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)

The amino acid sequences of different copper proteins containing coupled binuclear copper centers are compared. Hemocyanins from arthropods and molluscs and tyrosinases from three different species were found to share a highly homologous region in the C-terminal parts. This region contains three invariant histidines previously identified as ligands to Cu(B) in Panulirus interruptus hemocyanin by X-ray crystallography (Gaykema et al., Nature 309, 23-29 (1984]. In contrast, the ligand environment for the second copper, Cu(A), proved to be quite variable. It is proposed that hemocyanin and tyrosinase have arisen from a common mononuclear copper protein with the typical Cu(B) site. From this ancestral protein two types of binuclear proteins evolved independently into a tyrosinase and an arthropodan hemocyanin type. The amino acid sequence comparison between human ceruloplasmin and Neurospora crassa laccase together with the results from a preliminary X-ray structure analysis of zucchini ascorbate oxidase showed a close relationship in primary and most likely also in tertiary structure in the C-terminal parts of these enzymes. It is suggested that the multicopper oxidases have evolved from an ancestral copper protein which presumably contained all the ligands required for the binding of one binuclear and two additional mononuclear metal centers.
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PMID:Evolutionary relationships among copper proteins containing coupled binuclear copper sites. 313 63

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

A gene coding for the multi-copper phenol oxidase laccase has been isolated from the white-rot basidiomycete Trametes versicolor. The gene, which is preceded by a TATA box and a pyrimidine-rich region, is predicted to contain ten introns. The mature translation product, preceded by a 22-residue signal peptide, should consist of 498 residues. Comparisons with Edman degradation data of peptides from T. versicolor laccase strongly suggest that two disulfide bridges are formed by Cys-85/Cys-487 and Cys-117/Cys-205, respectively. The encoded protein contains five Cys, and the sequence surrounding the remaining Cys-452 is consistent with its involvement in the ligation of type-1 copper. Alignment of sequences indicates that T. versicolor laccase displays a Phe at the position corresponding to a residue (Met in ascorbate oxidase and azurin) considered important for the reduction potential of type-1 copper proteins.
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PMID:Characterization of a laccase gene from the white-rot fungus Trametes versicolor and structural features of basidiomycete laccases. 766 13

The thermal denaturation of laccase from the Japanese lacquer tree (Rhus vernicifera) was studied by differential scanning calorimetry. The endotherms of holo-laccase, type 2-Cu-depleted laccase and apo-laccase were deconvoluted into two independent two-state transitions, providing evidence for a domain structure of the protein. The correlation of the two transitions with the bleaching of copper optical bands and the decrease of the transitions' enthalpy on Cu removal show that the process involves the denaturation of Cu sites. No detectable unfolding of secondary structure was observed, since the thermal transitions, characterized by low overall specific enthalpy, did not modify either the laccase c.d. spectra in the beta-fold region or the maximum wavelength of the fluorescence emission. On chemical denaturation, however, the emission was red-shifted by about 20 nm. The laccase behaviour is substantially different from that of stellacyanin, a protein containing a single blue Cu ion, in which the thermal transition had higher specific enthalpy and induced a large change of the c.d. spectrum in the beta-fold region. The laccase denaturation behaviour is similar to that of ascorbate oxidase from zucchini (courgette; Cucurbita pepo) [Savini, D'Alessio, Giartosio, Morpurgo and Avigliano (1990) Eur. J. Biochem. 190, 491-495], suggesting a structural analogy. In both proteins heating may cause a change of tertiary structure through modifications of Cu co-ordination with loosening of the bonds between the structural domains at the interface of which the trinuclear Cu cluster is located.
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PMID:Stability of Japanese-lacquer-tree (Rhus vernicifera) laccase to thermal and chemical denaturation: comparison with ascorbate oxidase. 770 62

