RESUMO
The cutO gene of the photosynthetic purple bacterium Rhodobacter capsulatus codes for a multicopper oxidase as demonstrated by the ability of the recombinant Strep-tagged protein to oxidize several mono- and diphenolic compounds known as substrates of Escherichia coli CueO and multicopper oxidases from other organisms. The R. capsulatus cutO gene was shown to form part of a tri-cistronic operon, orf635-cutO-cutR. Expression of the cutO operon was repressed under low copper conditions by the product of the cutR gene. CutO conferred copper tolerance not only under aerobic conditions, as described for the well-characterized E. coli multicopper oxidase CueO, but also under anaerobic conditions.
Assuntos
Cobre/fisiologia , Oxirredutases/genética , Oxirredutases/fisiologia , Rhodobacter capsulatus/enzimologia , Rhodobacter capsulatus/fisiologia , Proteínas de Transporte/genética , Análise Mutacional de DNA/métodos , DNA Recombinante/genética , Tolerância a Medicamentos/fisiologia , Escherichia coli/enzimologia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Ordem dos Genes , Mutagênese Sítio-Dirigida/métodos , Oxirredutases/metabolismo , RNA Bacteriano/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rhodobacter capsulatus/genética , beta-Galactosidase/análiseRESUMO
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.
Assuntos
Escherichia coli/enzimologia , Escherichia coli/metabolismo , Oxirredutases/metabolismo , Sideróforos/metabolismo , Antibacterianos/farmacologia , Fusão Gênica Artificial , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Cobre/farmacologia , Farmacorresistência Bacteriana/fisiologia , Enterobactina/metabolismo , Enterobactina/farmacologia , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Genes Bacterianos/fisiologia , Genes Reporter , Hidroxibenzoatos/metabolismo , Oxirredução , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
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.
