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)

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.
...
PMID:Molecular cloning of the gene for bilirubin oxidase from Myrothecium verrucaria and its expression in yeast. 836 Jan 71

Bilirubin oxidase (EC:1.3.3.5) purified from a culture medium of Myrothecium verrucaria MT-1 (authentic enzyme) catalyzes the oxidation of bilirubin to biliverdin in vitro and recombinant enzyme (wild type) was obtained by using an overexpression system of the bilirubin oxidase gene with Aspergillus oryzae harboring an expression vector. The absorption and ESR spectra showed that both bilirubin oxidases are multicopper oxidases containing type 1, type 2, and type 3 coppers similar to laccase, ascorbate oxidase, and ceruloplasmin. Site-directed mutagenesis has been performed for the possible ligands of each type of copper. In some mutants, Cys457 --> Val, Ala, His94 --> Val, and His134.136 --> Val, type 1 and type 2 copper centers were perturbed completely and the enzyme activity was completely lost. Differing from the holoenzyme, these mutants showed type 3 copper signals. However, the optical and magnetic properties characteristic of type 1 copper were retained even by mutating one of the type 1 copper ligands, i.e., a mutant, Met467 --> Gly, showed a weak but apparent enzyme activity. A double mutant His456.458 --> Val had only type 1 Cu, showing a blue band at 600 nm (epsilon = 1.6 x 10(3)) and an ESR signal with very narrow hyperfine splitting (A parallel = 7.2 x 10(-)3 cm-1). Since the type 2 and type 3 coppers are not present, the mutant did not show enzyme activity. These results strongly imply that the peculiar sequence in bilirubin oxidase, His456-Cys457-His458, forms an intramolecular electron-transfer pathway between the type 1 copper site and the trinuclear center composed of the type 2 and type 3 copper sites.
...
PMID:Myrothecium verrucaria bilirubin oxidase and its mutants for potential copper ligands. 1007 56

Type 1 Cu centers in cupredoxins, nitrite reductases, and multi-copper oxidases utilize the same trigonal core ligation to His-Cys-His, with a weak axial ligand generally provided by a Met sulfur. In azurin, an additional axial ligand, a carbonyl oxygen from a Gly, is present. The importance of these axial ligands and in particular the Met has been debated extensively in terms of their role in fine-tuning the redox potential, spectroscopic properties, and rack-induced or entatic state properties of the copper sites. Extensive site-directed mutagenesis of the Met ligand has been carried out in azurin, but the presence of an additional carbonyl oxygen axial ligand has made it difficult to interpret the effects of these substitutions. Here, the axial methionine ligand (Met148) in rusticyanin is replaced with Leu, Gln, Lys, and Glu to examine the effect on the redox potential, acid stability, and copper site geometry. The midpoint redox potential varies from 363 (Met148Lys) to 798 mV (Met148Leu). The acid stability of the oxidized proteins is reduced except for the Met148Gln mutant. The Gln mutant remains blue at all pH values between 2.8 and 8, and has a redox potential of 563 mV at pH 3.2. The optical and rhombic EPR properties of this mutant closely resemble those of stellacyanin, which has the lowest redox potential among single-type 1 copper proteins (185 mV). The Met148Lys mutant exhibits type 2 Cu EPR and optical spectra in this pH range. The Met148Glu mutant exhibits a type 2 Cu EPR spectrum above pH 3 and a mixture of type 1 and type 2 Cu spectra at lower pH. The Met148Leu mutant exhibits the highest redox potential ( approximately 800 mV at pH 3.2) which is similar to the values in fungal laccase and in the type 1 Cu site of ceruloplasmin where this axial ligand is also a Leu.
...
PMID:Role of the axial ligand in type 1 Cu centers studied by point mutations of met148 in rusticyanin. 1050 37

In furtherance of our structure-activity relationship studies on the antitumor activity of indolo[2,3-b]quinolines, novel cytotoxic derivatives bearing methyl groups at N-5, C-11, C-2 and/or C-9, as well as methoxy-groups at C-2 and/or C-9, were synthesized by the modified Graebe-Ullmann reaction. To elucidate the metabolic pathways of these compounds, zygomycete fungus Cunninghamella elegans ATCC 9245 (which is known to produce drug metabolites that are also formed in mammals) was used as a mimetic organism. Simultaneously, biotransformation of the same substrates was carried out with a microsomal fraction of rat liver. Three forms of microbial conversion were observed: hydroxylation of the aromatic ring or hydroxylation of the methyl group, and O-demethylation. The reaction proceeded regioselectively, and only positions C-2 and C-9 were affected in the indolo[2,3-b]quinoline system. The products formed were found to be identical with the metabolites generated by rat liver microsomes. The metabolites obtained displayed a cytotoxic activity in vitro against colon adenocarcinoma SW-707 and lung carcinoma A-549 (ID50 in the range 0.27-3.04 microM), which was as strong as that of the substrates. In the course of the further metabolic pathway study of indolo[2,3-b]quinolines we found that metabolites with a hydroxyl group in the aromatic system were transformed to non-cytotoxic polymeric products by multicopper oxidases: human ceruloplasmin or fungal laccase (used as mimetic enzyme), whereas metabolites with a hydroxymethyl group did not undergo such bioconversion. The last mentioned compounds can be regarded as a novel type of cytotoxic indolo[2,3-b]quinoline derivatives formed in metabolic processes.
...
PMID:Microbial conversion of methyl- and methoxy- substituted derivatives of 5H-indolo[2,3-b]quinoline as a method of developing novel cytotoxic agents. 1065 30

