RESUMO
Mesothelioma is a rare and aggressive tumour with dismal prognosis arising in the pleura and associated with asbestos exposure. Its incidence is on the rise worldwide. In selected patients with early-stage MPM, a maximal surgical cytoreduction in combination with additional antitumour treatment may be considered in selected patients assessed by a multidisciplinary tumor board. In patients with unresectable or advanced MPM, chemotherapy with platinum plus pemetrexed is the standard of care. Currently, no standard salvage therapy has been approved yet, but second-line chemotherapy with vinorelbine or gemcitabine is commonly used. Novel therapeutic approaches based on dual immunotherapy or chemotherapy plus immunotherapy demonstrated promising survival benefit and will probably be incorporated in the future (AU)
Assuntos
Humanos , Neoplasias Pleurais/diagnóstico , Neoplasias Pleurais/terapia , Estadiamento de Neoplasias , Sociedades Médicas , EspanhaRESUMO
Mesothelioma is a rare and aggressive tumour with dismal prognosis arising in the pleura and associated with asbestos exposure. Its incidence is on the rise worldwide. In selected patients with early-stage MPM, a maximal surgical cytoreduction in combination with additional antitumour treatment may be considered in selected patients assessed by a multidisciplinary tumor board. In patients with unresectable or advanced MPM, chemotherapy with platinum plus pemetrexed is the standard of care. Currently, no standard salvage therapy has been approved yet, but second-line chemotherapy with vinorelbine or gemcitabine is commonly used. Novel therapeutic approaches based on dual immunotherapy or chemotherapy plus immunotherapy demonstrated promising survival benefit and will probably be incorporated in the future.
Assuntos
Mesotelioma Maligno/diagnóstico , Mesotelioma Maligno/terapia , Neoplasias Pleurais/diagnóstico , Neoplasias Pleurais/terapia , Antineoplásicos/uso terapêutico , Amianto/toxicidade , Carcinógenos/toxicidade , Terapia Combinada/métodos , Procedimentos Cirúrgicos de Citorredução , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoterapia/métodos , Oncologia , Mesotelioma Maligno/etiologia , Mesotelioma Maligno/patologia , Estadiamento de Neoplasias , Pemetrexede/uso terapêutico , Compostos de Platina/uso terapêutico , Neoplasias Pleurais/etiologia , Neoplasias Pleurais/patologia , Radioterapia/métodos , Sociedades Médicas , Espanha , Vinorelbina/uso terapêutico , GencitabinaRESUMO
Francisella tularensis is a Gram-negative bacterium causing tularaemia. Classified as possible bioterrorism agent, it may be transmitted to humans via animal infection or inhalation leading to severe pneumonia. Its virulence is related to iron homeostasis involving siderophore biosynthesis directly controlled at the transcription level by the ferric uptake regulator Fur, as presented here together with the first crystal structure of the tetrameric F. tularensis Fur in the presence of its physiological cofactor, Fe2+. Through structural, biophysical, biochemical and modelling studies, we show that promoter sequences of F. tularensis containing Fur boxes enable this tetrameric protein to bind them by splitting it into two dimers. Furthermore, the critical role of F. tularensis Fur in virulence and pathogenesis is demonstrated with a fur-deleted mutant showing an attenuated virulence in macrophage-like cells and mice. Together, our study suggests that Fur is an attractive target of new antibiotics that attenuate the virulence of F. tularensis.
RESUMO
Lung cancer is the most common cancer worldwide as well as the leading cause of cancer related deaths as reported by Torre et al (CA Cancer J Clin 65:87-108, 2015]. Non-small cell lung cancer (NSCLC) accounts for up to 85 % of all lung cancers. Multiple advances in the staging, diagnostic procedures, therapeutic options, as well as molecular knowledge have been achieved during the past years, although the overall outlook has not greatly changed for the majority of patients with the overall 5-year survival having marginally increased over the last decade from 15.7 to 17.4 % as reported by Howlader et al. (SEER Cancer Statistics Review 2015).
Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/terapia , Guias de Prática Clínica como Assunto/normas , Ensaios Clínicos como Assunto , Terapia Combinada , Gerenciamento Clínico , Detecção Precoce de Câncer , Humanos , Oncologia , Estadiamento de Neoplasias , Prognóstico , Sociedades MédicasRESUMO
Lung cancer is the most common cancer worldwide as well as the leading cause of cancer related deaths as reported by Torre et al (CA Cancer J Clin 65:87-108, 2015]. Non-small cell lung cancer (NSCLC) accounts for up to 85 % of all lung cancers. Multiple advances in the staging, diagnostic procedures, therapeutic options, as well as molecular knowledge have been achieved during the past years, although the overall outlook has not greatly changed for the majority of patients with the overall 5-year survival having marginally increased over the last decade from 15.7 to 17.4 % as reported by Howlader et al. (SEER Cancer Statistics Review 2015) (AU)
No disponible
Assuntos
Humanos , Masculino , Feminino , /normas , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Terapêutica/métodos , Doenças Transmissíveis/patologia , Doenças Transmissíveis/transmissão , Biópsia/métodos , Biomarcadores/metabolismo , Preparações Farmacêuticas/metabolismo , Fumar/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/terapia , Terapêutica/normas , Doenças Transmissíveis/complicações , Doenças Transmissíveis/metabolismo , Biópsia/instrumentação , Biomarcadores/análise , Preparações Farmacêuticas/provisão & distribuição , Fumar/prevenção & controleRESUMO
PURPOSE: To assess the radiation dose to the fetus of a pregnant patient undergoing high-dose-rate (HDR) (192)Ir interstitial breast brachytherapy, and to design a new patient setup and lead shielding technique that minimizes the fetal dose. METHODS: Radiochromic films were placed between the slices of an anthropomorphic phantom modeling the patient. The pregnant woman was seated in a chair with the breast over a table and inside a leaded box. Dose variation as a function of distance from the implant volume as well as dose homogeneity within a representative slice of the fetal position was evaluated without and with shielding. RESULTS: With shielding, the peripheral dose after a complete treatment ranged from 50 cGy at 5 cm from the caudal edge of the breast to <0.1 cGy at 30 cm. The shielding reduces absorbed dose by a factor of two near the breast and more than an order of magnitude beyond 20 cm. The dose is heterogeneous within a given axial plane, with variations from the central region within 50%. Interstitial HDR (192)Ir brachytherapy with breast shielding can be more advantageous than external-beam radiotherapy (EBRT) from a radiation protection point of view, as long as the distance to the uterine fundus is higher than about 10 cm. Furthermore, the weight of the shielding here proposed is notably lower than that needed in EBRT. CONCLUSIONS: Shielded breast brachytherapy may benefit pregnant patients needing localized radiotherapy, especially during the early gestational ages when the fetus is more sensitive to ionizing radiation.
Assuntos
Braquiterapia/instrumentação , Neoplasias da Mama/radioterapia , Feto/efeitos da radiação , Radioisótopos de Irídio/uso terapêutico , Complicações na Gravidez/radioterapia , Proteção Radiológica/instrumentação , Planejamento da Radioterapia Assistida por Computador , Adulto , Braquiterapia/efeitos adversos , Feminino , Humanos , Radioisótopos de Irídio/efeitos adversos , Gravidez , Radiometria , Dosagem RadioterapêuticaRESUMO
Ralstonia metallidurans CH34 (formerly Alcaligenes eutrophus CH34) is a soil bacterium characteristic of metal-contaminated biotopes, as it is able to grow in the presence of a variety of heavy metals. R. metallidurans CH34 is reported now to resist up to 6 mM selenite and to reduce selenite to elemental red selenium as shown by extended X-ray absorption fine-structure analysis. Growth kinetics analysis suggests an adaptation of the cells to the selenite stress during the lag-phase period. Depending on the culture conditions, the medium can be completely depleted of selenite. Selenium accumulates essentially in the cytoplasm as judged from electron microscopy and energy-dispersive X-ray analysis. Elemental selenium, highly insoluble, represents a nontoxic storage form for the bacterium. The ability of R. metallidurans CH34 to reduce large amounts of selenite may be of interest for bioremediation processes targeting selenite-polluted sites.
