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)

cDNA clones for ascorbate oxidase were isolated from a cDNA library made from cucumber (Cucumis sativus) fruit mRNA. The library was screened with synthetic oligonucleotides that encode the NH2-terminal sequence of this enzyme. Nucleotide sequence analysis of the cloned cDNA inserts revealed a 1761-base-pair open reading frame that encoded an NH2-terminal signal peptide of 33 amino acids and a mature enzyme of 554 amino acids (Mr, 62,258). The amino acid sequence deduced from nucleotide sequence analysis agrees with the NH2-terminal amino acid sequence of the purified ascorbate oxidase, as determined by microsequencing methods. Cucumber ascorbate oxidase contained four histidine-rich regions with striking sequence homology to the corresponding parts of the other multicopper oxidases such as Neurospora crassa laccase and human ceruloplasmin and, to some extent, to a low molecular weight copper protein such as plastocyanin. Moreover, these data further support the hypothesis that the small blue copper proteins and the multicopper oxidases have evolved from the same ancestral gene. By RNA blot hybridization analysis, the mRNA for the ascorbate oxidase was found to be abundant in cucumber fruit tissue while expressed at very low levels in leaf and root tissues.
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PMID:Primary structure of cucumber (Cucumis sativus) ascorbate oxidase deduced from cDNA sequence: homology with blue copper proteins and tissue-specific expression. 291 72

The laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) gene from Neurospora crassa was cloned and part of its nucleotide sequence corresponding to the carboxyl-terminal region of the protein has been determined. The gene was cloned by cDNA synthesis with a laccase-specific synthetic deoxyundecanucleotide as primer and poly(A) RNA isolated from cycloheximide-treated N. crassa cultures as template. Based on the nucleotide sequence of the cDNA obtained, a unique 21-mer was synthesized and used to screen a genomic DNA library from N. crassa. Five different positive clones were isolated and shown to share an overlapping DNA region with the same pattern of restriction sites. Sequence analysis of the common 1.36-kilobase Sal I fragment revealed an open reading frame of 726 nucleotides. The amino acid sequence deduced is in complete agreement with the primary structures of several tryptic peptides isolated previously from N. crassa laccase. The analyzed carboxyl-terminal region of laccase exhibits a striking sequence homology to the carboxyl-terminal part of the third homology unit of the multicopper oxidase ceruloplasmin and to a smaller extent, to the low molecular weight blue copper proteins plastocyanin and azurin. Based on amino acid sequence comparison between these proteins, putative copper ligands of N. crassa laccase are proposed. Moreover, these data further support the hypothesis that the small blue copper proteins and the multicopper oxidases have evolved from the same ancestral gene.
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PMID:Isolation and partial nucleotide sequence of the laccase gene from Neurospora crassa: amino acid sequence homology of the protein to human ceruloplasmin. 294 40

Reoxidation process of reduced cucumber ascorbate oxidase (1.10.3.3) with dioxygen was investigated in detail through absorption, circular dichroic (CD) and electron paramagnetic resonance (EPR) spectra. One of the three type I coppers and the type II copper were reoxidized more rapidly than other type I coppers. The principal active site of ascorbate oxidase was considered to be comprised of one type I, one type II and a pair of type III coppers similarly to the active sites in laccase and ceruloplasmin. Remaining two type I and a pair of type III coppers were also disclosed to contribute to the oxidation of L-ascorbate.
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PMID:Oxidation of reduced cucumber ascorbate oxidase. 299 19

The evolutionary relationships of blue copper proteins are reviewed. Five homologous families of small blue proteins are recognized. Despite differences in length their peptide chains can all be accommodated into the eight-stranded fold of plastocyanin with some adjustments at three of the loops and the two termini. The C-termini of the blue oxidases ceruloplasmin and Neurospora laccase also fit into this fold and they are suggested to be homologous to the small blue proteins. The alignment of their amino acid sequences suggest some of the histidines to be binding active site copper. A superposition of the structures of poplar plastocyanin and bovine Cu-Zn superoxide dismutase (SOD) showed that 68 out of 99 alpha-carbons in plastocyanin overlapped with corresponding atoms in SOD with a rms distance of 2.99 A. In addition three of the histidine residues that were proposed to be copper-binding in laccase and ceruloplasmin aligned with ligands to the Cu-Zn pair in a SOD. Thus also SOD might be related to the blue proteins.
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PMID:Evolution of blue copper proteins. 304 63

