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 cDNA library of the Japanese lacquer tree (Rhus vernicifera) was constructed by the reverse transcription of mRNA. A cDNA encoding laccase was amplified by PCR using primers based on the N-terminal amino acid sequences of the purified laccase and its peptide fragments formed by digestions with chymotrypsin and trypsin, and subcloned. The laccase cDNA clone contained a single, large open reading frame of 1599 nucleotides, encoding a protein of 533 amino acids with a calculated molecular mass of 58981 Da. The lacquer laccase was found to have 42 to 62% identity with other plant laccases and 20 to 24% identity with microorganism laccases at the deduced amino acid level. Differing from microorganism laccases the lacquer laccase utilizes a Met residue in addition to one Cys and two His residues to construct the type 1 Cu site. The secondary structure of the lacquer laccase was predicted to mainly consist of the beta-structure (28.7%) and loop and random structures (67.0%). The alpha-helix content was predicted to be only 4.3%. The location of these secondary structures was assumed to be very similar to those of ascorbate oxidase and fungal laccase, the crystal structures of which have been determined.
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PMID:Primary structure of a Japanese lacquer tree laccase as a prototype enzyme of multicopper oxidases. 1212 69

Various amino acids, their analogues and vitamins have shown stimulatory as well as inhibitory effects on laccase production by Cyathus bulleri. DL-methionine, DL-tryptophan, glycine and DL-valine stimulated laccase production, while L-cysteine monohydrochloride completely inhibited the enzyme production. Among vitamins tested biotin, riboflavin and pyridoxine hydrochloride were found to induce laccase production.
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PMID:Effect of amino acids and vitamins on laccase production by the bird's nest fungus Cyathus bulleri. 1213 66

A variety of spectroscopic techniques, combined with density functional calculations, are used to describe the electronic structure of the Leu513His variant of the type 1 Cu site in Myceliophthora thermophila laccase. This mutation changes the type 1 Cu from a blue to a green site. Electron paramagnetic resonance (EPR), optical absorption, circular dichroism, and magnetic circular dichroism (MCD) spectroscopies reveal that, relative to the trigonal planar blue type 1 Cu site in wild-type fungal laccase, the covalency and the ligand field strength at the Leu513His green type 1 Cu center decrease. Additionally, there is a significant reorientation of the d(x)()()2(-)(y)()()2( )singly occupied MO, such that the overlap with the Cys sulfur valence orbital changes from pi to sigma. A density functional study in which internal coordinates are systematically altered reveals that these changes are due to the increased strength of the axial ligand (none to His), leading to a tetragonal distortion and elongation of the equatorial Cu-ligand bonds. These calculations provide insight into the experimental differences in the EPR parameters, charge-transfer absorption spectrum, and ligand-field MCD spectrum between the axial-His variant and blue Cu centers (plastocyanin and the type 1 site in fungal laccase). There are also significant differences between the green site in the Leu513His variant and other naturally occurring, green type 1 Cu sites such as in nitrite reductase, which have short axial Cu-S(Met) bonds. The large difference in EPR parameters between these green type 1 sites derives from a change in ligand field excitation energies observed by MCD, which reflects a decrease in ligand field strength. This is associated with different steric interactions of a His vs an axial Met ligand in a tetragonally distorted type 1 site. Changes in the electronic structure of the Cu site correlate with the difference in reactivity of the green His variant relative to blue wild-type fungal laccase.
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PMID:Spectroscopic characterization of the Leu513His variant of fungal laccase: effect of increased axial ligand interaction on the geometric and electronic structure of the type 1 Cu site. 1281 56

