 160 Jan 46

The structure of Cu,Zn yeast superoxide dismutase has been determined to 2.5 A resolution. The enzyme crystallizes in the P2(1)2(1)2 space group with two dimeric enzyme molecules per asymmetric unit. The structure has been solved by molecular replacement techniques using the dimer of the bovine enzyme as the search model, and refined by molecular dynamics with crystallographic pseudo-energy terms, followed by conventional crystallographic restrained refinement. The R-factor for 32,088 unique reflections in the 10.0 to 2.5 A resolution range (98.2% of all possible reflections) is 0.158 for a model comprising two protein dimers and 516 bound solvent molecules, with a root-mean-square deviation of 0.016 A from the ideal bond lengths, and an average B-factor value of 29.9 A2. A dimeric molecule of the enzyme is composed of two identical subunits related by a non-crystallographic 2-fold axis. Each subunit (153 amino acid residues) has as its structural scaffolding a flattened antiparallel eight-stranded beta-barrel, plus three external loops. The overall three-dimensional structure is quite similar to the phylogenetically distant bovine superoxide dismutase (55% amino acid homology), the largest deviations can be observed in the regions of amino acid insertions. The major insertion site hosting residues Ser25A and Gly25B, occurs in the 2,3 beta-turn between strands 2b and 3c, resulting in the structural perturbations of the two neighbouring strands. The second insertion site, at the end of the 3c beta-strand in the wide Greek-key loop, hosts the Asn35A residue, having an evident effect on the structure of the loop and possibly on the neighbouring 5,4 beta-turn. The salt bridge Arg77-Asp99 and the disulphide bridge Cys55-Cys144 stabilize the loop regions containing the metal ligands. The stereochemistry of the two metal centres is conserved, with respect to the bovine enzyme. The Cu2+ ligands show an uneven distortion from a square plane, while Zn2+ co-ordination geometry is distorted tetrahedral. The imidazole ring of the His61 residue forms a bridge between Cu and Zn ions. A solvent peak compatible with a fifth ligand is observed 2.0 A away from the copper in the active site channel, which is filled by ordered water molecules that possibly contribute to the stability and function of the enzyme. The charged residues responsible for the electrostatic guidance of the substrate to the active site (Glu130, Glu131, Lys134 and Arg141) are fairly conserved in their positions, some of them showing different interactions in the four chains due to the intermolecular contacts between the dimers.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1992 Jun 05
PMID:Crystal structure of yeast Cu,Zn superoxide dismutase. Crystallographic refinement at 2.5 A resolution. 160 82

We have reproducibly crystallized the metal-dependent Class II fructose-1,6-bisphosphate aldolase from Escherichia coli. Crystals in the shape of truncated hexagonal bipyramids have unit cell dimensions of a = b = 78.4 A, c = 290.6 A and are suitable for a detailed structural analysis. The space group has been identified as P6(1)22 or enantiomorph. Data sets to approximately 2.9 A resolution have been recorded using both the Rigaku R-AXIS IIc image plate area detector coupled to a copper target rotating anode X-ray source and using the MAR image plate systems with synchrotron radiation at the EMBL outstation DESY in Hamburg, and at S.R.S. Daresbury. Diffraction beyond 2.5 A has been observed when large freshly grown crystals are used with the synchrotron beam. A data set to this resolution has been collected. Several putative heavy-atom derivative data sets have also been measured using synchrotron radiation facilities and analysis of these data sets is in progress.
J Mol Biol 1992 Jun 20
PMID:Initiating a crystallographic study of a class II fructose-1,6-bisphosphate aldolase. 161 97

The semisynthetic Co-substituted bovine erythrocyte superoxide dismutase (SOD) has been crystallized in a new crystalline form and the structure determined at 2.0 A (1 A = 0.1 nm) resolution. The crystals belong to space group P2(1)2(1)2(1) with cell constants: a = 51.0, b = 147.6, c = 47.5 A, and contain one dimeric molecule of 32,000 M(r) per asymmetric unit. The structure has been solved by molecular replacement techniques using the Cu,Zn bovine enzyme as a search model, and refined by molecular dynamics with the crystallographic pseudo-energy term, followed by conventional crystallographic refinement. The R-factor for the 18,964 unique reflections in the resolution range from 10.0 to 2.0 A is 0.176 for a model comprising 2188 protein atoms and 200 solvent molecules; the root-mean-square deviation from the ideal bond lengths is 0.010 A, and the average atomic temperature factor is 26.5 A2. The dimeric molecule of the enzyme is composed of two identical subunits related by a non-crystallographic 2-fold axis. The subunit has as its structural scaffolding the conventional SOD-flattened antiparallel eight-stranded beta-barrel, with three external loops. The co-ordination geometry of the metal center in the active site is fairly well preserved when compared with the native Cu,Zn bovine enzyme. Co2+ is in tetrahedral co-ordination, while the Cu2+ ligands show an uneven distortion from the square planar geometry. The least-squares superposition of the metals ligands and the catalytically important Arg141 of the native and Co-substituted enzyme yields a root-mean-square value of 0.401 A, the largest deviation occurring at the Co2+ ligand Asp81. An additional copper ligand, compatible with a water molecule, is observed at 2.38 A from Cu2+ in the active-site channel, at the supposed binding site of the O2- anion substrate. Several ordered water molecules have been observed on the protein surface and in the active-site channel; their structural locations coincide remarkably with those of related water molecules found in the crystal structure of the phylogenetically distant superoxide dismutase from yeast.
J Mol Biol 1992 Jul 05
PMID:Crystal structure solution and refinement of the semisynthetic cobalt-substituted bovine erythrocyte superoxide dismutase at 2.0 A resolution. 161 51

Apolipoprotein B100 (apoB), the only protein of low-density lipoprotein, is produced primarily in the liver and serves as a ligand for the low-density lipoprotein receptor. Hepatic cell-specific expression of the human apoB gene is controlled by at least two cis-acting positive elements located between positions-128 and -70 (H. K. Das, T. Leff, and J.L. Breslow, J. Biol. Chem. 263:11452-11458, 1988). The distal element (-128 to -85) appears to be liver specific since it shows positive activity in HepG2 cells and negative activity in HeLa cells. The proximal element (-84 to -70) acts as a positive element in both these cell lines, and two rat liver nuclear proteins, BRF-1 and C/EBP, bind to two overlapping sites (-84 to -60 and -70 to -50, respectively). By gel mobility shift assay, we have identified a rat liver nuclear protein (BRF-2) which binds to the distal element (-128 to -85) of the apoB gene. This putative trans-acting factor has been purified to apparent homogeneity by DEAE-cellulose, heparin-agarose, and DNA-specific affinity chromatography. The purified BRF-2 has an apparent molecular mass of 120 kDa and was found to specifically recognize sequence -128 to -85; BRF-2 also produced a strong hypersensitive site at nucleotide position -95 with copper-orthophenanthroline reagent. A double-stranded oligonucleotide (-128 to -85) containing a 3-nucleotide (TTC) insertion between position -95 and -94 was found to abolish DNA binding by BRF-2. This result suggests that the region surrounding the hypersensitive site -95 is important for protein-DNA interaction. By using apoB promoter fragments containing various internal deletions as templates for gel mobility shift assay, the region between -104 and -85 was identified to be crucial for binding by BRF-2. We propose that BRF-2 may play an important role in the tissue-specific regulation of apoB gene transcription.
Mol Cell Biol 1992 Jul
PMID:Transcriptional regulation of the apolipoprotein B100 gene: purification and characterization of trans-acting factor BRF-2. 162 Jan 25


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