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.
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
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
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
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
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)Assuntos
Veia Axilar , Síndrome da Costela Cervical/complicações , Síndrome da Costela Cervical/diagnóstico , Veia Subclávia , Síndrome do Desfiladeiro Torácico/complicações , Síndrome do Desfiladeiro Torácico/diagnóstico , Trombose/etiologia , Adulto , Síndrome da Costela Cervical/cirurgia , Feminino , Humanos , Reoperação , Trombose/diagnóstico , Trombose/cirurgiaAssuntos
Fraturas Ósseas/cirurgia , Traumatismos da Perna/cirurgia , Infecção da Ferida Cirúrgica/cirurgia , Adulto , Humanos , Isquemia/cirurgia , Perna (Membro)/irrigação sanguínea , Masculino , Meniscos Tibiais/cirurgia , Métodos , Complicações Pós-Operatórias/cirurgia , Transplante de Pele , Transplante AutólogoRESUMO
Iron is an essential element in all living cells. Solubilization, uptake and transport of iron by microorganisms is controlled by highly efficient and specific Fe(3+)-chelating agents named siderophores. However, mechanisms of mobilization of iron from ferrisiderophores are still enigmatic. Here, we demonstrate that Escherichia coli contains a powerful enzymatic system for the reduction of ferrisiderophores. Siderophores have a much lower affinity for ferrous iron, which then can be liberated. This system has been previously purified and characterized as a NAD(P)H:flavin oxidoreductase [Fontecave, M., Eliasson, R. and Reichard, P. (1987) J. Biol. Chem. 262, 12,325-12,331)]. It catalyzes the reduction of free flavins, FMN, FAD or riboflavin by NADH or NADPH. Reduced flavins, in turn, transfer their electrons to physiological ferric complexes: ferrisiderophores, ferric citrate and ferritins. The reaction is inhibited by molecular oxygen and greatly stimulated by Fe(2+)-acceptors such as ferrozine or the iron-free form of ribonucleotide reductase subunit R2. We suggest that the reduction and the mobilization of iron from ferrisiderophores in the cell might be regulated by the presence of physiological ferrous traps such as apoproteins.
Assuntos
Escherichia coli/enzimologia , Ferro/metabolismo , NADH NADPH Oxirredutases/metabolismo , Catálise , Transporte de Elétrons , FMN Redutase , Compostos Férricos/metabolismo , Ferricromo/metabolismo , Ferritinas/metabolismo , Compostos Ferrosos/metabolismo , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Concentração de Íons de Hidrogênio , Cinética , NAD/metabolismo , NADP/metabolismo , Oxirredução , Riboflavina/metabolismoRESUMO
A protein fraction from Escherichia Coli soluble extracts contain a NAD(P)H:hydrogen peroxide oxidoreductase activity. This activity is compared to and found to be distinct from well-known E. Coli enzymes involved in the protection from peroxides: hydroperoxidase I (HPI) and its o-dianisidine peroxidase component and the alkyl hydroperoxide reductase.
Assuntos
Escherichia coli/enzimologia , Peroxidases/metabolismo , Catalase/metabolismo , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , CinéticaRESUMO
We have demonstrated a lipid requirement for the UDPgalactose:1,2-diacylglycerol 3-beta-D-galactosyl-transferase (or monogalactosyldiacylglycerol synthase; EC 2.4.1.46), an enzyme involved in the biosynthesis of monogalactosyldiacylglycerol, solubilized from chloroplast envelope membranes and partially purified by hydroxyapatite chromatography. The enzyme fraction was highly delipidated (<0.1 mg of lipid per mg of protein), and addition of lipids extracted from chloroplast membranes was necessary to reveal the activity. Acidic glycerolipids, and especially phosphatidylglycerol, were the best activators of the enzyme. The preparation of a delipidated enzyme fraction and the development of optimal assay conditions were prerequisites for the determination of the kinetic parameters for the hydrophobic substrate of the enzyme, diacylglycerol. In addition, we have demonstrated the existence of two substrate-binding sites: a hydrophobic one for diacylglycerol and a hydrophilic one for UDP-galactose.
RESUMO
A soluble ferrisiderophore reductase activity of Escherichia coli was purified to homogeneity and identified as the sulfite reductase. The pure enzyme catalyzes the reduction of ferric citrate, ferriaerobactin, ferrioxamin, ferricrocin, ferrichrome and ferrifusarinin by NADPH. Free flavins, riboflavin, FMN, FAD were absolutely required, suggesting that this activity resides in the flavin reductase activity of sulfite reductase.
