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

---

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

From the peelings of cucumber Cucumis sativus and marrow squash Cucurbita pepo var. giramontia highly purified ascorbate oxidase preparations were obtained. Molecular weights, optical and EPR spectra, total copper contents and different type copper contents of the both proteins were similar. The effects of NaN3, KCN, I- and F- on the optical and EPR spectra of the proteins were studied. The incubation of ascorbate oxidase with these anions lead to the partial reduction of the copper. The data obtained indicate that F- is bound to the copper atoms of the type 2, and that N5- modifies surroundings of these copper atoms. The copper atoms of types 1 and 2 in both ascorbate oxidases, unlike fungal laccase, are completely reduced under effect of CN-. The bleaching of ascorbate oxidase, observed in alkaline media involves also increasing of the intensity of the band at 330 nm. The results show that three types of copper in ascorbate oxidase have various sensitivities to the inorganic anions. These data are compared with results observed for another blue copper-containing enzymes, such as laccases and ceruloplasmin.
...
PMID:[Interaction of ascorbate oxidase with inorganic anions]. 58 36

The ageing phenomenon exhibited by the ascomycetous fungus Podospora anserina can be either delayed or induced by either different carbon sources or effectors. As these effects seem to have analogy to catabolite-repression of respiratory genes, experiments concerning respiratory functions have been carried out. Ageing is parallelled by switching from cytochrome c-oxidase-mediated respiration to alternative, cyanide-resistant respiration for reasons still unknown. The latter is always accompanied by appearance of the phenol oxidizing enzyme laccase (EC 1.10.3.2), which seems to act as an alternative oxidase. The existence of a second, non-mitochondrially encoded respiratory pathway relieves the selective pressure on mitochondria leading to disintegrated, non-functional mtDNA and thereby whole mitochondria which accumulate in the hyphal cells. Mutants lacking cytochrome c-oxidase aa3 or laccase have stable mitochondrial populations and live eternally.
...
PMID:Oxidative stress and ageing in the fungus Podospora anserina. 133 31

We have studied the removal of the type-2 copper from tree laccase (Rhus vernicifera) by treatment with EDTA at pH 5.2 in the presence of a redox buffer containing ferri- and ferrocyanide. The efficiency with which the copper is removed depends on the Fe(CN) 6(4-)/Fe(CN) 6(3-) ratio. We have varied this ratio from approx. 2:1 to about 50:1 and the best results were obtained with the highest ratio, i.e., the most cathodic solution potential. Nevertheless, the presence of Fe(CN) 6(3-) is required for the procedure to be effective. Although we cannot exclude the possibility that a mixed-valence form of laccase is the reactive species, we believe the results are better explained by a model which assumes that the removal of the type-2 copper depends upon an ordered sequence of oxidation-reduction reactions. Specifically, we propose that the copper is released as the monovalent ion from previously reduced laccase and then reoxidized in solution and sequestered with EDTA. The reoxidation step drives the reaction because recombination with the protein is inhibited when copper is in the divalent form. In testing this model, we have also shown that the type-2 copper can be removed under strictly reducing conditions when 4,4'-dicarboxy-2,2'-biquinoline (BCA) is present to complex the copper(I) ion. Although the BCA method is effective, the reaction takes longer, perhaps because of the limited solubility of BCA at the pH values of interest. Finally, we have found that the best results are obtained with either method when a cyanometalate ion such as Fe(CN) 6(3-) or Co(CN) 6(3-) is present in the medium. The exact role of this factor has yet to be established, but there is no indication that free cyanide has a role in the process. The most likely interpretation is that some type of binding interaction with the protein facilitates copper release.
...
PMID:The removal of the type-2 copper from Rhus vernicifera laccase. 147 94

Although copper is quantitatively removed from fungal laccase (Polyporus versicolor) by extended dialysis against high concentrations of cyanide, we have been unable to reconstitute the protein by addition of Cu(I) ions. However, two new methods for reversibly removing the type 2 Cu centre have been developed. The visible absorption at 610 nm, which is attributable to type 1 Cu, is unaffected by the procedure, but the absorbance of the type 3 Cu at 330 nm is decreased by 60 +/- 10%. The decrease is due, at least in part, to partial reduction of the binuclear type 3 centre, although there may be some change in the molar absorptivity of the oxidized chromophore as well. The change in the c.d. spectrum that occurs at approx. 350 nm may be explained in the same way, but it may also reflect the loss of a signal due to the type 2 Cu. Upon removal of the type 2 Cu an absorbance increase appears at approx. 435 nm, and it is assigned to the semi-reduced form of the type 3 pair. In the e.p.r. spectrum of the type 2-depleted enzyme the type 1 Cu signal exhibits well-resolved ligand hyperfine splitting, which can be simulated on the basis of contributions from two N and two H nuclei (AH congruent to AN congruent to 25 MHz). The H atoms are assumed to be attached to the beta-carbon of the covalently bonded cysteine ligand. A signal from a semi-reduced form(s) of the type 3 site can also be resolved in the spectrum of the type 2-depleted enzyme, and on the basis of the second integral of the e.p.r. spectrum 40% of the type 3 pairs are believed to be in a partially reduced state. The semi-reduced type 3 site is remarkably stable and is not readily oxidized by H2O2 or IrCl6(2-) or reduced by Fe(CN)6(4-). Intramolecular electron transfer is apparently quite slow in at least some forms of the type 2-depleted enzyme, and this may explain why the activity is at best 5% of that of the native enzyme. Full activity returns when type 2 copper is restored.
...
PMID:Type 2-depleted fungal laccase. 284 23