The small blue copper protein rusticyanin from Thiobacillus ferrooxidans contains a type 1 Cu centre with a single axial ligand, Met148, which together with the His-Cys-His trigonal planar ligands produces a distorted trigonal pyramidal coordination geometry to copper. Type 1 Cu sites are found in cupredoxins and several multicopper proteins, including oxidases and nitrite reductases. The role of the axial ligand has been extensively debated in terms of its function in the fine tuning of the redox potential and spectroscopic properties of type 1 Cu sites. Numerous mutations of the Met ligand in azurins have been studied, but interpretation of the results has been complicated by the presence of the additional carbonyl oxygen ligand from Gly45, a neighbouring residue to the coordinating His46. The importance of the axial ligand has been further emphasized by the finding that the type 1 centre in Rhus vernicifera stellacyanin, with the lowest redox potential in a type 1 Cu site of 184 mV, has Gln as the axial ligand, whilst fungal laccase and ceruloplasmin, which have redox potentials of 550-800 mV, have a Leu in this position. Here, the crystal structure of the M148Q mutant of rusticyanin at 1.5 A resolution is presented. This is a significantly higher resolution than that of the structures of native rusticyanin. In addition, the M148Q structure is that of the oxidized protein while the native structures to date are of the reduced protein. The mutant protein crystallizes with two molecules per asymmetric unit, in contrast to the one present in the native crystal form. This mutant's redox potential (550 mV at pH 3.2) is lowered compared with that of the native protein ( approximately 670 mV at pH 3.2) by about 120 mV. The type 1 Cu site of M148Q closely mimics the structural characteristics of the equivalent site in non-glycosylated cucumber stellacyanin (redox potential approximately 260 mV) and, owing to the absence in rusticyanin of the fifth, carbonyl ligand present in azurin, may provide a better model for the R. vernicifera stellacyanin (redox potential approximately 184 mV) type 1 Cu site, which also lacks the fifth ligand. Furthermore, the presence of two molecules in the asymmetric unit cell indicates a potential binding region of the redox partners.
...
PMID:Structure of the M148Q mutant of rusticyanin at 1.5 A: a model for the copper site of stellacyanin. 1122 11

Fet3p is a multicopper oxidase recently isolated from the yeast, Saccharomyces cerevisiae. Fet3p is functionally homologous to ceruloplasmin (Cp) in that both are ferroxidases. However, by sequence homology Fet3p is more similar to fungal laccase, and both contain a type 1 Cu site that lacks the axial methionine ligand present in the functional type 1 sites of Cp. To determine the contribution of the electronic structure of the type 1 Cu site of Fet3p to the ferroxidase mechanism, we have examined the absorption, circular dichroism, magnetic circular dichroism, electron paramagnetic resonance, and resonance Raman spectra of wild-type Fet3p and type 1 and type 2 Cu-depleted mutants. The spectroscopic features of the type 1 Cu site of Fet3p are nearly identical to those of fungal laccase, indicating a very similar three-coordinate geometry. We have also examined the reactivity of the type 1 Cu site by means of redox titrations and stopped-flow kinetics. From poised potential redox titrations, the E degrees of the type 1 Cu site is 427 mV, which is low for a three-coordinate type 1 Cu site. The kinetics of reduction of the type 1 Cu sites of four different multicopper oxidases with two different substrates were compared. The type 1 site of a plant laccase (Rhus vernicifera) is reduced moderately slowly by both Fe(II) and a bulky organic substrate, 1,4-hydroquinone (with 6 equiv of substrate, k(obs) = 0.029 and 0.013 s(-)(1), respectively). On the other hand, the type 1 site of a fungal laccase (Coprinus cinereus) is reduced very rapidly by both substrates (k(obs) > 23 s(-)(1)). In contrast, both Fet3p and Cp are rapidly reduced by Fe(II) (k(obs) > 23 s(-)(1)), but only very slowly by 1,4-hydroquinone (10- and 100-fold more slowly than plant laccase, respectively). Semiclassical theory is used to analyze the origin of these differences in reactivity in terms of type 1 Cu site accessibility to specific substrates.
...
PMID:Spectroscopy and reactivity of the type 1 copper site in Fet3p from Saccharomyces cerevisiae: correlation of structure with reactivity in the multicopper oxidases. 1138 33