Assuntos
Cupriavidus necator/metabolismo , Selenito de Sódio/metabolismo , Absorciometria de Fóton/métodos , Biodegradação Ambiental , Meios de Cultura , Cupriavidus necator/efeitos dos fármacos , Cupriavidus necator/crescimento & desenvolvimento , Microanálise por Sonda Eletrônica , Microscopia Eletrônica , Selenito de Sódio/farmacologiaRESUMO
Escherichia coli sulfite reductase (SiR) is a large and soluble enzyme with an alpha(8)beta(4) quaternary structure. Protein alpha (or sulfite reductase flavoprotein) contains both FAD and FMN, whereas protein beta (or sulfite reductase hemoprotein (SiR-HP)) contains an iron-sulfur cluster coupled to a siroheme. The enzyme is set up to arrange the redox cofactors in a FAD-FMN-Fe(4)S(4)-Heme sequence to make an electron pathway between NADPH and sulfite. Whereas alpha spontaneously polymerizes, we have been able to produce SiR-FP60, a monomeric but fully active truncated version of it, lacking the N-terminal part (Zeghouf, M., Fontecave, M., Macherel, D., and Covès, J. (1998) Biochemistry 37, 6114-6123). Here we report the cloning, overproduction, and characterization of the beta subunit. Pure recombinant SiR-HP behaves as a monomer in solution and is identical to the native protein in all its characteristics. Moreover, we demonstrate that the combination of SiR-FP60 and SiR-HP produces a functional 1:1 complex with tight interactions retaining about 20% of the activity of the native SiR. In addition, fully active SiR can be reconstituted by incubation of the octameric sulfite reductase flavoprotein with recombinant SiR-HP. Titration experiments and spectroscopic properties strongly suggest that the holoenzyme should be described as an alpha(8)beta(8) with equal amounts of alpha and beta subunits and that the alpha(8)beta(4) structure is probably not correct.
Assuntos
Escherichia coli/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Sequência de Bases , Primers do DNA , Espectroscopia de Ressonância de Spin Eletrônica , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/isolamento & purificação , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Escherichia coli NADPH-sulfite reductase (SiR) is a 780 kDa multimeric hemoflavoprotein composed of eight alpha-subunits (SiR-FP) and four beta-subunits (SiR-HP) that catalyses the six electron reduction of sulfite to sulfide. Each beta-subunit contains a Fe4S4 cluster and a siroheme, and each alpha-subunit binds one FAD and one FMN as prosthetic groups. The FAD gets electrons from NADPH, and the FMN transfers the electrons to the metal centers of the beta-subunit for sulfite reduction. We report here the 1.94 A X-ray structure of SiR-FP60, a recombinant monomeric fragment of SiR-FP that binds both FAD and FMN and retains the catalytic properties of the native protein. The structure can be divided into three domains. The carboxy-terminal part of the enzyme is composed of an antiparallel beta-barrel which binds the FAD, and a variant of the classical pyridine dinucleotide binding fold which binds NADPH. These two domains form the canonic FNR-like module, typical of the ferredoxin NADP+ reductase family. By analogy with the structure of the cytochrome P450 reductase, the third domain, composed of seven alpha-helices, is supposed to connect the FNR-like module to the N-terminal flavodoxine-like module. In four different crystal forms, the FMN-binding module is absent from electron density maps, although mass spectroscopy, amino acid sequencing and activity experiments carried out on dissolved crystals indicate that a functional module is present in the protein. Our results clearly indicate that the interaction between the FNR-like and the FMN-like modules displays lower affinity than in the case of cytochrome P450 reductase. The flexibility of the FMN-binding domain may be related, as observed in the case of cytochrome bc1, to a domain reorganisation in the course of electron transfer. Thus, a movement of the FMN-binding domain relative to the rest of the enzyme may be a requirement for its optimal positioning relative to both the FNR-like module and the beta-subunit.
