Linkage between catecholate siderophores and the multicopper oxidase CueO in Escherichia coli

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.     

A labile regulatory copper ion lies near the T1 copper site in the multicopper oxidase CueO

CueO, a multicopper oxidase, is part of the copper-regulatory cue operon in Escherichia coli, is expressed under conditions of copper stress and shows enhanced oxidase activity when additional copper is present. The 1.7-A resolution structure of a crystal soaked in CuCl2 reveals a Cu(II) ion bound to the protein 7.5 A from the T1 copper site in a region rich in methionine residues. The trigonal bipyramidal coordination sphere is unusual, containing two methionine sulfur atoms, two aspartate carboxylate oxygen atoms, and a water molecule. Asp-439 both ligates the labile copper and hydrogen-bonds to His-443, which ligates the T1 copper. This arrangement may mediate electron transfer from substrates to the T1 copper. Mutation of residues bound to the labile copper results in loss of oxidase activity and of copper tolerance, confirming a regulatory role for this site. The methionine-rich portion of the protein, which is similar to that of other proteins involved in copper homeostasis, does not display additional copper binding. The type 3 copper atoms of the trinuclear cluster in the structure are bridged by a chloride ion that completes a square planar coordination sphere for the T2 copper atom but does not affect oxidase activity.     

Enhancement of laccase activity through the construction and breakdown of a hydrogen bond at the type I copper center in Escherichia coli CueO and the deletion mutant Δα5-7 CueO

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.     

Genes involved in copper homeostasis in Escherichia coli

Recently, genes for two copper-responsive regulatory systems were identified in the Escherichia coli chromosome. In this report, data are presented that support a hypothesis that the putative multicopper oxidase CueO and the transenvelope transporter CusCFBA are involved in copper tolerance in E. coli.     

Dimethoxyphenol oxidase activity of different microbial blue multicopper proteins

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.     

Trade-off between iron uptake and protection against oxidative stress: deletion of cueO promotes uropathogenic Escherichia coli virulence in a mouse model of urinary tract infection

The periplasmic multicopper oxidase (CueO) is involved in copper homeostasis and protection against oxidative stress. Here, we show that the deletion of cueO in uropathogenic Escherichia coli increases its colonization of the urinary tract despite its increased sensitivity to hydrogen peroxide. The cueO deletion mutant accumulated iron with increased efficiency compared to its parent strain; this may account for its advantage in the iron-limited environment of the urinary tract.     

A Multicopper oxidase (Cj1516) and a CopA homologue (Cj1161) are major components of the copper homeostasis system of Campylobacter jejuni

Metal ion homeostasis mechanisms in the food-borne human pathogen Campylobacter jejuni are poorly understood. The Cj1516 gene product is homologous to the multicopper oxidase CueO, which is known to contribute to copper tolerance in Escherichia coli. Here we show, by optical absorbance and electron paramagnetic resonance spectroscopy, that purified recombinant Cj1516 contains both T1 and trinuclear copper centers, which are characteristic of multicopper oxidases. Inductively coupled plasma mass spectrometry revealed that the protein contained approximately six copper atoms per polypeptide. The presence of an N-terminal “twin arginine” signal sequence suggested a periplasmic location for Cj1516, which was confirmed by the presence of p-phenylenediamine (p-PD) oxidase activity in periplasmic fractions of wild-type but not Cj1516 mutant cells. Kinetic studies showed that the pure protein exhibited p-PD, ferroxidase, and cuprous oxidase activities and was able to oxidize an analogue of the bacterial siderophore anthrachelin (3,4-dihydroxybenzoate), although no iron uptake impairment was observed in a Cj1516 mutant. However, this mutant was very sensitive to increased copper levels in minimal media, suggesting a role in copper tolerance. This was supported by increased expression of the Cj1516 gene in copper-rich media. A mutation in a second gene, the Cj1161c gene, encoding a putative CopA homologue, was also found to result in copper hypersensitivity, and a Cj1516 Cj1161c double mutant was found to be more copper sensitive than either single mutant. These observations and the apparent lack of alternative copper tolerance systems suggest that Cj1516 (CueO) and Cj1161 (CopA) are major proteins involved in copper homeostasis in C. jejuni.     