Laccase (EC 1.10.3.2) is an enzyme with p-diphenol oxidase activity that is a member of a group of proteins collectively known as multicopper, or blue copper, oxidases. Laccase is hypothesized to play an important role in insect cuticle sclerotization by oxidizing catechols in the cuticle to their corresponding quinones, which then catalyze protein cross-linking reactions. To facilitate studies of the structure, function and regulation of insect laccases, we have cloned two cDNAs for laccases from the tobacco hornworm, Manduca sexta (MsLac1 and 2), and one from the malaria mosquito, Anopheles gambiae (AgLac1). The MsLac1 and 2 cDNAs encode proteins of 801 amino acids (aa) and 760 aa, respectively, while the AgLac1 cDNA encodes a protein of 1009 aa. All three cDNAs contain putative secretion signal sequences, and the 10 histidines and one cysteine that form the copper-binding centers, as well as a methionine in the T1 copper center. Novel to the insect laccases, relative to both fungal and plant laccases, is a longer amino-terminal sequence characterized by a unique domain consisting of several conserved cysteine, aromatic, and charged residues. Northern blot analyses identified single transcripts of approximately 3.6, 3.5, and 4.4 kb for MsLac1, MsLac2, and AgLac1, respectively, and also showed that AgLac1 was expressed in all life stages of the mosquito. RT-PCR revealed that the MsLac1 transcript was most abundant in the midgut, Malpighian tubules, and epidermis, whereas the MsLac2 transcript was most abundant in the epidermis. MsLac2 showed strong expression in the pharate pupal and reduced expression in the early pupal epidermis, consistent with the laccases' presumed role in cuticle sclerotization.
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PMID:Characterization of cDNAs encoding putative laccase-like multicopper oxidases and developmental expression in the tobacco hornworm, Manduca sexta, and the malaria mosquito, Anopheles gambiae. 1472 95

The relative Cu(2+)/Cu(+) reduction potentials of six type-1 copper sites (cucumber stellacyanin, P. aeruginosa azurin, poplar plastocyanin, C. cinereus laccase, T. ferrooxidans rusticyanin, and human ceruloplasmin), which lie in a reduction potential range from 260 mV to over 1000 mV, have been studied by quantum mechanical calculations. The range and relative orderings of the reduction potentials are reproduced very well compared to experimental values. The study suggests that the main structural determinants of the relative reduction potentials of the blue copper sites are located within 6 A of the Cu atoms. Further analysis suggests that the reduction potential differences of type-1 copper sites are caused by axial ligand interactions, hydrogen bonding to the S(Cys), and protein constraint on the inner sphere ligand orientations. The low reduction potential of cucumber stellacyanin is due mainly to a glutamine ligand at the axial position, rather than a methionine or a hydrophobic residue as in the other proteins. A stronger interaction with a backbone carbonyl group is a prime contributor to the lower reduction potential of P. aeruginosa azurin as compared to poplar plastocyanin, whereas the reverse is true for C. cinereus laccase and T. ferrooxidans rusticyanin. The lack of an axial methonine ligand also contributes significantly to the increased reduction potentials of C. cinereus laccase and human ceruloplasmin. However, in the case of C. cinereus laccase, this increase is attenuated by the presence of only one amide NH hydrogen bond to the S(Cys) rather than two in the other proteins. In human ceruloplasmin the reduction potential is further increased by the structural distortion of the equatorial ligand orientation.
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PMID:Determinants of the relative reduction potentials of type-1 copper sites in proteins. 1521 51

CueO is a multicopper oxidase (MCO) that is involved in the homeostasis of Cu in Escherichia coli and is the sole cuprous oxidase to have ever been found. Differing from other MCOs, the substrate-binding site of CueO is deeply buried under a methionine-rich helical region including alpha-helices 5, 6, and 7 that interfere with the access of organic substrates. We deleted the region Pro357-His406 and replaced it with a Gly-Gly linker. The crystal structures of a truncated mutant in the presence and in the absence of excess Cu(II) indicated that the scaffold of the CueO molecule and metal-binding sites were reserved in comparison with those of CueO. In addition, the high thermostability of the protein molecule and its spectroscopic and magnetic properties due to four Cu centers were also conserved after truncation. As for functions, the cuprous oxidase activity of the mutant was reduced to ca 10% that of recombinant CueO owing to the decrease in the affinity of the labile Cu site for Cu(I) ions, although activities for laccase substrates such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), p-phenylenediamine, and 2,6-dimethoxyphenol increased due to changes in the access of these organic substrates to the type I Cu site. The present engineering of CueO indicates that the methionine-rich alpha-helices function as a barrier to the access of bulky organic substrates, which provides CueO with specificity as a cuprous oxidase.
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PMID:Structure and function of the engineered multicopper oxidase CueO from Escherichia coli--deletion of the methionine-rich helical region covering the substrate-binding site. 1780 14