Assuntos
Escherichia coli/enzimologia , NADH NADPH Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Cromatografia , Cromatografia em Gel , Cromatografia por Troca Iônica , Durapatita , Hidroxiapatitas , Cinética , NADH NADPH Oxirredutases/isolamento & purificação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/isolamento & purificação , Especificidade por SubstratoRESUMO
The haemoglobin-like protein (HMP) of E. coli previously isolated as a dihydropteridine reductase was shown to be also a ferric citrate reductase. We demonstrate that, in fact, HMP is a flavin reductase and that its ferric reductase activity is a result of its ability to reduce free flavins. However, when compared to the two main ferric/flavin reductases of E. coli, i.e., the NAD(P)H: flavin oxidoreductase and the sulfite reductase, one can conclude that the contribution of HMP to iron reduction is negligible.
Assuntos
Proteínas de Bactérias/metabolismo , Di-Hidropteridina Redutase , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Hemeproteínas/metabolismo , NADH NADPH Oxirredutases/metabolismo , Proteínas de Bactérias/isolamento & purificação , Cromatografia em Gel , Cromatografia por Troca Iônica , FMN Redutase , Mononucleotídeo de Flavina/metabolismo , Flavinas/metabolismo , Hemeproteínas/isolamento & purificação , Cinética , NADH NADPH Oxirredutases/isolamento & purificação , OxirreduçãoRESUMO
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
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.
Assuntos
Escherichia coli/enzimologia , Flavoproteínas/metabolismo , NADH NADPH Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Cromatografia DEAE-Celulose , Transporte de Elétrons , FMN Redutase , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Flavoproteínas/química , Flavoproteínas/isolamento & purificação , Cinética , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/isolamento & purificação , NADP/metabolismo , NADP/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrofotometria , Especificidade por SubstratoRESUMO
Protein R2, the small subunit of ribonucleotide reductase of Escherichia coli, contains an essential free radical localized to tyrosine 122 of its polypeptide chain. When this radical is scavenged by hydroxyurea, the enzyme is transformed into an inactive form, metR2. E. coli contains a NAD(P)H:flavin oxidoreductase, named Fre, absolutely required for the regeneration of the radical and the activation of metR2 into R2. Consequently, an E. coli mutant strain lacking an active fre gene is more sensitive to hydroxyurea during growth, demonstrating the physiological protective function of Fre from the loss of the radical. However, this gene is not essential, and we found that E. coli contains a second tyrosyl radical generating activity, also residing in a flavin reductase. The enzyme has been purified 200-fold to homogeneity and found to be identical to sulfite reductase. Pure sulfite reductase has the ability to catalyze the reduction of free riboflavin, FMN, or FAD by NADPH and thus, as Fre, to transfer electrons to the iron center of metR2, a key step during the activation reaction.
Assuntos
Escherichia coli/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Ribonucleotídeo Redutases/metabolismo , Sequência de Aminoácidos , Western Blotting , Catálise , Transporte de Elétrons , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , FMN Redutase , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Radicais Livres , Hidroxiureia/farmacologia , Dados de Sequência Molecular , Peso Molecular , Mutação , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , NADP/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/isolamento & purificação , Sulfito Redutase (NADPH) , Tirosina/metabolismoRESUMO
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
Assimilation of iron by microorganisms requires the presence of ferric reductases which participate in the mobilization of iron from ferrisiderophores. The common structural and catalytic properties of these enzymes are described and shown to be identical to those of flavin reductases. This strongly suggests that, in general, the reduction of iron depends on reduced flavins provided by flavin reductases.
Assuntos
Proteínas de Bactérias/química , NADH NADPH Oxirredutases/classificação , Transporte de Elétrons , Escherichia coli/enzimologia , Escherichia coli/genética , FMN Redutase , Genes Bacterianos , Ferro/metabolismo , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/fisiologia , NADP/metabolismo , Oxirredução , Sideróforos/metabolismo , Especificidade por SubstratoRESUMO
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
Ribonucleotide reductase has been demonstrated to be inhibited by NO synthase product(s). The experiments reported here show that nitric oxide generated from sodium nitroprusside, S-nitrosoglutathione and the sydnonimine SIN-1 inhibits ribonucleotide reductase activity present in cytosolic extracts of TA3 mammary tumor cells. Stable derivatives of these nitric oxide donors were either inactive or much less inhibitory. EPR experiments show that the tyrosyl radical of the small subunit of E. Coli or mammalian ribonucleotide reductase is efficiently scavenged by these NO donors.