Etudes structurales de la protéine PerR (Peroxide resistance Regulator): une métalloprotéine senseur de H2O2

Bacterial defense mechanisms towards oxidative stress are mainly based on expression of enzymes able to eleminate the reactive oxygen species such as superoxide anion, hydrogen peroxide and hydroxyl radical. The PerR protein, which belongs to the Fur family, was identified as a sensor of the hydrogen peroxide in Bacillus subtilis. PerR is a dimeric zinc protein with a regulatory metal binding site that coordinates either Fe2+ or Mn2+. Unlike other peroxide sensors such as OxyR from Escherichia coli and Orp1-Yap1 from Saccharomyces cerevisiaie, either the structural or the biochemical characterization of PerR was not as complete. The present work reports the structural studies of the PerR protein by X-ray crystallography and X-ray absorption spectroscopy (XAS). The resolved structures (the apoprotein, metal bound and the inactivated forms) further characterize the PerR protein. The DNA binding mode of PerR is also discussed in the manuscript. Interestingly, while most of the peroxide sensors use redox-active cysteines to detect H2O2, the reaction of PerR with H2O2 involve two histidine residues. The oxidation of PerR is catalyzed by the regulatory metal (Fe2+) and leads to the formation of a 2-oxo-histidine residue. The four cystein residues of the protein that are involved in the coordination of the zinc ion do not participate to the hydrogen peroxide detection. The crystal structure of the oxidized protein showing a 2-oxo-histidine residue is the first example of a 2-oxo-histidine containing structure reported in the PDB.Les systèmes de défense bactériens face à un stress oxydant reposent essentiellement sur l'expression d'enzymes capables de dégrader les espèces réactives de l'oxygène telles que le peroxyde d'hydrogène, le radical anion superoxyde et le radical hydroxyle. La protéine PerR a été identifiée chez Bacillus subtilis comme senseur de H2O2 appartenant à la famille des métallo-régulateurs Fur. La protéine PerR est un homodimère contenant deux sites métalliques par sous unité : un site à zinc structural et un site de régulation pouvant coordiner un ion Fe2+ ou Mn2+. La protéine PerR se fixe à l'ADN en présence de ses deux métaux pour réprimer la transcription de certains gènes. Contrairement à d'autres senseurs du peroxyde d'hydrogène comme la protéine OxyR chez Escherichia coli et le complexe Orp1-Yap1 chez Saccharomyces cerevisiaie, la caractérisation structurale et biochimique de la protéine PerR n'était pas aussi avancée. Les travaux présentés dans ce manuscrit rapportent les études structurales menées sur la protéine PerR par cristallographie aux rayons X et par spectroscopie d'absorption des rayons X (SAX). Les différentes structures résolues au cours de ce travail (l'apoprotéine, les formes active et inactive de la protéine) permettent de mieux caractériser la protéine PerR. Le mode de fixation à l'ADN de la protéine PerR est également discuté. De façon intéressante, tandis que les protéines OxyR et Orp1-Yap1 utilisent des résidus cystéine pour détecter le peroxyde, le mécanisme d'activation de PerR fait intervenir un résidu histidine qui est oxydé en 2-oxo-histidine. Cette oxydation est catalysée par le métal de régulation (Fe2+). Les quatre cystéines de la protéine coordinent l'ion zinc et ne participent pas à la détection du peroxyde. La structure de la forme oxydée de la protéine est également présentée : cette structure met en évidence, pour la première fois dans la PDB, une 2-oxo-histidine dans une structure cristallographique

Etudes structurales de la protéine PerR (Peroxide resistance Regulator): une métalloprotéine senseur de H2O2