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

Using laser flash photolysis of lumiflavin/EDTA solutions containing ascorbate oxidase, we find that the rate constant for intramolecular electron transfer varies from one enzyme preparation to another and is generally a more sensitive measure of the state of the active site than are steady-state assays. Thus, type I copper is initially reduced in a second-order reaction followed by first-order reoxidation by the type II-III trinuclear copper center. The observed rate constant for this intramolecular process in presumably native enzyme is 160 s-1 at pH 7, whereas an enzyme preparation which had less than 20% activity had a rate constant of 2.6 s-1. Other samples of relatively active enzyme showed biphasic intramolecular kinetics intermediate between the above values. The inactive enzyme sample could be reactivated by dialysis against ascorbate or by treatment with ferricyanide, resulting in a corresponding increase in the intramolecular rate constant for type I copper reoxidation to a value comparable to that of native enzyme. Using this same methodology, we have determined that the type I copper in Japanese lacquer tree laccase is reoxidized by the type II-III trinuclear copper center in a first-order (intramolecular) process with rate constants of 1 s-1 at pH 7.0 and 4.9 s-1 at pH 6.0, values which are approximately two orders of magnitude smaller than for ascorbate oxidase. The intramolecular rate constant and enzyme activity for laccase also increased, but only by a factor of 2-6, when the enzyme was treated with ascorbate or ferricyanide, respectively. We further found that intramolecular electron transfer in laccase was completely inhibited by fluoride ion, in contrast to ascorbate oxidase which is unaffected by this ion. These differences in behavior for these two very similar enzymes are rather remarkable, when it is considered that the distance between copper atoms is constrained by the location of the protein-derived copper ligands in the three-dimensional structure, and that the redox potentials of the enzymes are similar. Our results may be interpreted in terms of an interconversion between active and inactive enzyme in which there is a rearrangement of the type II-III trinuclear copper center, resulting in a lowering of the redox potential and a block in electron transfer. Turnover restores the active enzyme conformation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Oxidative turnover increases the rate constant and extent of intramolecular electron transfer in the multicopper enzymes, ascorbate oxidase and laccase. 826 95

Myrothecium verrucaria bilirubin oxidase (EC 1.3.3.5) is an enzyme catalyzing the oxidation of bilirubin to biliverdin and other substrates. We have purified bilirubin oxidase from the medium of M. verrucaria and determined its partial amino acid sequence and isolated cDNA fragment amplified by polymerase chain reaction using oligonucleotide primers designed on the basis of the partial amino acid sequence. The gene for bilirubin oxidase has been cloned from a genomic library using the cDNA fragment as a probe. The gene encodes a precursor of bilirubin oxidase consisting of 572 amino acid residues, which comprises the prepro-region of 38 amino acid residues and the mature enzyme of 534 amino acid residues containing one cysteine. Five introns were found within the coding region. Sequence comparison of bilirubin oxidase with other blue copper proteins (laccase, ascorbate oxidase, human ceruloplasmin, plastocyanin, and azurin) revealed the presence of four domains corresponding to potential copper ligands. We have expressed this bilirubin oxidase gene in Saccharomyces cerevisiae under the repressible acid phosphatase promotor and found an active recombinant bilirubin oxidase, establishing the functional identity of the gene.
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PMID:Molecular cloning of the gene for bilirubin oxidase from Myrothecium verrucaria and its expression in yeast. 836 Jan 71

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

A gas-phase oxygen biosensor based on blue copper-containing oxidases was developed. Blue-oxidase enzymes, including laccase and ascorbate oxidase, have a blue chromophore prosthetic group, type 1 Cu+2, which can be reduced and decolorized with reducing substrates. When the enzyme is reoxidized with molecular oxygen, there is a concomitant return of the blue color. The oxygen biosensor consisted of the Rhus vernicifera laccase and ascorbate as substrate enclosed in pouches of low-density polyethylene under nitrogen gas. Operational stability of the biosensor was established by exposing it to different oxygen/nitrogen gas mixtures at 5 degrees C. Gas-phase oxygen concentrations were measured by keeping it under nitrogen gas and subsequently recording the rate of reappearance of the enzyme blue color, both visually and spectrophotometrically at 610 nm. The oxygen biosensor was able to detect a wide range of oxygen concentrations. The time required to recover the blue color, namely the biosensor response time, at the optimized assay conditions of 5 degrees C and a high-water activity level, was determined. This research describes the development of an oxygen biosensor with adequate activity and stability to measure gas-phase oxygen concentrations at 5 degrees C and high-water activity levels. The oxygen biosensor could be used to indicate oxygen concentrations above acceptable levels in headspace oxygen concentration which could affect the quality and safety of products packaged under initial low levels of oxygen concentration.
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PMID:Development of a gas-phase oxygen biosensor using a blue copper-containing oxidase. 888 2


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