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1.
Francisco Solano Patricia Lucas-El&#x;o Daniel López-Serrano Eva Fernández Antonio Sanchez-Amat 《FEMS microbiology letters》2001,204(1):175-181
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. 相似文献
2.
The putative multi-copper oxidase CueO had previously been implicated in intrinsic copper resistance in Escherichia coli. In this report we showed that the presence of CueO in the periplasm protected alkaline phosphatase from copper-induced damage. CueO contained four copper atoms per molecule and displayed spectroscopic properties typical of blue copper oxidases. CueO catalyzed the oxidation of p-phenylenediamine (pPD), 2,6-dimethoxyphenol (DMP) and exhibited ferroxidase activity in vitro. 相似文献
3.
CueO is a multicopper oxidase involved in a copper efflux system of Escherichia coli and has high cuprous oxidase activity but little or no oxidizing activity toward various organic substances. However, its activity toward oxidization of organic substrates was found to be considerably increased by the removal of the methionine-rich helical segment that covers the substrate-binding site (Δα5-7 CueO) [Kataoka, K., et al. (2007) J. Mol. Biol. 373, 141]. In the study presented here, mutations at Pro444 to construct a second NH-S hydrogen bond between the backbone amide and coordinating Cys500 thiolate of the type I copper are shown to result in positive shifts in the redox potential of this copper center and enhanced oxidase activity in CueO. Analogous enhancement of the activity of Δα5-7 CueO has been identified only in the Pro444Gly mutant because Pro444 mutants limit the incorporation of copper ions into the trinuclear copper center. The activities of both CueO and Δα5-7 CueO were also enhanced by mutations to break down the hydrogen bond between the imidazole group of His443 that is coordinated to the type I copper and the β-carboxy group of Asp439 that is located in the outer sphere of the type I copper center. A synergetic effect of the positive shift in the redox potential of the type I copper center and the increase in enzyme activity has been achieved by the double mutation of Pro444 and Asp439 of CueO. Absorption, circular dichroism, and resonance Raman spectra indicate that the characteristics of the Cu(II)-S(Cys) bond were only minimally perturbed by mutations involving formation or disruption of a hydrogen bond from the coordinating groups to the type I copper. This study provides widely applicable strategies for tuning the activities of multicopper oxidases. 相似文献
4.
Laccases produced by white rot fungi are capable of rapidly oxidizing benzo[a]pyrene. We hypothesize that the polycyclic aromatic hydrocarbon (PAH)-degrading bacteria producing laccase can enhance the degree of benzo[a]pyrene mineralization. However, fungal laccases are glycoproteins which cannot be glycosylated in bacteria, and there is no evidence to show that bacterial laccases can oxidize benzo[a]pyrene. In this study, the in vitro oxidation of PAHs by crude preparations of the bacterial laccase, CueO, from Escherichia coli was investigated. The results revealed that the crude CueO catalyzed the oxidation of anthracene and benzo[a]pyrene in the same way as the fungal laccase from Trametes versicolor, but showed specific characteristics such as thermostability and copper dependence. In the presence of 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid), high amounts of anthracene and benzo[a]pyrene, 80% and 97%, respectively, were transformed under optimal conditions of 60°C, pH 5, and 5 mmol l(-1) CuCl(2) after a 24-h incubation period. Other PAHs including fluorene, acenaphthylene, phenanthrene, and benzo[a]anthracene were also oxidized by the crude CueO. These findings indicated the potential application of prokaryotic laccases in enhancing the mineralization of benzo[a]pyrene by PAH-degrading bacteria. 相似文献
5.
Melanocarpus albomyces laccase crystals were soaked with 2,6-dimethoxyphenol, a common laccase substrate. Three complex structures from different soaking times were solved. Crystal structures revealed the binding of the original substrate and adducts formed by enzymatic oxidation of the substrate. The dimeric oxidation products were identified by mass spectrometry. In the crystals, a 2,6-dimethoxy-p-benzoquinone and a C-O dimer were observed, whereas a C-C dimer was the main product identified by mass spectrometry. Crystal structures demonstrated that the substrate and/or its oxidation products were bound in the pocket formed by residues Ala191, Pro192, Glu235, Leu363, Phe371, Trp373, Phe427, Leu429, Trp507 and His508. Substrate and adducts were hydrogen-bonded to His508, one of the ligands of type 1 copper. Therefore, this surface-exposed histidine most likely has a role in electron transfer by laccases. Based on our mutagenesis studies, the carboxylic acid residue Glu235 at the bottom of the binding site pocket is also crucial in the oxidation of phenolics. Glu235 may be responsible for the abstraction of a proton from the OH group of the substrate and His508 may extract an electron. In addition, crystal structures revealed a secondary binding site formed through weak dimerization in M. albomyces laccase molecules. This binding site most likely exists only in crystals, when the Phe427 residues are packed against each other. 相似文献
6.