Assuntos
Escherichia coli/enzimologia , Flavodoxina/química , Flavodoxina/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Cristalização , Cristalografia por Raios X , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Movimento (Física) , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/metabolismo , NADP/metabolismo , NADPH-Ferri-Hemoproteína Redutase , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Maleabilidade , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Eletricidade Estática , Sulfito Redutase (NADPH)RESUMO
SiR-FP43, the NADPH- and FAD-binding domain of the Escherichia coli sulphite reductase flavoprotein component (SiR-FP), has been overexpressed and characterized. It folds independently, retaining FAD as a cofactor and the catalytic properties associated with the presence of this cofactor. Iodonium diphenyl chloride (IDP) was shown to be a very efficient inhibitor of SiR-FP43 and SiR-FP60, the monomeric form of SiR-FP, containing both FMN and FAD as cofactors (K(i) = 18.5 +/- 5 microM, maximal inactivation rate = 0.053 +/- 0.005 s(-1)). In both cases, inactivation was shown to result from covalent binding of a phenyl group to FAD exclusively, in marked contrast with previous results obtained with cytochrome P450 reductase (CPR), where FMN and a tryptophan were phenylated, but not FAD. However, our kinetic analyses are in agreement with the inhibition mechanism demonstrated with CPR [Tew (1993) Biochemistry 32, 10209-10215]. Nine different FAD phenylated adducts were isolated and, for the first time, two FAD phenylated adducts were identified directly after extraction from a protein. Taken together, our results have shown that flavoprotein inactivation by IDP is not a reliable indicator for a flavin radical intermediate in catalysis.
Assuntos
Escherichia coli/enzimologia , Flavina-Adenina Dinucleotídeo/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Sequência de Bases , Sítios de Ligação , Compostos de Bifenilo/farmacologia , Cromatografia Líquida de Alta Pressão , Primers do DNA/genética , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Expressão Gênica , Espectrometria de Massas , Oniocompostos/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfito Redutase (NADPH)RESUMO
SiR-FP60, the monomeric form of the Escherichia coli sulfite reductase flavoprotein component (SiR-FP), has been analysed by 31P-NMR spectroscopy. This protein was reported previously as a reliable simplified model for native SiR-FP [Zeghouf, M., Fontecave, M., Macherel, D., & Covès, J. (1998) Biochemistry 37, 6117-6123]. SiR-FP60 was examined in its native form, as a complex with NADP+ and after monoelectronic reduction either with NADPH or dithionite. In these latter cases, the stabilized FMN semiquinone radical offers a natural and internal paramagnetic probe. The paramagnetic effect of added manganese was also studied. In each case, the NMR parameters were extracted from digitalized data by a deconvolution procedure and compared with those obtained previously with cytochrome P450 reductase. Evolution of the NMR parameters and of calculated relaxation rate constants upon biochemical modifications of SiR-FP60 led us to propose that the reactive center is more compact than the one of cytochrome P450 reductase, with the redox components, FMN, FAD and NADPH, in a tighter spatial arrangement, close to the protein surface. This underlies some subtle differences between the two proteins for which a very similar overall structure is likely considering their common genetic origin and common operating cycle.
