RESUMO
l-cysteine is the source of all bacterial sulfurous biomolecules. However, the cytoplasmic level of l-cysteine must be tightly regulated due to its propensity to reduce iron and drive damaging Fenton chemistry. It has been proposed that in Escherichia coli the component of cytochrome bd-I terminal oxidase, the CydDC complex, shuttles excessive l-cysteine from the cytoplasm to the periplasm, thereby maintaining redox homeostasis. Here, we provide evidence for an alternative function of CydDC by demonstrating that the cydD phenotype, unlike that of the bona fide l-cysteine exporter eamA, parallels that of the l-cystine importer tcyP. Chromosomal induction of eamA, but not of cydDC, from a strong pLtetO-1 promoter (Ptet) leads to the increased level of extracellular l-cysteine, whereas induction of cydDC or tcyP causes the accumulation of cytoplasmic l-cysteine. Congruently, inactivation of cydD renders cells resistant to hydrogen peroxide and to aminoglycoside antibiotics. In contrast, induction of cydDC sensitizes cells to oxidative stress and aminoglycosides, which can be suppressed by eamA overexpression. Furthermore, inactivation of the ferric uptake regulator (fur) in Ptet-cydDC or Ptet-tcyP cells results in dramatic loss of survival, whereas catalase (katG) overexpression suppresses the hypersensitivity of both strains to H2O2 These results establish CydDC as a reducer of cytoplasmic cystine, as opposed to an l-cysteine exporter, and further elucidate a link between oxidative stress, antibiotic resistance, and sulfur metabolism.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Cisteína/metabolismo , Grupo dos Citocromos b/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/metabolismo , NADH NADPH Oxirredutases/metabolismo , Estresse Oxidativo/fisiologia , Oxirredutases/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Aminoglicosídeos/metabolismo , Aminoglicosídeos/farmacologia , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Grupo dos Citocromos b/genética , Citoplasma/enzimologia , Citoplasma/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Proteínas de Escherichia coli/genética , Peróxido de Hidrogênio/metabolismo , NADH NADPH Oxirredutases/genética , Estresse Oxidativo/genética , Oxirredutases/genética , Periplasma/metabolismoRESUMO
Endogenous hydrogen sulfide (H2S) renders bacteria highly resistant to oxidative stress, but its mechanism remains poorly understood. Here, we report that 3-mercaptopyruvate sulfurtransferase (3MST) is the major source of endogenous H2S in Escherichia coli Cellular resistance to H2O2 strongly depends on the activity of mstA, a gene that encodes 3MST. Deletion of the ferric uptake regulator (Fur) renders ∆mstA cells hypersensitive to H2O2 Conversely, induction of chromosomal mstA from a strong pLtetO-1 promoter (P tet -mstA) renders ∆fur cells fully resistant to H2O2 Furthermore, the endogenous level of H2S is reduced in ∆fur or ∆sodA ∆sodB cells but restored after the addition of an iron chelator dipyridyl. Using a highly sensitive reporter of the global response to DNA damage (SOS) and the TUNEL assay, we show that 3MST-derived H2S protects chromosomal DNA from oxidative damage. We also show that the induction of the CysB regulon in response to oxidative stress depends on 3MST, whereas the CysB-regulated l-cystine transporter, TcyP, plays the principle role in the 3MST-mediated generation of H2S. These findings led us to propose a model to explain the interplay between l-cysteine metabolism, H2S production, and oxidative stress, in which 3MST protects E. coli against oxidative stress via l-cysteine utilization and H2S-mediated sequestration of free iron necessary for the genotoxic Fenton reaction.
Assuntos
Sulfeto de Hidrogênio/metabolismo , Sulfurtransferases/metabolismo , Antibacterianos/metabolismo , Cisteína/metabolismo , Cistina/metabolismo , Dano ao DNA/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Peróxido de Hidrogênio/metabolismo , Ferro/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Sulfurtransferases/fisiologiaRESUMO
Uridine phosphorylase catalyzes the phosphorolysis of ribonucleosides, with the nitrogenous base and ribose 1-phosphate as products. Additionally, it catalyzes the reverse reaction of the synthesis of ribonucleosides from ribose 1-phosphate and a nitrogenous base. However, the enzyme does not catalyze the synthesis of nucleosides when the substrate is a nitrogenous base substituted at the 6-position, such as 6-methyluracil (6-MU). In order to explain this fact, it is essential to investigate the three-dimensional structure of the complex of 6-MU with uridine phosphorylase. 6-MU is a pharmaceutical agent that improves tissue nutrition and enhances cell regeneration by normalization of nucleotide exchange in humans. 6-MU is used for the treatment of diseases of the gastrointestinal tract, including infectious diseases. Here, procedures to obtain the uridine phosphorylase from the pathogenic bacterium Vibrio cholerae (VchUPh), purification of this enzyme, crystallization of the complex of VchUPh with 6-MU, and X-ray data collection and preliminary X-ray analysis of the VchUPh-6-MU complex at atomic resolution are reported.
Assuntos
Uracila/análogos & derivados , Uridina Fosforilase/química , Vibrio cholerae/enzimologia , Sítios de Ligação , Biocatálise , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Uracila/químicaRESUMO
A high-resolution structure of the complex of Vibrio cholerae uridine phosphorylase (VchUPh) with its physiological ligand thymidine is important in order to determine the mechanism of the substrate specificity of the enzyme and for the rational design of pharmacological modulators. Here, the expression and purification of VchUPh and the crystallization of its complex with thymidine are reported. Conditions for crystallization were determined with an automated Cartesian Dispensing System using The Classics, MbClass and MbClass II Suites crystallization kits. Crystals of the VchUPh-thymidine complex (of dimensions â¼200-350â µm) were grown by the sitting-drop vapour-diffusion method in â¼7â d at 291â K. The crystallization solution consisted of 1.5â µl VchUPh (15â mgâ ml(-1)), 1â µl 0.1â M thymidine and 1.5â µl reservoir solution [15%(w/v) PEG 4000, 0.2â M MgCl(2).6H2O in 0.1â M Tris-HCl pH 8.5]. The crystals diffracted to 2.12â Å resolution and belonged to space group P2(1) (No. 4), with unit-cell parameters a=91.80, b=95.91, c=91.89â Å, ß=119.96°. The Matthews coefficient was calculated as 2.18â Å3â Da(-1); the corresponding solvent content was 43.74%.