16-O- Acetylvindoline (1a) was oxidatively transformed into an iminium derivative (2a) by copper oxidases (laccase and human ceruloplasmin), an unknown enzyme system(s) of Streptomyces griseus, and the chemical oxidizing agent 2,3-dichloro-5,6- dicyano -1,4-benzoquinone ( DDQ ). The iminium derivative (2a) was isolated from enzymatic and chemical oxidation mixtures and was identified by spectral and chemical techniques. Reduction of the iminium compound with sodium borodeuteride provided monodeuterated 16-O- acetylvindoline (1b) as the sole product. Mass spectral analysis indicated that the deuterium atom was introduced into position C-3 of the piperidine portion of the alkaloid structure. The location and stereochemistry of the deuterium atom were confirmed by high-field 1H and 2H NMR analyses of the deuterated product to be in the 2H alpha orientation. Hydrolysis of the 16-O-acetyl functional group from the iminium derivative (2a) resulted in the production of a previously identified dimer (5), which forms by intramolecular etherification through the reactive enamine (3). The iminium derivative (2a) reacts with cyanide to provide complex mixtures of products, one of which was identified by mass spectrometry as a cyanide addition product. The results confirm the existence of a reactive iminium intermediate formed by all of the biochemical and chemical systems examined.
...
PMID:Formation of a reactive iminium derivative by enzymatic and chemical oxidations of 16-O-acetylvindoline. 673 16

We have improved a method for the removal of the type 2 copper from tree laccase under anaerobic reducing conditions and developed a mechanistic model. We identify two key steps in the reaction: (i) dissociation of copper(I) catalyzed by trace levels of cyanide in a weakly acidic medium and (ii) sequestration of the released metal by an appropriate chelator such as 2,9-dimethyl-1,10-phenanthroline. We maintain a steady-state concentration of cyanide in a pH 5.5 acetate buffer under a constantly exchanging nitrogen atmosphere by introducing a cyanometalate ion as a cofactor or by continuously injecting the ion into the protein solution. The type 2-depleted product is identical to previous preparations as regards its spectral properties, activity level and ability to recombine with copper(I). The mechanistic insights appear to be quite general and should form the basis for the development of methods for removing the type 2 copper from other related systems.
...
PMID:The role of cyanide in the removal of type 2 copper from laccase. 762 34

Detailed investigations of the EPR-active copper ion in the trinuclear type 2/type 3 cluster site of T1Hg Rhus vernicifera laccase suggest that at least some inhibitor anions bind to what was an EPR-silent copper center of the resting enzyme. The key observation is that with [15N]azide the adduct exhibits remarkably well resolved ligand hyperfine structure indicative of splitting from three protein (histidine) nitrogens and one azide nitrogen. This accords nicely with recent X-ray diffraction studies of adducts of the related enzyme, ascorbate oxidase (A. Messerschmidt, H. Luecke, and R. Huber, 1993, J. Mol. Biol. 230, 997-1014). We have also characterized a previously unknown dicyanide adduct that exhibits an EPR signal with ligand hyperfine structure from two protein nitrogens and two cyanide carbons. Cyanide may bind to the same copper center as azide, but not without a structural reorganization of the cluster. The results also imply that the protonation of a bridging ligand within the type 2/type 3 cluster explains the pH dependence of anion binding. Imidazole interacts with the protein but does not bind to the EPR-active copper. In keeping with the function of the dioxygen reduction site, the type 2/type 3 cluster in laccase proves to be an extremely flexible host capable of accommodating a variety of ligands.
...
PMID:EPR studies of ligand binding to the type 2/type 3 cluster in tree laccase. 797 82

