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)

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

In order to reveal the detailed structure of the trinuclear site composed of type 2 copper and a pair of type 3 copper centers in multicopper oxidases, the action of inhibitors such as azide, thiocyanate, and fluoride on laccase and ascorbate oxidase has been investigated by absorption, CD, and EPR spectroscopies. Anaerobic reactions of inhibitor-treated laccase and ascorbate oxidase with pyrocatechol and L-ascorbate, respectively, gave EPR signals originating from the inhibitor-bound type 3 copper, except for the case of F(-)-laccase. The hyperfine splittings of these EPR signals (Az = 10.10(-3)-18.10(-3) cm-1) were smaller than those of type 2 copper centers (ca. 20.10(-3) cm-1), indicating that type 3 copper has a tetragonal geometry with tetrahedral distortion. The facile detection of a series of the inhibitor-bound type 3 copper centers indicates that the action of the exogenous anionic inhibitors is not only to interfere the access of dioxygen to the trinuclear site, but also to restrain the reduction of type 3 copper by lowering its reduction potential.
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PMID:EPR spectra of type 3 copper centers in Rhus vernicifera laccase and Cucumis sativus ascorbate oxidase. 774 96

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.
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PMID:EPR studies of ligand binding to the type 2/type 3 cluster in tree laccase. 797 82

A series of fungal laccases (Polyporus pinsitus, Rhizoctonia solani, Myceliophthora thermophila, Scytalidium thermophilum) and one bilirubin oxidase (Myrothecium verrucaria) have been studied to determine their redox potential, specificity, and stability. Polyporus and Rhizoctonia laccases possess potentials near 0.7-0.8 V (vs. NHE), while other oxidases have potentials near 0.5 V. It is observed that higher redox potential correlates with higher activity. By EPR, no significant change in the geometry of type 1 copper (II) site is observed over this series. At the optimal pH, the two substrates studied, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine, show Km values ranging form 10 to 120 and from 1 to 45 microM; and kcat values ranging from 50 to 16 000 and 200 to 3000 per min, respectively. The enzymes are more stable in the neutral-alkaline pH range. The thermal stability is in the order of bilirubin oxidase equivalent to Myceliophthora laccase equivalent to Scytalidium laccase > Polyporus laccase > Rhizoctonia laccase. Based on these results and the sequence alignments made against Zucchini ascorbate oxidase it is speculated that structural differences in the substrate-activation site (a 'blue', type 1 copper center) control the redox potential range as well as substrate specificity, and the cystine content contributes to stability.
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PMID:A study of a series of recombinant fungal laccases and bilirubin oxidase that exhibit significant differences in redox potential, substrate specificity, and stability. 859 77

Extracellular laccases from submerged cultures of Coriolus versicolor BKM F-116, Panus tigrinus 8/18, Phlebia radiata 79 (ATCC 64658), Phlebia tremellosa 77-51 and from cultures of Pa. tigrinus 8/18, Ph. radiata 79 and Agaricus bisporus D-649 grown on wheat straw (solid-state fermentation) were purified. All enzymes from submerged cultures had a blue colour and characteristic absorption and EPR spectra. Laccases from the solid-state cultures were yellow-brown and had no typical blue oxidase spectra and also showed atypical EPR spectra. Comparison of N-terminal amino acid sequences of purified laccases showed high homology between blue and yellow-brown laccase forms. Formation of yellow laccases as a result of binding of lignin-derived molecules by enzyme protein is proposed.
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PMID:Blue and yellow laccases of ligninolytic fungi. 936 54

Mono-azide adduct of Rhus vernicifera laccase, a multicopper oxidase containing one type-1 (blue) copper, one type-2 (non-blue normal) copper, and a pair of type-3 (binuclear and EPR silent) coppers, of which type-2 and type-3 coppers constitute a trinuclear site, was investigated with resonance Raman (RR) and Fourier transform infrared (FT-IR) spectroscopies as a step toward elucidation of the structure and function of the trinuclear site. The Cu-N3- stretching (vCu-N3-) RR band was observed for azide-bound multicopper oxidases for the first time. The vCu-N3- band was located at 400 cm-1 for mono-14N3- laccase, which shifted to 396 cm-1 with the 15N14N14N3- analog. The N3- asymmetric stretching (v(N3-)asym) band was observed by FT-IR spectroscopy at 2035 cm-1 for mono-14N3- laccase and at 2025 cm-1 for the 15N14N14N3- analog. The vCu-N3- and v(N3-)asym frequencies and their 15N14N14N- isotope shifts for azido laccase correspond well with those of metazido hemocyanin, indicating that both derivatives should have a similar binding geometry of azide.
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PMID:Observation of Cu-N3- stretching and N3- asymmetric stretching bands for mono-azide adduct of Rhus vernicifera laccase. 948 Aug 26