Structure and function of the engineered multicopper oxidase CueO from Escherichia coli--deletion of the methionine-rich helical region covering the substrate-binding site

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.     

Crystal structures of multicopper oxidase CueO bound to copper(I) and silver(I): functional role of a methionine-rich sequence

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 k_cat 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.     

CueO is a multi-copper oxidase that confers copper tolerance in Escherichia coli

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.     

Crystal structure and dimerization equilibria of PcoC, a methionine-rich copper resistance protein from Escherichia coli

PcoC is a soluble periplasmic protein encoded by the plasmid-born pco copper resistance operon of Escherichia coli. Like PcoA, a multicopper oxidase encoded in the same locus and its chromosomal homolog CueO, PcoC contains unusual methionine rich sequences. Although essential for copper resistance, the functions of PcoC, PcoA, and their conserved methionine-rich sequences are not known. Similar methionine motifs observed in eukaryotic copper transporters have been proposed to bind copper, but there are no precedents for such metal binding sites in structurally characterized proteins. The high-resolution structures of apo PcoC, determined for both the native and selenomethionine-containing proteins, reveal a seven-stranded beta barrel with the methionines unexpectedly housed on a solvent-exposed loop. Several potential metal-binding sites can be discerned by comparing the structures to spectroscopic data reported for copper-loaded PcoC. In the native structure, the methionine loop interacts with the same loop on a second molecule in the asymmetric unit. In the selenomethionine structure, the methionine loops are more exposed, forming hydrophobic patches on the protein surface. These two arrangements suggest that the methionine motifs might function in protein-protein interactions between PcoC molecules or with other methionine-rich proteins such as PcoA. Analytical ultracentrifugation data indicate that a weak monomer-dimer equilibrium exists in solution for the apo protein. Dimerization is significantly enhanced upon binding Cu(I) with a measured delta(deltaG degrees )相似文献   

Cuprous oxidase activity of CueO from Escherichia coli

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).     

Escherichia coli mechanisms of copper homeostasis in a changing environment

Escherichia coli is equipped with multiple systems to ensure safe copper handling under varying environmental conditions. The Cu(I)-translocating P-type ATPase CopA, the central component in copper homeostasis, is responsible for removing excess Cu(I) from the cytoplasm. The multi-copper oxidase CueO and the multi-component copper transport system CusCFBA appear to safeguard the periplasmic space from copper-induced toxicity. Some strains of E. coli can survive in copper-rich environments that would normally overwhelm the chromosomally encoded copper homeostatic systems. Such strains possess additional plasmid-encoded genes that confer copper resistance. The pco determinant encodes genes that detoxify copper in the periplasm, although the mechanism is still unknown. Genes involved in copper homeostasis are regulated by MerR-like activators responsive to cytoplasmic Cu(I) or two-component systems sensing periplasmic Cu(I). Pathways of copper uptake and intracellular copper handling are still not identified in E. coli.     

Gene cloning, protein purification, and enzymatic properties of multicopper oxidase, from Klebsiella sp. 601

A gene encoding a putative multicopper oxidase (MCO) was cloned from the soil bacterium Klebsiella sp. 601 and its corresponding enzyme was overexpressed in an Escherichia coli strain. Klebsiella sp. 601 MCO is composed of 536 amino acids with a molecular mass of 58.2 kDa. Theoretical calculation gave a pI value of 6.11. The amino acid sequence of Klebsiella sp. 601 MCO is strongly homologous to that of E. coli CueO with a similarity of 90% and an identity of 78%. Unlike E. coli CueO, Klebsiella sp. 601 MCO contains an extra 20 amino acids close to its C-terminus. The enzyme was purified to homogeneity by Ni-affinity chromatography. The purified enzyme was capable of using DMP (2,6-dimethoxyphenol), ABTS (2,2'-azino-bis(3-ethylbenzthiazolinesulfonic acid)), and SGZ (syringaldazine) as substrates with an optimal pH of 8.0 for DMP, 3.0 for ABTS, and 7.0 for SGZ. Klebsiella sp. 601 MCO was quite stable at pH 7.0 in which its activity was constant for 25 h without any significant change. Kinetic studies gave Km, kcat, and kcat//Km values of 0.49 mmol/L, 1.08 x 103 s-1, and 2.23 x 103 s-1.mmol/L-1, respectively, for DMP, 5.63 mmol/L, 6.64 x 103 s-1, and 1.18 x 103 s-1.mmol/L-1 for ABTS, and 0.023 mmol/L, 11 s-1, and 4.68 x 102 s-1.mmol/L-1 for SGZ.     