The axial ligand of the catalytic mononuclear T1 copper site (Met(502)) of the CotA laccase was replaced by a leucine or phenylalanine residue to increase the redox potential of the enzyme. These mutations led to an increase in the redox potential by approx. 100 mV relative to the wild-type enzyme but the catalytic constant k(cat) in the mutant enzymes was severely compromised. This decrease in the catalytic efficiency was unexpected as the X-ray analysis of mutants has shown that replacement of methionine ligand did not lead to major structural changes in the geometry of the T1 centre or in the overall fold of the enzyme. However, the mutations have a profound impact on the thermodynamic stability of the enzyme. The fold of the enzyme has become unstable especially with the introduction of the larger phenylalanine residue and this instability should be related to the decrease in the catalytic efficiency. The instability of the fold for the mutant proteins resulted in the accumulation of an intermediate state, partly unfolded, in-between native and unfolded states. Quenching of tryptophan fluorescence by acrylamide has further revealed that the intermediate state is partly unfolded.
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PMID:Insight into stability of CotA laccase from the spore coat of Bacillus subtilis. 1803 Dec 70

A new method is proposed for the removal of the phenylhydrazide protecting group by the action of peroxidase or laccase, the enzymes attributed to the class of oxidoreductases. The deblocking procedure is performed under mild oxidative conditions, i.e., aqueous solution and neutral or close to neutral pH. Such mild oxidizing agents as 1 mM H(2)O(2) and air oxygen are used for unmasking. The method is available for the deblocking of both alpha- and gamma-carboxyl groups. The enzyme-catalyzed removal of the phenylhydrazide protecting group causes no oxidative modification nor destruction of methionine or tryptophan side chains.
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PMID:Peroxidase and laccase as catalysts for removal of the phenylhydrazide protecting group under mild conditions. 1860 61

Laccase-catalyzed oxidation was able to induce intermolecular cross-links in beta-lactoglobulin, and ferulic acid-mediated laccase-catalyzed oxidation was able to induce intermolecular cross-links in alpha-casein, whereas transglutaminase cross-linked only alpha-casein. In addition, different patterns of laccase-induced oxidative modifications were detected, including dityrosine formation, formation of fluorescent tryptophan oxidation products, and carbonyls derived from histidine, tryptophan, and methionine. Laccase-catalyzed oxidation as well as transglutaminase induced only minor changes in surface tension of the proteins, and the changes could not be correlated to protein cross-linking. The presence of ferulic acid was found to influence the effect of laccase, allowing laccase to form irreducible intermolecular cross-links in beta-lactoglobulin and resulting in proteins exercising higher surface tensions due to cross-linking as well as other oxidative modifications. The outcome of using ferulic acid-mediated laccase-catalyzed oxidation to modify the functional properties of proteinaceous food components or other biosystems is expected to be highly dependent on the protein composition, resulting in different changes of the functional properties.
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PMID:Cross-linking proteins by laccase-catalyzed oxidation: importance relative to other modifications. 1905 90

Phenoloxidase (PO) is a crucial component of the immune system of echinoderms. In the present study, the full-length cDNA of PO (AjPO) was cloned from coelomocytes of the sea cucumber Apostichopus japonicus using 3'- and 5'-rapid amplification of cDNA ends (RACE) PCR method, which is 2508 bp, with an open reading frame (ORF) of 2040 bp encoding 679 amino acids. AjPO contains a transmembrane domain, and three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine respectively. Phylogenetic analysis revealed that AjPO was clustered with laccase-type POs of invertebrates. Using the isolated membrane proteins as crude AjPO, the enzyme could catalyze the substrates catechol, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine. The results described above collectively proved that AjPO was a membrane-binding laccase-type PO. The quantitative real-time PCR (qRT-PCR) analysis revealed that AjPO mRNA was expressed in muscle, body wall, coelomocytes, tube feet, respiratory tree and intestine with the highest expression level in coelomocytes. AjPO could be significantly induced by lipopolysaccharide (LPS), peptidoglycan (PGN), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C), suggesting AjPO is closely involved in the defense against the infection of bacteria, fungi and double-stranded RNA viruses.
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PMID:Phenoloxidase from the sea cucumber Apostichopus japonicus: cDNA cloning, expression and substrate specificity analysis. 2435 5


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