Assuntos
Escherichia coli/enzimologia , Flavoproteínas/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Sítios de Ligação , Sistema Enzimático do Citocromo P-450/química , Ditionita/farmacologia , Mononucleotídeo de Flavina/análogos & derivados , Mononucleotídeo de Flavina/química , Espectroscopia de Ressonância Magnética , Manganês/química , NAD/química , NADP/química , Sulfito Redutase (NADPH)RESUMO
The FNR-like domain of the Escherichia coli sulfite reductase flavoprotein subunit was crystallized using the hanging-drop technique, with PEG 4000 as precipitant. The crystals belong to space group P3112 or enantiomorph, with unit-cell parameters a = b = 171.0, c = 152.1 A. A solvent content of 75% was determined by a calibrated tetrachloromethane/toluene gradient which corresponds to three monomers per asymmetric unit. A 3 A resolution native data set was collected at beamline W32 of LURE, Orsay, France.
Assuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Flavoproteínas/química , Proteínas Ferro-Enxofre/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Domínio Catalítico , Cristalografia por Raios X , Sulfito Redutase (NADPH)RESUMO
The flavoprotein component (SiR-FP) of the E. coli sulfite reductase was found to support 17 alpha-hydroxylation of pregnenolone in the presence of cytochrome P450c17. Half maximum activity is obtained for a 1:1 ratio of SiR-FP, expressed as monomer concentration, to P450c17. When compared to bovine NADPH-cytochrome P450 reductase, SiR-FP is about 12-15 times less efficient. P450c17 was demonstrated to interact specifically with the FMN-binding domain of the protein and the N-terminal part of SiR-FP is suspected to play a role in electron transfer. A cluster of negatively charged residues was found in SiR-FP by amino acid sequence comparison with rat cytochrome P450 reductase. These results argue in favour of the flavodoxin origin of the FMN-binding domain of SiR-FP.
Assuntos
Escherichia coli/enzimologia , Flavoproteínas/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Esteroide 17-alfa-Hidroxilase/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Evolução Molecular , Mononucleotídeo de Flavina , Flavodoxina/genética , Flavoproteínas/genética , Dados de Sequência Molecular , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Fragmentos de Peptídeos/metabolismo , Pregnenolona/metabolismo , Ligação Proteica , Esteroide 17-alfa-Hidroxilase/genética , Especificidade por Substrato , Sulfito Redutase (NADPH)RESUMO
The flavoprotein component (SiR-FP) of the sulfite reductase from Escherichia coli is an octamer containing one FAD and one FMN per polypeptide chain. SiR-FP60, a SiR-FP fragment starting with alanine-52, was overexpressed in E. coli and purified as a monomer. The N-terminal part of the native protein contains thus all the determinants required for the polymerization. SiR-FP60 retains both FAD and FMN with comparable contributions of the two flavins and the catalytic properties of SiR-FP. Thus, SiR-FP60 can be considered as a reliable simplified model of the sulfite reductase flavoprotein component. The formation and the stabilization of the neutral FMN semiquinone is thermodynamically favorable in SiR-FP60 upon reduction with photoreduced deazaflavin, dithionite, or NADPH. Generation of FMNH* is explained from a disproportionation of electrons between the reduced and oxidized FMN moieties during an intermolecular reaction, as shown with SiR-FP23, the FMN-binding domain of SiR-FP. The neutral FAD semiquinone can be observed only within SiR-FP43, the isolated FAD-binding domain. NADPH was used as a titrant or in excess to demonstrate that electron transfer is possible only because the FMN cofactor is coupled to FAD as an electron acceptor in the protein. The electron distribution within the various reduced forms of SiR-FP60 has been compared with that of the reduced forms of cytochrome P450 reductase, bacterial cytochrome P450, and nitric-oxide synthase. Despite the conservation of the bi-flavin-domain structure between these proteins over evolutionary time, each of them provides significantly different flavin reactivities.