Bacterial defense mechanisms towards oxidative stress are mainly based on expression of enzymes able to eleminate the reactive oxygen species such as superoxide anion, hydrogen peroxide and hydroxyl radical. The PerR protein, which belongs to the Fur family, was identified as a sensor of the hydrogen peroxide in Bacillus subtilis. PerR is a dimeric zinc protein with a regulatory metal binding site that coordinates either Fe2+ or Mn2+. Unlike other peroxide sensors such as OxyR from Escherichia coli and Orp1-Yap1 from Saccharomyces cerevisiaie, either the structural or the biochemical characterization of PerR was not as complete. The present work reports the structural studies of the PerR protein by X-ray crystallography and X-ray absorption spectroscopy (XAS). The resolved structures (the apoprotein, metal bound and the inactivated forms) further characterize the PerR protein. The DNA binding mode of PerR is also discussed in the manuscript. Interestingly, while most of the peroxide sensors use redox-active cysteines to detect H2O2, the reaction of PerR with H2O2 involve two histidine residues. The oxidation of PerR is catalyzed by the regulatory metal (Fe2+) and leads to the formation of a 2-oxo-histidine residue. The four cystein residues of the protein that are involved in the coordination of the zinc ion do not participate to the hydrogen peroxide detection. The crystal structure of the oxidized protein showing a 2-oxo-histidine residue is the first example of a 2-oxo-histidine containing structure reported in the PDB.Les systèmes de défense bactériens face à un stress oxydant reposent essentiellement sur l'expression d'enzymes capables de dégrader les espèces réactives de l'oxygène telles que le peroxyde d'hydrogène, le radical anion superoxyde et le radical hydroxyle. La protéine PerR a été identifiée chez Bacillus subtilis comme senseur de H2O2 appartenant à la famille des métallo-régulateurs Fur. La protéine PerR est un homodimère contenant deux sites métalliques par sous unité : un site à zinc structural et un site de régulation pouvant coordiner un ion Fe2+ ou Mn2+. La protéine PerR se fixe à l'ADN en présence de ses deux métaux pour réprimer la transcription de certains gènes. Contrairement à d'autres senseurs du peroxyde d'hydrogène comme la protéine OxyR chez Escherichia coli et le complexe Orp1-Yap1 chez Saccharomyces cerevisiaie, la caractérisation structurale et biochimique de la protéine PerR n'était pas aussi avancée. Les travaux présentés dans ce manuscrit rapportent les études structurales menées sur la protéine PerR par cristallographie aux rayons X et par spectroscopie d'absorption des rayons X (SAX). Les différentes structures résolues au cours de ce travail (l'apoprotéine, les formes active et inactive de la protéine) permettent de mieux caractériser la protéine PerR. Le mode de fixation à l'ADN de la protéine PerR est également discuté. De façon intéressante, tandis que les protéines OxyR et Orp1-Yap1 utilisent des résidus cystéine pour détecter le peroxyde, le mécanisme d'activation de PerR fait intervenir un résidu histidine qui est oxydé en 2-oxo-histidine. Cette oxydation est catalysée par le métal de régulation (Fe2+). Les quatre cystéines de la protéine coordinent l'ion zinc et ne participent pas à la détection du peroxyde. La structure de la forme oxydée de la protéine est également présentée : cette structure met en évidence, pour la première fois dans la PDB, une 2-oxo-histidine dans une structure cristallographique

Biochemical and Structural Characterization of the Subclass B1 Metallo-β-Lactamase VIM-4 ▿

The metallo-β-lactamase VIM-4, mainly found in Pseudomonas aeruginosa or Acinetobacter baumannii, was produced in Escherichia coli and characterized by biochemical and X-ray techniques. A detailed kinetic study performed in the presence of Zn2+ at concentrations ranging from 0.4 to 100 μM showed that VIM-4 exhibits a kinetic profile similar to the profiles of VIM-2 and VIM-1. However, VIM-4 is more active than VIM-1 against benzylpenicillin, cephalothin, nitrocefin, and imipenem and is less active than VIM-2 against ampicillin and meropenem. The crystal structure of the dizinc form of VIM-4 was solved at 1.9 Å. The sole difference between VIM-4 and VIM-1 is found at residue 228, which is Ser in VIM-1 and Arg in VIM-4. This substitution has a major impact on the VIM-4 catalytic efficiency compared to that of VIM-1. In contrast, the differences between VIM-2 and VIM-4 seem to be due to a different position of the flapping loop and two substitutions in loop 2. Study of the thermal stability and the activity of the holo- and apo-VIM-4 enzymes revealed that Zn2+ ions have a pronounced stabilizing effect on the enzyme and are necessary for preserving the structure

Structural and functional characterization of 2-oxo-histidine in oxidized PerR protein.

