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:1.10.3.2 (laccase)
4,656 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Formation of the mRNA specific for the inducible forms of laccase was evidenced in Coriolus versicolor, Pleurotus ostreatus and Pholiota mutabilis. The half-life time of these mRNAs in the fungi species studied were, respectively, 30, 37 and 24 min. Molecular weight of the newly synthesized mRNA in Pleurotus ostreatus was about 4.5X10(5), consistently with the size of the inducible laccase protein. The polysome obtained from the ferulic acid-treated mycelium, synthesized in vitro a polypeptide with the electrophoretic mobility similar to that of laccase.
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PMID:Induction of laccase in Basidiomycetes: the laccase-coding messenger. 10 48

Golgi complex and endoplasmic reticulum (ER) were isolated from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) by stepwise sucrose density gradient centrifugation using protoplasts as starting material. The purity of the two organelle fractions isolated was assessed by measuring marker enzyme activities. Localization of glycolipid and glycoprotein glycosyltransferase activities in the isolated Golgi and ER fractions was examined; three glycosyltransferases, i.e., galactosyltransferase, fucosyltransferase, and xylosyltransferase, proved to be almost exclusively confined to the Golgi, whereas the ER fractions contained glycolipid glycosyltransferase. The Golgi complex was further subfractionated on a discontinuous sucrose density gradient into two components, migrating at densities of 1.118 and 1.127 g/cm3. The two fractions differed in their compositional polypeptide bands discernible from Na-dodecylsulfate gel electrophoresis. Galactosyltransferase distributed nearly equally between the two protein peaks and xylosyltransferase activities using the endogenous acceptor also appeared to be localized in the two subcompartments. By contrast, fucosyltransferase, engaged in the terminal stage of glycosylation, banded in the lower density fractions. Golgi-specific alpha-mannosidase, which is presumably engaged in the sugar trimming of Asn-N-linked glycoprotein carbohydrate core, was enriched fourfold in specific activity in the fractions of the higher density. The overall experimental results indicate that the cotranslational glycosylation of Asn-N-linked glycoproteins, e.g., polyphenol oxidase (laccase), takes place in the ER, while subsequent post-translational processing of the oligosaccharide moiety proceeds successively in the two physically separable compartments of the Golgi complex.
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PMID:Golgi-specific localization of transglycosylases engaged in glycoprotein biosynthesis in suspension-cultured cells of sycamore (Acer pseudoplatanus L.). 309 42

The interaction of the radicals OH, t-BuO, Eaq, CO2 and O2 with the copper oxidase, laccase, from Polyporus, has been studied by the pulse-radiolysis technique. Each of these radicals formed transient adducts with a broad absorption maximum around 310 nm. Analysis of the optical properties and of the very fast rates of formation of these compounds shows that each radical interacts with a limited number of sites on the polypeptide part of the protein amongst R-S-S-R, histidine and aromatic residues. Interaction with the carbonyl group of some of the peptides bonds is also possible. The few target sites are probably hit simultaneously and electron transfer between these sites may also occur. In all cases, ina subsequent step, intramolecular electron transfer from the polypeptide radical adducts leads to a partial reduction of the blue type-1 Cu2+ with rates varying between 10(3) adn 10(4) s-1. Further reduction of the type-1 CU2+ occurs through a slow intermolecular reaction between two laccase radical transient adducts. In the case of CO2 and O2, this slow reduction could alternatively be due to an intermolecular reaction between laccase and CO2 or O2. The oxidation radicals OH, Br2 and (SCN)2, which formed radical adducts with fully ascorbate-reduced laccase, did not induce any type-1 copper reoxidation.
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PMID:Radical scavenging and electron-transfer reactions in Polyporus versicolor laccase a pulse radiolysis study. 628 3