Christian Bukh 《Journal of inorganic biochemistry》2010,104(10):1029-1037
The specific activities of crude and purified Coprinus cinereus laccase preparations could be enhanced by a factor of 10-12 by activation with copper ions. The copper to protein contents of purified non-activated laccase were 2.3 ± 0.1 compared to 3.3 ± 0.1 in purified activated laccase indicating that only a fraction of the laccase can be activated. Purified laccase not activated with copper ions shows in isoelectric focusing four bands in order of decreasing pI in a ratio 1/5/3/1 where only bands I and II had laccase activity. Purified activated laccase showed only three bands (I, II and III) in the ratio 5/4/1 all with some laccase activity. The pH profile of the activity for activated and non-activated laccase showed identical behavior indicating that the active forms were the same. The change in UV-Vis around 330 nm following the depletion and reconstitution of the enzyme combined with activity measurements supports the reversibility of the selective removal and insertion of copper ions at the type 2 site. The circular dichroism spectrum of activated purified laccase has characteristic changes around 350 nm relative to non-activated laccase indicative of changes at the type 2/type 3 sites. The difference between the electron paramagnetic resonance spectra of non-activated and activated C. cinereus laccase indicates that a fraction of the non-activated purified laccase contained a copper(II) signal with a coupling constant between a type 1 and a type 2 copper(II). This electron paramagnetic resonance signal could be explained by an induced asymmetry in the type 3 site due to a missing type 2 copper ion. 相似文献
7.
We have found CueO from Escherichia coli to have a robust cuprous oxidase activity, severalfold higher than any homologue. These data suggest that a functional role for CueO in protecting against copper toxicity in vivo includes the removal of Cu(I). 相似文献
8.
Further characterization of peptidylglycine alpha-amidating monooxygenase from bovine neurointermediate pituitary 总被引:2,自引:0,他引:2
The ability of purified bovine neurointermediate pituitary peptidyl glycine alpha-amidating monooxygenase to catalyze the conversion of peptide substrates (D-Tyr-X-Gly) into amidated product peptides (D-Tyr-X-NH2) was evaluated. The pH optimum of the reaction was pH 8.5 when X was Val, Trp, or Pro, but 5.5 to 6.0 when X was Glu. Similar maximum velocity (Vmax) values were obtained for the Val, Trp, and Pro substrates while the Glu substrate had a substantially higher Vmax. The Michaelis-Menten constant (Km) of the enzyme for the peptide substrate increased in the order Trp less than Val less than Pro much less than Glu. Increasing levels of ascorbate brought about parallel increases in Km and Vmax, suggesting the presence of an irreversible step separating the interaction of the enzyme with the two substrates. The effect of copper on enzyme activity was dependent on the peptide substrate and the reaction pH. With the Val substrate, exogenous copper was required for optimal activity; no other metal ion tested could substitute for copper. With the Glu substrate, exogenous copper was not required for optimal activity; however, diethyldithiocarbamate, a copper chelator, inhibited activity and only copper could reverse this inhibitory effect. The ability of various cofactors to stimulate alpha-amidating monooxygenase activity was also dependent on assay conditions. With the Val or Glu substrate in the presence of exogenous copper, a variety of cofactors in addition to ascorbate were capable of supporting activity. With the Glu substrate in the absence of exogenous copper, the requirement of the enzyme for ascorbate was more strict. In keeping with the proposed reaction mechanism, nearly 1 mol ascorbate was consumed for each mole of D-Tyr-Glu-NH2 produced. 相似文献
9.
Regulation of Laccase Gene Transcription in Trametes versicolor 总被引:2,自引:0,他引:2
10.
Kataoka K Komori H Ueki Y Konno Y Kamitaka Y Kurose S Tsujimura S Higuchi Y Kano K Seo D Sakurai T 《Journal of molecular biology》2007,373(1):141-152
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. 相似文献
11.