Sequential reconstitution of copper sites in the multicopper oxidase CueO

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.     

CuiD is a crucial gene for survival at high copper environment in Salmonella enterica serovar Typhimurium

Copper ion is an essential micronutrient but it is also extremely cytotoxic when it exists in excess. Our studies have shown that Salmonella enterica serovar Typhimurium can survive potentially lethal copper exposures by the way of copper efflux system. A copper ion inducible gene was identified in virulent S. typhimurium by using the technique of MudJ (Km, lac)-directed lacZYA operon fusions. A copper ion inducible strain LF153 (cuiD::MudJ) has been identified. The cuiD mutant exhibits a copper sensitive phenotype but possesses normal resistance to other metal ions, and lost DMP oxidase activity. Therefore, we suggest that cuiD is an important gene for copper homeostasis and the copper resistance response. The copper sensitive phenotype was complemented by pYL3.0 carrying cuiD+. Sequence analysis showed cuiD contains 1,614 bp encoding a 536 amino acid with a 27 amino acid signal peptide and a 509 amino acid residues comprising the mature peptide. The CuiD shows 81% homology to YacK, a putative multicopper oxidases which extrudes copper in Escherichia coli. This ORF contains four conserved regions that contain 12 copper ligands (types 1, 2, and 3) present in various copper homeostasis responsible proteins. The H2O2 sensitive phenotype of the cuiD mutant indicates that cuiD may be involved in oxidative stress response.     

Crystal structures of E. coli laccase CueO at different copper concentrations

CueO protein is a hypothetical bacterial laccase and a good laccase candidate for large scale industrial application. Four CueO crystal structures were determined at different copper concentrations. Low copper occupancy in apo-CueO and slow copper reconstitution process in CueO with exogenous copper were demonstrated. These observations well explain the copper dependence of CueO oxidase activity. Structural comparison between CueO and other three fungal laccase proteins indicates that Glu106 in CueO constitutes the primary counter-work for reconstitution of the trinuclear copper site. Mutation of Glu106 to a Phe enhanced CueO oxidation activity and supported this hypothesis. In addition, an extra alpha-helix from Leu351 to Gly378 covers substrate biding pocket of CueO and might compromises the electron transfer from substrate to type I copper.     

Enhanced archaeal laccase production in recombinant Escherichia coli by modification of N-terminal propeptide and twin arginine translocation motifs

Laccases are multicopper oxidases that couple the oxidation of phenolic polymers to the reduction of molecular oxygen. While an archaeal laccase has only recently been described (LccA from the culture broth of Haloferax volcanii), this enzyme appears promising for biotechnology applications based on its robust bilirubin oxidase and laccase activities as well as its ability to withstand prolonged exposure to extreme conditions. To further optimize LccA productivity and develop an option for LccA purification from whole cells, the encoding gene was modified through deletion of the twin-arginine translocation motif and N-terminal propeptide, and the modified genes were expressed in Escherichia coli. With this approach, LccA was readily purified (overall yield up to 54?%) from the soluble fraction of E. coli as a 74-kDa monomer with syringaldazine oxidizing activity as high as 33?U?mg(-1). LccA proteins prepared from H. volcanii culture broth and the soluble fraction of E. coli cells were compared by ICP-AES, EPR, DSC, CD, and UV-Vis spectroscopy and found to have a similar folding pattern with T (m) values and a rich β-sheet structure analogous to other multicopper oxidases. However, in contrast to the H. volcanii-purified LccA, which was loaded with copper, copper was not fully incorporated into the type-I Cu center of E. coli purified LccA, thus, providing insight into avenues for further optimization.