Pleurotus florida (ITCC 3308) produces two laccase enzymes (L1 and L2) in potato-dextrose media containing 0.5% yeast extract. Concentrated culture filtrate was separated on DEAE-Sephadex (A-50) column into two enzyme peaks, subsequently named L1 and L2. The L1 enzyme has been purified to homogeneity by ion-exchange and gel-permeation chromatography. L1 is a monomeric glycoprotein with a molar mass of 77 and 82 kDa as determined by SDS-PAGE and gel-filtration chromatography, respectively. The pI value of L1 has been determined to be 4.1. The optimum reaction temperature of the enzyme is 50 degrees C. The Km and some other kinetic parameters of L1 have been determined. Cyanide and azide completely inhibit the enzyme activity. The enzyme was fully active in 1:1 (V/V) buffer-chloroform for at least 2 h. Spectroscopic analysis revealed that the enzyme has four copper atoms, a type 1 copper, a type 2 copper and a type 3 binuclear copper.
...
PMID:Purification and characterization of laccase-1 from Pleurotus florida. 1134 72

A recently isolated basidiomycete, Trametes sp. strain AH28-2, can be induced to produce a high level of laccases when grown on a cellobiose-asparagine liquid medium. After induction by kraft lignin, two major isozymes were detected in the fermentation supernatant of the fungus. The principal component laccase A, which accounts for about 85% of the total activity, can be purified to electrophoretic homogeneity by three chromatographic steps: DEAE-Sepharose FF, Superdex-200 and Mono-Q. The solution containing purified laccase is blue in color, and the ratio of absorbance at 280 nm to that at 600 nm is 22. The molecular mass of laccase A is estimated to be 62 kDa by SDS-PAGE, 57 kDa by FPLC, and measured as 58522 Da by MALDI mass spectrum. Laccase A is a monomeric glycoprotein with a carbohydrate content of 11-12% and an isoelectric point of 4.2. The optimum pH and temperature for oxidizing guaiacol are 4.5 and 50 degrees C, respectively. The half-life of the enzyme at 75 degrees C is 27 min. The enzyme shows a good stability from pH 4.2 to pH 8.0. The K(m) values of the enzyme toward substrates 2,2'-azino-bis (3-ethylbenzothazoline-6-sulfonate) (ABTS), guaiacol and 2,6-dimethoxyphenol are 25, 420 and 25.5 microM, respectively, and the corresponding V(max) values are 670, 66.8, and 79 microM min(-1) x mg(-1), respectively. Laccase A activity is strongly inhibited by 0.1 mM NaN(3) or 0.1 mM cyanide. Two units of laccase A alone is able to completely oxidize 100 micromol 2,6-chlorophenol in 6 h. In the presence of 1 mM ABTS and 1-hydroxybenzotriazole, 15.0 U laccase A is able to oxidize 45% and 70% of 50 micromol fluorene in 12 and 18 h, respectively. The laccase A gene was cloned by a PCR method, and preliminary analysis of its sequence indicates 87.0% similarity to the corresponding segment in the phenoloxidase gene from Coriolus hirsutus.
...
PMID:Purification, molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete Trametes sp. strain AH28-2. 1266 49

Constant laccase activities were detected in culture supernatant of newly isolated basidiomycete Trametes gallica. Tryptone and glucose have great effects on the production of laccase. Two laccase isoenzymes (Lac I and Lac II) produced by T. gallica were purified to homogeneity (51- and 50-fold, respectively) by gel filtration chromatography, anion exchange chromatography, and improved native PAGE, with an overall yield of 24.8%. Lac I and Lac II from this fungus are glycoproteins with 3.6% and 4% carbohydrate content, the same molecular masses (by SDS-PAGE) of 60 kDa, and the pI of 3.1 and 3.0, respectively. Native gel electrophoresis indicates that the two laccases have different migration ratios. Lac I and Lac II have the same optimal pH of 3.0 on 2,6-dimethoxyphenol (DMP), pH 2.2 on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and of pH 4.0 on guaiacol. The highest rate of ABTS oxidation for both laccases was reached at 70 degrees C. Both laccases are stable from pH 6 to 9, retaining 88-90% activity after 24 hr incubation, and show good stability when incubated at temperatures lower than 40 degrees C. The Km values of Lac I for ABTS, DMP, and guaiacol are 0.118 x 10(-2), 0.420, and 0.405 mM, respectively; the Km values of Lac II for ABTS, DMP, and guaiacol are 0.086 x 10(-2), 0.41, and 0.40 mM, respectively. Their N-terminal sequences are determined and show strong similarity with those from other basidiomycetes. Graphite-furnace atomic absorption analysis revealed that both laccases have four copper atoms per protein molecule, but they have no type I copper signal at around 600 nm and a type III copper signal near 330 nm. Cyanide, azide, and halides completely inhibit the enzyme activity, whereas EDTA has less inhibition.
...
PMID:Purification and characterization of two laccase isoenzymes from a ligninolytic fungus Trametes gallica. 1519 12


1 2 3 Next >>

---