《Process Biochemistry》2014,49(8):1266-1273
Biological processes for the degradation of intractable materials are still not considered to be practical due to the slow rates of enzymatic degradation. Cellulosomes are complexed enzyme systems with great degradative potential and one of the strategies for overcoming this problem. In this study, the laccase CueO from Escherichia coli was fused to the dockerin domain of a cellulosome system and further assembled with the scaffoldin miniCbpA, forming a laccase–miniCbpA complex. Compared to the individual subunits, laccase–miniCbpA complex caused a noticeable 2.1-fold increase in enzyme activity levels and enhanced degradation of various synthetic dyes, showing an increase of approximately 1.6-fold. Also, pretreated barley straw by laccase complexes was efficiently converted to bioethanol using a cellulase producing Saccharomyces cerevisiae strain. The laccase complexes caused a 2.6-fold increase in the amount of reduced sugar with an insoluble substrate in conditions with an identical amount of enzymes. The cellulolytic yeast with the aid of laccase complexes produced 2.34 g/L ethanol after 72 h, indicating an increase of approximately 2.1-fold compared to fermentation without the laccase complexes. This demonstrates the feasibility of developing an efficient laccase complex based on the cellulosome and this strategy may be used to degrade recalcitrant materials. 相似文献
12.
Enguita FJ Martins LO Henriques AO Carrondo MA 《The Journal of biological chemistry》2003,278(21):19416-19425
Endospores produced by the Gram-positive soil bacterium Bacillus subtilis are shielded by a proteinaceous coat formed by over 30 structural components, which self-assemble into a lamellar inner coat and a thicker striated electrodense outer coat. The 65-kDa CotA protein is an abundant component of the outer coat layer. CotA is a highly thermostable laccase, assembly of which into the coat is required for spore resistance against hydrogen peroxide and UV light. Here, we report the structure of CotA at 1.7-A resolution, as determined by x-ray crystallography. This is the first structure of an endospore coat component, and also the first structure of a bacterial laccase. The overall fold of CotA comprises three cupredoxin-like domains and includes one mononuclear and one trinuclear copper center. This arrangement is similar to that of other multicopper oxidases and most similar to that of the copper tolerance protein CueO of Escherichia coli. However, the three cupredoxin domains in CotA are further linked by external interdomain loops, which increase the packing level of the structure. We propose that these interdomain loops contribute to the remarkable thermostability of the enzyme, but our results suggest that additional factors are likely to play a role. Comparisons with the structure of other monomeric multicopper oxidases containing four copper atoms suggest that CotA may accept the largest substrates of any known laccase. Moreover, and unlike other laccases, CotA appears to have a flexible lidlike region close to the substrate-binding site that may mediate substrate accessibility. The implications of these findings for the properties of CotA, its assembly and the properties of the bacterial spore coat structure are discussed. 相似文献
13.
CueO belongs to the family of multicopper oxidases which are characterized by the presence of multiple copper-binding sites with different structural and functional properties. These enzymes share the ability to couple the one-electron oxidation of substrate to reduction of oxygen to water by way of a functional unit composed of a mononuclear type 1 blue copper site, which is the entry site for electrons, and of a trinuclear copper cluster formed by type 2 and binuclear type 3 sites, where oxygen binding and reduction take place. The mechanism of copper incorporation in CueO has been investigated by optical and EPR spectroscopy. The results indicate unambiguously that the process is sequential, with type 1 copper being the first to be reconstituted, followed by type 2 and type 3 sites. 相似文献
14.
Linkage between catecholate siderophores and the multicopper oxidase CueO in Escherichia coli
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Grass G Thakali K Klebba PE Thieme D Müller A Wildner GF Rensing C 《Journal of bacteriology》2004,186(17):5826-5833
The multicopper oxidase CueO had previously been demonstrated to exhibit phenoloxidase activity and was implicated in intrinsic copper resistance in Escherichia coli. Catecholates can potentially reduce Cu(II) to the prooxidant Cu(I). In this report we provide evidence that CueO protects E. coli cells by oxidizing enterobactin, the catechol iron siderophore of E. coli, in the presence of copper. In vitro, a mixture of enterobactin and copper was toxic for E. coli cells, but the addition of purified CueO led to their survival. Deletion of fur resulted in copper hypersensitivity that was alleviated by additional deletion of entC, preventing synthesis of enterobactin. In addition, copper added together with 2,3-dihydroxybenzoic acid or enterobactin was able to induce a Phi(cueO-lacZ) operon fusion more efficiently than copper alone. The reaction product of the 2,3-dihydroxybenzoic acid oxidation by CueO that can complex Cu(II) ions was determined by gas chromatography-mass spectroscopy and identified as 2-carboxymuconate. 相似文献
15.