International audienceIn Bacillus subtilis, PerR is a metal-dependent sensor of hydrogen peroxide. PerR is a dimeric zinc protein with a regulatory site that coordinates either Fe(2+) (PerR-Zn-Fe) or Mn(2+) (PerR-Zn-Mn). Though most of the peroxide sensors use cysteines to detect H(2)O(2), it has been shown that reaction of PerR-Zn-Fe with H(2)O(2) leads to the oxidation of one histidine residue. Oxidation of PerR leads to the incorporation of one oxygen atom into His37 or His91. This study presents the crystal structure of the oxidized PerR protein (PerR-Zn-ox), which clearly shows a 2-oxo-histidine residue in position 37. Formation of 2-oxo-histidine is demonstrated and quantified by HPLC-MS/MS. EPR experiments indicate that PerR-Zn-H37ox retains a significant affinity for the regulatory metal, whereas PerR-Zn-H91ox shows a considerably reduced affinity for the metal ion. In spite of these major differences in terms of metal binding affinity, oxidation of His37 and/or His91 in PerR prevents DNA binding

The Challenge of Improving Soil Fertility in Yam Cropping Systems of West Africa

Yam (Dioscorea spp.) is a tuber crop grown for food security, income generation, and traditional medicine. This crop has a high cultural value for some of the groups growing it. Most of the production comes from West Africa where the increased demand has been covered by enlarging cultivated surfaces while the mean yield remained around 10 t tuber ha−1. In West Africa, yam is traditionally cultivated without input as the first crop after a long-term fallow as it is considered to require a high soil fertility. African soils, however, are being more and more degraded. The aims of this review were to show the importance of soil fertility for yam, discuss barriers that might limit the adoption of integrated soil fertility management (ISFM) in yam-based systems in West Africa, present the concept of innovation platforms (IPs) as a tool to foster collaboration between actors for designing innovations in yam-based systems and provide recommendations for future research. This review shows that the development of sustainable, feasible, and acceptable soil management innovations for yam requires research to be conducted in interdisciplinary teams including natural and social sciences and in a transdisciplinary manner involving relevant actors from the problem definition, to the co-design of soil management innovations, the evaluation of research results, their communication and their implementation. Finally, this research should be conducted in diverse biophysical and socio-economic settings to develop generic rules on soil/plant relationships in yam as affected by soil management and on how to adjust the innovation supply to specific contexts

Improving malaria control in West Africa: interruption of transmission as a paradigm shift.

With the paradigm shift from the reduction of morbidity and mortality to the interruption of transmission, the focus of malaria control broadens from symptomatic infections in children ≤5 years of age to include asymptomatic infections in older children and adults. In addition, as control efforts intensify and the number of interventions increases, there will be decreases in prevalence, incidence and transmission with additional decreases in morbidity and mortality. Expected secondary consequences of these changes include upward shifts in the peak ages for infection (parasitemia) and disease, increases in the ages for acquisition of antiparasite humoral and cellular immune responses and increases in false-negative blood smears and rapid diagnostic tests. Strategies to monitor these changes must include: (1) studies of the entire population (that are not restricted to children ≤5 or ≤10 years of age), (2) study sites in both cities and rural areas (because of increasing urbanization across sub-Saharan Africa) and (3) innovative strategies for surveillance as the prevalence of infection decreases and the frequency of false-negative smears and rapid diagnostic tests increases

Sahel, savana, riverine and urban malaria in West Africa: Similar control policies with different outcomes.

The study sites for the West African ICEMR are in three countries (The Gambia, Senegal, Mali) and are located within 750 km of each other. In addition, the National Malaria Control Programmes of these countries have virtually identical policies: (1) Artemisinin Combination Therapies (ACTs) for the treatment of symptomatic Plasmodium falciparum infection, (2) Long-Lasting Insecticide-treated bed Nets (LLINs) to reduce the Entomololgic Inoculation Rate (EIR), and (3) sulfadoxine-pyrimethamine for the Intermittent Preventive Treatment of malaria during pregnancy (IPTp). However, the prevalence of P. falciparum malaria and the status of malaria control vary markedly across the four sites with differences in the duration of the transmission season (from 4-5 to 10-11 months), the intensity of transmission (with EIRs from unmeasurably low to 4-5 per person per month), multiplicity of infection (from a mean of 1.0 to means of 2-5) and the status of malaria control (from areas which have virtually no control to areas that are at the threshold of malaria elimination). The most important priority is the need to obtain comparable data on the population-based prevalence, incidence and transmission of malaria before new candidate interventions or combinations of interventions are introduced for malaria control