2,6-Dimethoxyphenol is a versatile substrate for Pyricularia oryzae laccase, PpoA from Marinomonas mediterranea, phenoxazinone synthase from Streptomyces antibioticus and mammalian ceruloplasmin. In addition, in cellular extracts of microorganisms expressing other blue multicopper proteins with no enzymatic activity previously described, such as Escherichia coli (copper resistance CueO), Pseudomonas syringae and Xanthomonas campestris (copper resistance CopA), Bacillus subtilis (sporulation protein CotA) and Saccharomyces cerevisiae (iron transporter Fet3p), laccase activity is detected under appropriate conditions. This oxidase activity can be spectrophotometrically followed by the oxidation of 2,6-dimethoxyphenol. Specific staining after SDS-PAGE is also possible for some of these proteins. This detection assay can facilitate the study of the multiple functions that such proteins seem to carry out in a variety of microorganisms.
...
PMID:Dimethoxyphenol oxidase activity of different microbial blue multicopper proteins. 1168 98

The crystal structures of the Met148Leu and Ser86Asp mutants of rusticyanin are presented at 1.82 and 1.65 A resolution, respectively. Both of these structures have two molecules in the asymmetric unit compared to the one present in the crystal form of the native protein. This provides an opportunity to investigate intramolecular electron transfer pathways in rusticyanin. The redox potential of the Met148Leu mutant ( approximately 800 mV) is elevated compared to that of the native protein ( approximately 670 mV at pH 3.2) while that of the Ser86Asp mutant ( approximately 623 mV at pH 3.2) is decreased. The effect of the Ser86Asp mutation on the hydrogen bonding near the type 1 Cu site is discussed and hence its role in determining acid stability is examined. The type 1 Cu site of Met148Leu mimics the structural and biochemical characteristics of those found in domain II of ceruloplasmin and fungal laccase. Moreover, the native rusticyanin's cupredoxin core and the type 1 Cu site closely resemble those found in ascorbate oxidase and nitrite reductase. Structure based phylogenetic trees have been re-examined in view of the additional structural data on rusticyanin and fungal laccase. We confirm that rusticyanin is in the same class as nitrite reductase domain 2, laccase domain 3 and ceruloplasmin domains 2, 4 and 6.
...
PMID:Crystal structures of the Met148Leu and Ser86Asp mutants of rusticyanin from Thiobacillus ferrooxidans: insights into the structural relationship with the cupredoxins and the multi copper proteins. 1207 84

Laccase is a polyphenol oxidase, which belongs to the family of blue multicopper oxidases. These enzymes catalyze the one-electron oxidation of four reducing-substrate molecules concomitant with the four-electron reduction of molecular oxygen to water. Laccases oxidize a broad range of substrates, preferably phenolic compounds. In the presence of mediators, fungal laccases exhibit an enlarged substrate range and are then able to oxidize compounds with a redox potential exceeding their own. Until now, only one crystal structure of a laccase in an inactive, type-2 copper-depleted form has been reported. We present here the first crystal structure of an active laccase containing a full complement of coppers, the complete polypeptide chain together with seven carbohydrate moieties. Despite the presence of all coppers in the new structure, the folds of the two laccases are quite similar. The coordination of the type-3 coppers, however, is distinctly different. The geometry of the trinuclear copper cluster in the Trametes versicolor laccase is similar to that found in the ascorbate oxidase and that of mammalian ceruloplasmin structures, suggesting a common reaction mechanism for the copper oxidation and the O(2) reduction. In contrast to most blue copper proteins, the type-1 copper in the T. versicolor laccase has no axial ligand and is only 3-fold coordinated. Previously, a modest elevation of the redox potential was attributed to the lack of an axial ligand. Based on the present structural data and sequence comparisons, a mechanism is presented to explain how laccases could tune their redox potential by as much as 200 mV.
...
PMID:Crystal structure of a laccase from the fungus Trametes versicolor at 1.90-A resolution containing a full complement of coppers. 1216 89

Human ceruloplasmin is a copper containing serum glycoprotein with multiple functions. The crystal structure shows that its six domains are arranged in three pairs with a pseudo-ternary axis. Both the holo and apo forms of human ceruloplasmin were studied by size exclusion chromatography and small angle x-ray scattering in solution. The experimental curve of the holo form displays conspicuous differences with the scattering pattern calculated from the crystal structure. Once the carbohydrate chains and flexible loops not visible in the crystal are accounted for, remaining discrepancies suggest that the central pair of domains may move as a whole with respect to the rest of the molecule. The quasisymmetrical crystal structure therefore appears to be stabilized by crystal packing forces. Upon copper removal, the scattering pattern of human ceruloplasmin exhibits very large differences with that of the holoprotein, which are interpreted in terms of essentially preserved domains freely moving in solution around flexible linkers and exploring an ensemble of open conformations. This model, which is supported by the analysis of domain interfaces, provides a structural explanation for the differences in copper reincorporation into the apoprotein and activity recovery between human ceruloplasmin and two other multicopper oxidases, ascorbate oxidase and laccase. Our results demonstrate that, beyond catalytic activity, the three-copper cluster at the N-terminal-C-terminal interface plays a crucial role in the structural stability of human ceruloplasmin.
...
PMID:A key structural role for active site type 3 copper ions in human ceruloplasmin. 1217 70


<< Previous 1 2 3 4 Next >>

---