Martins LO Soares CM Pereira MM Teixeira M Costa T Jones GH Henriques AO 《The Journal of biological chemistry》2002,277(21):18849-18859
The Bacillus subtilis endospore coat protein CotA shows laccase activity. By using comparative modeling techniques, we were able to derive a model for CotA based on the known x-ray structures of zucchini ascorbate oxidase and Cuprinus cereneus laccase. This model of CotA contains all the structural features of a laccase, including the reactive surface-exposed copper center (T1) and two buried copper centers (T2 and T3). Single amino acid substitutions in the CotA T1 copper center (H497A, or M502L) did not prevent assembly of the mutant proteins into the coat and did not alter the pattern of extractable coat polypeptides. However, in contrast to a wild type strain, both mutants produced unpigmented colonies and spores unable to oxidize syringaldazine (SGZ) and 2'2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The CotA protein was purified to homogeneity from an overproducing Escherichia coli strain. The purified CotA shows an absorbance and a EPR spectra typical of blue multicopper oxidases. Optimal enzymatic activity was found at < or =pH 3.0 and at pH 7.0 for ABTS or SGZ oxidation, respectively. The apparent K(m) values for ABTS and SGZ at 37 degrees C were of 106 +/- 11 and 26 +/- 2 microm, respectively, with corresponding k(cat) values of 16.8 +/- 0.8 and 3.7 +/- 0.1 s(-1). Maximal enzyme activity was observed at 75 degrees C with ABTS as substrate. Remarkably, the coat-associated or the purified enzyme showed a half-life of inactivation at 80 degrees C of about 4 and 2 h, respectively, indicating that CotA is intrinsically highly thermostable. 相似文献
16.
Redox-induced protonation state changes of the Glu residue in the multicopper oxidases, CueO and bilirubin oxidase (BO), were studied by attenuated total reflectance-Fourier transform infrared spectroscopy. By monitoring IR bands of the carboxylic acid CO stretch in the wild-type and Glu-to-Gln mutant enzymes the Glu506 of CueO (Glu463 of BO) was found to be unprotonated in the oxidised and protonated in the reduced forms. The results provided direct evidence for proton uptake by the Glu, suggesting it plays a key role in the proton donation to the activated oxygen species in the catalytic cycle. 相似文献
17.
Singh SK Roberts SA McDevitt SF Weichsel A Wildner GF Grass GB Rensing C Montfort WR 《The Journal of biological chemistry》2011,286(43):37849-37857
The multicopper oxidase CueO oxidizes toxic Cu(I) and is required for copper homeostasis in Escherichia coli. Like many proteins involved in copper homeostasis, CueO has a methionine-rich segment that is thought to be critical for copper handling. How such segments function is poorly understood. Here, we report the crystal structure of CueO at 1.1 Å with the 45-residue methionine-rich segment fully resolved, revealing an N-terminal helical segment with methionine residues juxtaposed for Cu(I) ligation and a C-terminal highly mobile segment rich in methionine and histidine residues. We also report structures of CueO with a C500S mutation, which leads to loss of the T1 copper, and CueO with six methionines changed to serine. Soaking C500S CueO crystals with Cu(I), or wild-type CueO crystals with Ag(I), leads to occupancy of three sites, the previously identified substrate-binding site and two new sites along the methionine-rich helix, involving methionines 358, 362, 368, and 376. Mutation of these residues leads to a ∼4-fold reduction in kcat for Cu(I) oxidation. Ag(I), which often appears with copper in nature, strongly inhibits CueO oxidase activities in vitro and compromises copper tolerance in vivo, particularly in the absence of the complementary copper efflux cus system. Together, these studies demonstrate a role for the methionine-rich insert of CueO in the binding and oxidation of Cu(I) and highlight the interplay among cue and cus systems in copper and silver homeostasis. 相似文献
18.