Assuntos
Escherichia coli/enzimologia , Mononucleotídeo de Flavina/química , Flavina-Adenina Dinucleotídeo/química , Flavoproteínas/biossíntese , Flavoproteínas/isolamento & purificação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/biossíntese , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/isolamento & purificação , Catálise , Ditionita , Espectroscopia de Ressonância de Spin Eletrônica , Endopeptidases , Transferência de Energia , Flavinas/química , Flavoproteínas/química , Hidrólise , Peso Molecular , NADP , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Espectrofotometria , TitulometriaRESUMO
The flavoprotein component (SiR-FP) of the sulfite reductase from Escherichia coli is an octamer containing one FAD and one FMN as cofactors per polypeptide chain. We have constructed an expression vector containing the DNA fragment encoding for the FMN-binding domain of SiR-FP. The overexpressed protein (SiR-FP23) was purified as a partially flavin-depleted polymer. It could incorporate FMN exclusively upon flavin reconstitution to reach a maximum flavin content of 1.2 per polypeptide chain. Moreover, the protein could stabilize a neutral air-stable semiquinone radical over a wide range of pHs. During photoreduction, the flavin radical accumulated first, followed by the fully reduced state. The redox potentials, determined at room temperature [E'1 (FMNH./FMN) = -130 +/- 10 mV and E'2 (FMNH2/FMNH.) = -335 +/- 10 mV], were very close to those previously reported for Salmonella typhimurium SiR-FP [Ostrowski, J., Barber, M. J., Rueger, D. C., Miller, B. E., Siegel, L. M., & Kredich, N. M. (1989) J. Biol. Chem. 264, 15796-15808]. Both the radical and fully reduced forms of SiR-FP23 were able to transfer their electrons to cytochrome c quantitatively. Altogether, the results presented herein demonstrate that the N-terminal end of E. coli SiR-FP forms the FMN-binding domain. It folds independently, thus retaining the chemical properties of the bound FMN, and provides a good model of the FAD-depleted form of native SiR-FP. Moreover, the FMN prosthetic group in SiR-FP23 and native SiR-FP is compared to that of cytochrome P450 reductase and bacterial cytochrome P450, which also contain one FAD and one FMN per polypeptide chain.
Assuntos
Escherichia coli/enzimologia , Escherichia coli/metabolismo , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Flavoproteínas/química , Flavoproteínas/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Sítios de Ligação , Escherichia coli/química , Mononucleotídeo de Flavina/biossíntese , Mononucleotídeo de Flavina/isolamento & purificação , Flavina-Adenina Dinucleotídeo/metabolismo , Flavoproteínas/biossíntese , Flavoproteínas/isolamento & purificação , NADH Desidrogenase/metabolismo , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/isolamento & purificação , Potenciometria , Estrutura Terciária de Proteína , EspectrofotometriaRESUMO
Ribonucleotide reductase catalyzes a key step in DNA biosynthesis and repair, supplying the cell with the four common deoxyribonucleotides. It is thus the target of antiproliferative agents. The enzyme consists of two subunits named protein R1 and protein R2. R1 provides the sites for the nucleotide substrates and redox-active cysteines required for catalysis. R2 harbors a tyrosyl radical essential for activity. We show here that 2'-deoxy-2'-mercaptouridine 5'-diphosphate, a substrate analog, is a very efficient inactivator of ribonucleotide reductase (Ki = 35 microM, Kinact = 0.18 s-1). Inactivation is due to specific scavenging of the protein R2 tyrosyl radical. This unique feature sets this compound apart from other mechanism-based inhibitors such as 2'-azido-or 2'-chloro-2'-deoxyribonucleotide which induce partial or total protein R1 inactivation. During reaction, a transient organic radical was detected by EPR spectroscopy. Its g anisotropy (gz = 2.0620, gy = 2.0265, and gx = 2.0019) and its hyperfine structure are consistent with a perthiyl RSS. radical. The loss of the hyperfine structure by deuterium labeling of the beta protons of R1 cysteines unambiguously shows that the perthiyl radical is located on protein R1. We thus conclude that inactivation of ribonucleotide reductase by 2'-deoxy-2'-mercaptouridine 5'-diphosphate is due to an irreversible transfer of the radical located on protein R2 to a cysteine residue of protein R1.