In Podospora anserina, self-lysis resulting from the combination of the R and V incompatibility genes is accompanied by the appearance, in lysing cells, of specific enzyme activities, among which is a laccase exoenzyme, and by a quenching of ribonucleic acid synthesis. Present results show that the occurrence of the laccase is the result of de novo synthesis. By means of two-dimensional gel electrophoresis it was shown that the onset of self-lysis is accompanied by the immediate shut-off of more than 60% of the pre-existing normal polypeptide synthesis and the occurrence of at least 20 new polypeptides. The synthesis of these new polypeptides is active for several hours after the cessation of RNA synthesis, concurrently with the synthesis of about 30 normal polypeptides which is maintained. These modifications of protein synthesis are not accompanied by a concomitant variation in the level of polysomes. It is deduced that incompatibility genes are involved in the control of both transcription and translation.
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PMID:Polypeptide synthesis during protoplasmic incompatibility in the fungus Podospora anserina. 722 93

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

Two different bands with laccase activity were obtained after nondenaturing PAGE of the culture filtrate of Pleurotus ostreatus. Immunoblot analysis revealed that antisera raised against laccase I were not reactive to laccase II. Laccase I, which exhibited faster mobility on nondenaturing polyacrylamide gel, was purified 42.9-fold with an overall yield of 10.8%. Gel filtration and SDS-PAGE revealed that laccase I is a single polypeptide with a molecular mass of approximately 64 kDa. Laccase I contained 12.5% carbohydrate by weight and 3.9 mol copper (mol protein)-1. The absorption spectrum of laccase I showed a type 1 signal at 605 nm and EPR spectra showed that the parameters of the type 1 and type 2 Cu signals were g parallel = 2.197 and A parallel = 0.009 cm-1, and g parallel = 2.263 and A parallel = 0.0176 cm-1, respectively. The data obtained from the pH profiles suggested that two ionization groups, whose pKa values were 5.60-5.70 and 6.70-6.85, may play an important role in the active site of laccase I as the ligand of copper metal. The optimal pH and temperature for the activity of laccase I were 6.0-6.5 and 30-35 degrees C, respectively. The enzyme had affinity for various lignin-related phenolic compounds: the Km values for ferulic acid and syringic acid were 48 and 89 microM, respectively. EPR spectroscopic study of the action of laccase I on 3,5-dimethoxy-5-hydroxyacetophenone indicated that this enzyme catalyses single electron transfer with the formation of the phenoxy radical as an intermediate.
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PMID:Single electron transfer by an extracellular laccase from the white-rot fungus Pleurotus ostreatus. 770 70

Agaricus bisporus secretes abundant laccase activity into the medium during mycelial growth. SDS-PAGE analysis of extracellular laccase protein, purified from compost extract, showed a predominant band of 65 kDa molecular mass, together with lesser amounts of smaller polypeptides. The main polypeptide was purified electrophoretically. Amino acid sequence analysis of the N-terminal region of the main polypeptide was used to specify the sequence of a 15-residue chemically synthesized peptide (N-terminal peptide). Rabbit antibodies were raised against pure laccase, electrophoretically purified main polypeptide and the synthetic N-terminal peptide. Electrophoretically purified main polypeptide antibody was further purified by affinity chromatography on laccase-CNBr-Sepharose. Western blot analysis showed that the antigenic behaviour of laccase in compost extract, culture filtrate from malt-extract culture, and the purified enzyme from both sources, differed. The patterns of bands revealed are most simply explained by generation of (proteolytically) partially cleaved enzyme molecules in the culture medium, possibly combined with differences in extent of glycosylation. [35S]Methionine incorporation and immunoprecipitation were used to follow laccase synthesis in cultures grown on malt extract. After short-term labelling, a single polypeptide of 68 kDa apparent molecular mass was immunoprecipitated from both mycelial extracts and the culture medium. When poly(A)-containing RNA from malt-extract-grown mycelium was translated in vitro in rabbit reticulocyte lysate, a single polypeptide of about 57 kDa molecular mass was immunoprecipitated, consistent with the previously measured carbohydrate content of 15% for the pure enzyme. After treatment with N-glycanase, the polypeptide showed an increase in mobility during SDS-PAGE consistent with a reduction in molecular mass of about 5 kDa, indicating about equal amounts of N- and O-linked carbohydrate. C-terminal labelling of pure laccase was attempted by transpeptidation with carboxypeptidase Y. Although some minor bands were labelled, the main polypeptide was not, indicating that the C-terminus of the enzyme may be blocked.
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PMID:The structure of laccase protein and its synthesis by the commercial mushroom Agaricus bisporus. 809 17