The Tat (twin-arginine translocation) system from Escherichia coli transports folded proteins with N-terminal twin-arginine signal peptides across the cytoplasmic membrane. The influence of general chaperones on Tat substrate targeting has not been clarified so far. Here we show that the chaperones SlyD and DnaK bind to a broad range of different Tat signal sequences in vitro and in vivo. Initially, SlyD and GroEL were purified from DnaK-deficient extracts by their affinity to various Tat signal sequences. Of these, only SlyD bound Tat signal sequences also in the presence of DnaK. SlyD and DnaK also co-purified with Tat substrate precursors, demonstrating the binding to Tat signal sequences in vivo. Deletion of dnaK completely abolished Tat-dependent translocation of CueO, but not of DmsA, YcdB, or HiPIP, indicating that DnaK has an essential role specifically for CueO. DnaK was not required for stability of the CueO precursor and thus served in some essential step after folding. A CueO signal sequence fusion to HiPIP was Tat-dependently transported without the need of DnaK, indicating that the mature domain of CueO is responsible for the DnaK dependence. The overall results suggest that SlyD and DnaK are in the set of chaperones that can serve as general Tat signal-binding proteins. DnaK has additional functions that are indispensable for the targeting of CueO. 相似文献
19.
Purification and characterization of a novel laccase from Coprinus cinereus and decolorization of different chemically dyes 总被引:1,自引:0,他引:1
Yaqiu Lin Zhen Zhang Yongsheng Tian Wei Zhao Bo Zhu Zhisheng Xu Rihe Peng Quanhong Yao 《Molecular biology reports》2013,40(2):1487-1494
Laccase is a blue copper oxidase with multiple copper ions and widely distributed in higher plant and fungi. To date, numerous fungal laccases have been reported by many researchers. In present work, a new laccase gene, named CcLCC5I, from Coprinus cinereus was synthesized chemically according to the yeast bias codon and integrated into Pichia pastoris GS115 genome by electroporation. SDS-PAGE analysis showed that the recombinant laccase has a molecular mass of approximately 56.8 kDa. Its biochemical properties was carried out using substrate 2-2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was showed that the optimum pH and temperature of the laccase is 3.0 and 55 °C, respectively. Except for copper ions, most metal ions inhibited the laccase activity at a high concentration about 10 mM. Sodium sulfite can also highly inhibit laccase activity whereas EDTA had no inhibitory effect on the laccase activity. The CcLCC5I have high ability to decolor not only azo but also aryl methane dyes. The recombinant laccase decolored 44.6 % orange G, 54.8 % Crystal Violet, and 87.2 % Malachite green at about 2.6 h. The novel laccase may be a good candidate for breeding engineering strains used in the treatment of industrial effluent containing azo and aryl methane dyes. 相似文献
20.
Differential regulation by organic compounds and heavy metals of multiple laccase genes in the aquatic hyphomycete Clavariopsis aquatica 总被引:2,自引:0,他引:2
Solé M Müller I Pecyna MJ Fetzer I Harms H Schlosser D 《Applied and environmental microbiology》2012,78(13):4732-4739
To advance the understanding of the molecular mechanisms controlling microbial activities involved in carbon cycling and mitigation of environmental pollution in freshwaters, the influence of heavy metals and natural as well as xenobiotic organic compounds on laccase gene expression was quantified using quantitative real-time PCR (qRT-PCR) in an exclusively aquatic fungus (the aquatic hyphomycete Clavariopsis aquatica) for the first time. Five putative laccase genes (lcc1 to lcc5) identified in C. aquatica were differentially expressed in response to the fungal growth stage and potential laccase inducers, with certain genes being upregulated by, e.g., the lignocellulose breakdown product vanillic acid, the endocrine disruptor technical nonylphenol, manganese, and zinc. lcc4 is inducible by vanillic acid and most likely encodes an extracellular laccase already excreted during the trophophase of the organism, suggesting a function during fungal substrate colonization. Surprisingly, unlike many laccases of terrestrial fungi, none of the C. aquatica laccase genes was found to be upregulated by copper. However, copper strongly increases extracellular laccase activity in C. aquatica, possibly due to stabilization of the copper-containing catalytic center of the enzyme. Copper was found to half-saturate laccase activity already at about 1.8 μM, in favor of a fungal adaptation to low copper concentrations of aquatic habitats. 相似文献