Assuntos
Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Compostos de Sulfidrila/química , Tionucleotídeos/farmacologia , Difosfato de Uridina/análogos & derivados , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Oxigênio/química , Ribonucleotídeo Redutases/química , Difosfato de Uridina/farmacologiaRESUMO
The active form of protein R2, the small subunit of ribonucleotide reductase, contains a diferric center and a free radical localized at Tyr122. Hydroxyurea scavenges this radical but leaves the iron center intact. The resulting metR2 protein is inactive. The introduction of a radical into metR2 is dependent on the reduction of the iron center. In Escherichia coli, this is achieved by an enzyme system consisting of a NAD(P)H:flavin oxidoreductase and a poorly defined protein fraction, fraction b. Assuming that the iron center is deeply buried within the protein, electron transfer is suggested to occur over long distances. Site-directed mutagenesis allowed us to identify two invariant residues, Tyr356 at the C-terminal part of the protein and Tyr122 located 0.5 nm away from the closest iron atom, as mediators of this electron transfer. We also found that deazaflavins were excellent catalysts in the photoreduction of the iron center of metR2 and generation of the tyrosyl radical, providing the simplest and most efficient model for the physiological flavin reductase/fraction b activating system. The properties of the model reaction are described.
Assuntos
Escherichia coli/enzimologia , Ribonucleotídeo Redutases/química , Sítios de Ligação , Transporte de Elétrons , Escherichia coli/genética , Radicais Livres/química , Ferro/química , Estrutura Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Fotoquímica , Conformação Proteica , Ribonucleotídeo Redutases/genética , Tirosina/químicaRESUMO
The flavoprotein component (SiR-FP) of the sulfite reductase of E. coli is an octamer of the 66 kDa alpha subunit. It was shown to be cleaved in two peptide fragments. The 23 kDa fragment has been purified as a polymer of 8-10 subunits. It corresponds to the N-terminal part of the native protein and was shown to contain essentially FMN as cofactor. The 43 kDa fragment is monomeric. It contains exclusively FAD and remains able to catalyze efficiently NADPH-dependent reductions. One can conclude that each alpha-chain of SiR-FP is composed of two distinct domains, one binding FAD and the other FMN and that the FMN-binding domains cooperate for a head-to-head subunit interaction.
Assuntos
Escherichia coli/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Flavoproteínas/metabolismo , Fragmentos de Peptídeos/metabolismo , Sulfito Redutase (NADPH)RESUMO
Sulfite reductase (SiR) from Escherichia coli has a alpha 8 beta 4 subunit structure, where alpha 8 is a flavoprotein (SiR-FP) containing both FAD and FMN as prosthetic groups. It also exhibits a NADPH:flavin oxidoreductase activity with exogenous riboflavin, FMN, and FAD serving as substrates. The flavin reductase activity may function during activation of ribonucleotide reductase or during ferrisiderophore reduction. A plasmid containing cysJ gene, coding for the alpha subunit, overexpresses flavin reductase activity by 100-fold, showing that alpha is the site of free flavin reduction. The overproducer allows a fast and simple preparation of large amounts of the flavoprotein. Kinetic studies of its flavin reductase activity demonstrates a ping-pong bisubstrate-biproduct reaction mechanism. NADP+ inhibition studies show that both substrates, NADPH and free flavins, bind to the same site. While the FAD cofactor mediates the electron transfer between NADPH and free flavins, the FMN cofactor is not essential since a FMN-depleted SiR-FP retains a large proportion of activity. In contradiction with previous reports, SiR-FP is found to contain 1.6-1.7 flavin per alpha subunit. This result, together with the sequence homology between SiR-FP and NADPH-cytochrome P-450 reductase, suggests a new model for the structure of the protein with one FMN and one FAD prosthetic group per alpha subunit.