We have isolated and characterized the cDNA and genomic DNA coding for a phenoloxidase, laccase I, previously purified from culture supernatant of the newly isolated ligninolytic basidiomycete PM1 (CECT 2971). A cDNA library from basidiomycete PM1 was constructed, and laccase-encoding cDNAs were identified by screening with antiserum raised against the purified enzyme. The lac1 gene coding for the laccase was identified in a partial genomic library by using the isolated cDNA as a probe. Nucleotide sequence determination of the full-length cDNA revealed an open reading frame of 1,551 bp encoding a polypeptide of 517 amino acid residues with a putative signal peptide of 21 amino acid residues. Ten small introns interrupted the genomic DNA. A single 1.8-kb transcript mRNA was detected by Northern (RNA) blot analysis, and its 5' end maps to a position 51 bp upstream from the site of initiation of protein synthesis. Eukaryotic regulatory sequences, CAAT and TATA, were observed in the 5' flanking region, which also contains sequences similar to those of copper-regulated proteins. Comparative analysis of the predicted amino acid sequence showed that basidiomycete PM1 laccase I had great similarity to the laccases from Coriolus versicolor, Coriolus hirsutus, and Phlebia radiata.
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PMID:Characterization and structural analysis of the laccase I gene from the newly isolated ligninolytic basidiomycete PM1 (CECT 2971). 828 10

Melanin production is a major virulence factor for Cryptococcus neoformans, an organism causing life-threatening infections in an estimated 10% of AIDS patients. In order to characterize the events involved in melanin synthesis, an enzyme having diphenol oxidase activity was purified and its gene was cloned. The enzyme was purified as a glycosylated 75-kDa protein which migrated at 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after deglycosylation by endoglycosidase F. Substrate specificity resembled that of a laccase in that it oxidized multiple diphenolic and diamino compounds. Dopamine was shown by mass spectroscopy to be oxidized to decarboxy dopachrome, an intermediate of melanin synthesis. The enzyme contained 4.1 +/- 0.1 mol of copper per mol. It resembled a laccase in its absorbance spectrum, containing a peak of 610 nm and the shoulder at 320 nm, corresponding to the absorbance of a type I and type III copper, respectively. The cloned gene of C. neoformans laccase (CNLAC1) contained a single open reading frame encoding a polypeptide 624 amino acids in length. The encoded polypeptide contained a presumptive leader sequence, on the basis of its relative hydrophobicity and by comparison of the sequence to that of the N-terminal sequence of the purified enzyme. CNLAC1 also contained 14 introns ranging from 52 to 340 bases long. Transcriptional activity of CNLAC1 was found to be derepressed in the absence of glucose and to correspond to an increase in enzymatic activity.
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PMID:Biochemical and molecular characterization of the diphenol oxidase of Cryptococcus neoformans: identification as a laccase. 830 May 20

A cDNA library was constructed in lambda gt11 using mRNA from 11-d-old mycelium of Agaricus bisporus. Three clones containing laccase sequence were identified using an affinity-purified anti-laccase antibody. From one of these clones, a 333 bp sequence was used to identify further cDNA clones (including one which is close to full length) and a genomic clone. The coding sequences found were of two similar but not identical versions with differences at 36 out of 520 residues of deduced amino acid sequence. The laccase genes each encode a sequence expressed as a 2.3 kb mRNA, specifying a 520 residue polypeptide including a 19 amino acid residue signal peptide that is absent from the N terminus of the mature (extracellular) protein. The coding sequence of lcc1 is interrupted by 14 short introns. The lcc1 and lcc2 genes are not allelic as they do not segregate in uninucleate spores derived from a four-spored basidium. Comparison of the deduced amino acid sequences with that of the other fungal laccases that have been cloned, and with the very similar ascorbate oxidases from higher plants shows that whilst some sequence is absolutely conserved at and around the amino acid residues involved in copper binding, the overall sequence similarities are low.
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PMID:Identification of two laccase genes in the cultivated mushroom Agaricus bisporus. 836 Jun 14


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