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

alpha-Carboline substituted at C-6 with hydroxy-group is oxidized by human ceruloplasmin and fungal laccase into reactive intermediate which dimerizes, while 6-amino-alpha-carboline subjected to action of both enzymes yields the polymeric products. To prepare sufficient quantities of compounds needed for structure elucidation studies, laccase, immobilised in polyacrylamide gel was employed as a convenient reagent. The resulting products are suggested to be metabolities of 6-hydroxy- and 6-amino-alpha-carbolines formed in vivo.
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PMID:Microbial transformation of azacarbazoles. VI. Conversion of 6-hydroxy- and 6-amino-alpha-carbolines with copper oxidases. 344 28

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.
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PMID:Formation of a reactive iminium derivative by enzymatic and chemical oxidations of 16-O-acetylvindoline. 673 16

Human ceruloplasmin was attached to activated thiol-Sepharose via its thiol groups and was then digested with pepsin. After appropriate washings the thiol peptides were eluted by reduction and were carboxymethylated and purified by column chromatography and electrophoresis. Amino acid sequencing showed that the peptides were derived from five different areas in the molecule and together accounted for 92 residues, six of which were cysteines. Since one of the peptides contained two cysteines it seemed evident that, prior to the reductive elution of the peptides, one of these had been paired in a disulfide bridge with one of the four remaining thiol peptides present in the mixture. The disulfide was isolated and identified by digesting the immobilized protein with pepsin followed by trypsin. The second (tryptic) digestion released the disulfide peptide. Three of the true thiol peptides obtained occur in regions of sequence that have already been reported and which account for 564 of the approximately 1050 residues present in the protein. Three of them also show about 40% identity with each other, whereas no relatedness is observed with the fourth. The three related peptides are, moreover, clearly homologous to the copper-binding areas in the small blue plant and bacterial proteins plastocyanin and azurin. Homologous regions are also evident when the peptides are compared to the two sequences reported for the blue oxidase, fungal laccase, one of which contains a disulfide bridge.
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PMID:Identification of the thiol groups in human ceruloplasmin. 684 89

The phenoxazinone chromophore occurs in a variety of biological systems, including numerous pigments and certain antibiotics. It also appears to form as part of a mechanism to protect mammalian tissue from oxidative damage. During cultivation of the basidiomycete, Pycnoporus cinnabarinus, a red pigment was observed to accumulate in the culture medium. It was identified as the phenoxazinone derivative, cinnabarinic acid (CA). Laccase was the predominant extracellular phenoloxidase activity in P. cinnabarinus cultures. In vitro studies showed that CA was formed after oxidation of the precursor, 3-hydroxyanthranilic acid (3-HAA), by laccases. Moreover, oxidative coupling of 3-HAA to form CA was also demonstrated for the mammalian counterpart of laccase, the blue copper oxidase, ceruloplasmin.
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PMID:Laccase-mediated formation of the phenoxazinone derivative, cinnabarinic acid. 749 42

The copper oxidases human ceruloplasmin and Polyporous anceps laccase catalyze the oxidative coupling of mithramycin (1) and its aglycone chromomycinone (2) with p-hydroquinone to form new mithramycin-hydroquinone (3) and chromomycinone-hydroquinone adducts (4), respectively. Similar adducts could be formed by the nonenzymatic mimic of this reaction using benzoquinone and these aureolic acids in buffer solutions. FABMS of 3 indicated that the hydroquinone moiety was attached to the aureolic acid aglycone. Acid hydrolysis of 3 yielded a compound with the same chromatographic and spectroscopic characteristics as 4. Structure elucidation of 4 by NMR and MS revealed that the hydroquinone was attached to the C-5 position of the aglycone. NMR evidence indicated that 4 consisted of a mixture of ortho-substituted biphenyl rotamers. The mechanism of the copper oxidase catalyzed adduct formation reaction is presumed to involve radical formation through hydrogen removal at the 8-phenolic position, radical isomerization, and coupling with semiquinone radical also formed during enzymatic and nonenzymatic incubations. Identification of the covalent-hydroquinone adduct provides evidence that aureolic acid antibiotics can be metabolically converted to reactive radical intermediates, and it establishes the C-5 position of aureolic acid as an enzymatically reactive site. Unlike mithramycin, the mithramycin-hydroquinone adducts was inactive in the in vivo P388 leukemic antitumor test system.
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PMID:Oxidative coupling of mithramycin and hydroquinone catalyzed by copper oxidases and benzoquinone. Implications for the mechanism of action of aureolic acid antibiotics. 800 Aug 77


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