The trinuclear centers in Rhus vernicifera laccase and Cucumis sativus ascorbate oxidase have been studied by EPR spectroscopy and magnetic susceptibility measurements over the wide range of 5 K to 300 K. The EPR spectra showed that type II copper receives increasing tetrahedral distortion with raising temperature. Magnetic susceptibilities of laccase showed that both of type I and type II coppers are almost fully paramagnetic since the antiferromagnetic interaction between type III coppers is extremely strong from 5 K to 300 K. On the other hand, the effective magnetic moment of ascorbate oxidase is contributed by ca. 1.7 Cu2+ even below ca. 100 K, since type II Cu is partly in the reduced form. The effective magnetic moment continuously increased with raising temperature because the antiferromagnetic interaction between type III coppers is not as strong as in the case of laccase. The simulation of the SQUID measurement results suggested that the conformational change of the ascorbate oxidase molecule caused the temperature dependence of the antiferromagnetic interaction. The type II Cu EPR signals in laccase and ascorbate oxidase were conspicuously broadened with raising temperature because of the increasing contribution of the triplet state by type III Cu's and/or of the rapid relaxation which finally led to only ca. 30% detection of the type II Cu signals at room temperature. The stepwise binding of azide to the trinuclear center made one of type III Cu's to be EPR detectable. SQUID measurements indicated that only one Cu in the trinuclear center is paramagnetic and other two Cu's are antiferromagnetically coupled for both of the one- and two-azide bound forms. The binding mode of azide to the trinuclear center was discussed based on some models.
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PMID:Magnetic studies of the trinuclear center in laccase and ascorbate oxidase approached by EPR spectroscopy and magnetic susceptibility measurements. 960 7

Two chromatographic forms of laccase c1 and c2 were purified approximately 225-fold from the extracellular culture fluid of ligninolytic cultures of Dichomitus squalens, using DEAE-Sepharose and Mono-Q fast protein liquid chromatography. Each homogeneous laccase (c1 and c2) has a molecular mass of approximately 66 kDa as determined by SDS-PAGE. Both forms are glycoproteins, and each contains four copper atoms per molecule of protein. The first 20 amino acids of the N-terminal sequences of these two laccases are identical and are similar to those of laccases from other lignin-degrading fungi. The electronic absorption spectra of these laccases exhibit bands at 610 and 330 nm, indicative of type I and type III copper. The EPR spectrum of laccase c1 exhibits bands indicative of type I and type II copper. Each laccase oxidizes a variety of phenolic substrates, has a pH optimum of 3.0 for the oxidation of 2,6-dimethoxyphenol, and is inhibited strongly by fluoride and azide.
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PMID:Purification and characterization of laccases from the white-rot basidiomycete Dichomitus squalens. 960 69

An isolate of Coriolus hirsutus constitutively expresses substantial amounts of extracellular laccase on a defined growth medium. The most efficient inducer of extracellular laccase synthesis was syringaldazine, which increased the enzyme yield by 1000% at a concentration of 0.11 microM. The constitutive form of the enzyme was purified 312-fold. Laccase from C. hirsutus, with an estimated molecular mass of 55 kDa and pI of 4.0, is a monomeric glycoprotein containing 12% carbohydrate consisting of mannose and N-acetylglucosamine. The laccase was found to contain 3.9-4.1 copper atoms per molecule. The absorption spectrum shows a maximum at 610 nm and a shoulder at 330 nm, which is typical of laccase possessing type 1 and type 3 copper atoms. The parameters of the first type of copper were determined by EPR as g perpendicular=2.046 and g parallel=2.200, A parallel=8.103 x 10(-3) cm-1. Laccase was found to be a pH-stable and thermostable enzyme. With organic substrates it exhibits a pH optimum of 4.5, but with the inorganic substrate K4[Fe(CN)6] this decreased to 3.5. The highest efficiency of catalysis was observed with sinapinic acid as the substrate. The kinetic constants kcat and Km of this reaction were 578 s-1 and 24 microM respectively. It was established that the kinetics of the assayed reaction shows a Ping Pong mechanism.
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PMID:Purification and characterization of the constitutive form of laccase from the basidiomycete Coriolus hirsutus and effect of inducers on laccase synthesis. 969 88

Trametes villosa laccase was mutated on a tetrapeptide segment near the type 1 site. The mutations F463M and F463L were at the position corresponding to the type 1 copper axial methionine (M517) ligand in Zucchini ascorbate oxidase. The mutations E460S and A461E were near the T1 copper site. The mutated Trametes laccases were expressed in an Aspergillus oryzae host and characterized. The E460S mutation failed to produce a transformant with meaningful expression. The F463L and A461E mutations did not significantly alter the molecular and enzymological properties of the laccase. In contrast, the F463M mutation resulted in a type 1 copper site with an EPR signal intermediate between that of the wild type laccase and plastocyanin, an altered UV-visible spectrum, and a decreased redox potential (by 0.1 V). In oxidizing phenolic substrate, the mutation led to a more basic optimal pH as well as an increase in kcat and Km. These effects are attributed to a significant perturbation of the T1 copper center caused by the coordination of the axial methionine (M463) ligand.
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PMID:Targeted mutations in a Trametes villosa laccase. Axial perturbations of the T1 copper. 1021 9


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