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Jennifer L. Martin - One of the best experts on this subject based on the ideXlab platform.
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NMR fragment screening reveals a novel small molecule binding site near the catalytic surface of the disulfide–dithiol Oxidoreductase Enzyme DsbA from Burkholderia pseudomallei
Journal of Biomolecular NMR, 2020Co-Authors: Stefan Nebl, Wesam S. Alwan, Martin L. Williams, Gaurav Sharma, Ashley Taylor, Bradley C. Doak, Karyn L. Wilde, Róisín M. Mcmahon, Maria A. Halili, Jennifer L. MartinAbstract:The presence of suitable cavities or pockets on protein structures is a general criterion for a therapeutic target protein to be classified as ‘druggable’. Many disease-related proteins that function solely through protein–protein interactions lack such pockets, making development of inhibitors by traditional small-molecule structure-based design methods much more challenging. The 22 kDa bacterial thiol Oxidoreductase Enzyme, DsbA, from the gram-negative bacterium Burkholderia pseudomallei (BpsDsbA) is an example of one such target. The crystal structure of oxidized BpsDsbA lacks well-defined surface pockets. BpsDsbA is required for the correct folding of numerous virulence factors in B. pseudomallei , and genetic deletion of dsbA significantly attenuates B. pseudomallei virulence in murine infection models. Therefore, BpsDsbA is potentially an attractive drug target. Herein we report the identification of a small molecule binding site adjacent to the catalytic site of oxidized BpsDsbA. ^1H^N CPMG relaxation dispersion NMR measurements suggest that the binding site is formed transiently through protein dynamics. Using fragment-based screening, we identified a small molecule that binds at this site with an estimated affinity of K _D ~ 500 µM. This fragment inhibits BpsDsbA enzymatic activity in vitro. The binding mode of this molecule has been characterized by NMR data-driven docking using HADDOCK. These data provide a starting point towards the design of more potent small molecule inhibitors of BpsDsbA.
Robert L. Barbieri - One of the best experts on this subject based on the ideXlab platform.
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Human ovarian 17-ketosteroid Oxidoreductase: unique characteristics of the granulosa-luteal cell and stromal Enzyme.
American Journal of Obstetrics and Gynecology, 1992Co-Authors: Robert L. BarbieriAbstract:Objective: We attempted to test the hypothesis that distinct forms of the 17-ketosteroid Oxidoreductase exist in the human ovary and to compare its activity in stroma obtained from normally cycling women and from hyperandrogenic women. Study Design: Human ovarian granulosa-luteal cell and stromal 17-ketosteroid Oxidoreductase were examined in cell incubations and subcellular homogenates. Results: In subcellular homogenates of granulosa-luteal cells 17-ketosteroid Oxidoreductase activity was greater in the cytosol fraction than in the membrane fraction. In contrast, in homogenates of both ovarian stroma and Leydig cells its activity was greater in the membrane fraction than in the cytosol fraction. At the substrate concentrations used estrone was a better substrate than androstenedione for the granulosa-luteal cell 17-ketosteroid Oxidoreductase. In contrast, androstenedione was a better substrate than estrone for that in ovarian stromal and Leydig cell membranes. In incubations of ovarian stroma from hyperandrogenic women, significantly more testosterone accumulated in the medium per milligram of tissue than in the medium of incubations of ovarian stroma from normally cycling women (142 ± 48 vs 7.9 ± 7.5 pg testosterone per milligram of tissue per 48 hours, mean ± SD, p p p Conclusion: The localization (cytosol fraction) and substrate specificity (estrone) of the granulosa-luteal cell 17-ketosteroid Oxidoreductase Enzyme resembles that seen in human placenta. The localization (membrane fraction) and substrate specificity (androstenedione) of the ovarian stromal 17-ketosteroid Oxidoreductase Enzyme resembles that seen in Leydig cells. It may be one Enzyme that exists in multiple forms or it may be two (or more) Enzymes. In some hyperandrogenic women the ovarian stromal 17-ketosteroid Oxidoreductase may be more active than in normally cycling women, contributing to an abnormally increased testosterone production rate.
Angela Duilio - One of the best experts on this subject based on the ideXlab platform.
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The thiol-disulfide Oxidoreductase system in the cold-adapted bacterium Pseudoalteromonas haloplanktis TAC 125: discovery of a novel disulfide Oxidoreductase Enzyme
Extremophiles, 2006Co-Authors: Stefania Madonna, Maria Luisa Tutino, Gennaro Marino, Rosanna Papa, Leila Birolo, Giovanni Sannia, Flavia Autore, Nunzianna Doti, Eric Quemeneur, Angela DuilioAbstract:In prokaryotes, protein disulfide bond oxidation, reduction and isomerization are catalyzed by members of the thioredoxin superfamily, characterized by the conserved C–X–X–C motif in their active site. Thioredoxins and glutaredoxins contribute to the reducing power in the cytoplasm, while the Dsb system catalyzes disulfide bonds formation in the periplasmic space. This paper addresses the question of disulfide bonds formation in a cold-adapted micro-organism, Pseudoalteromonas haloplanktis TAC 125 ( Ph TAC125) by characterizing the DsbA system. We found distinctive features respect mesophilic counterparts that highlighted for the first time the occurrence of two adjacent chromosomal DsbA genes organised in a functional operon. The sophisticated transcriptional regulation mechanism that controls the expression of these two genes was also defined. The two DsbA proteins, named Ph DsbA and Ph DsbA2, respectively, were expressed in Escherichia coli and characterized. Results reported in this paper provide some insights into disulfide bonds formation in a micro organism isolated in the Antarctic sea water.
Charlene M Kahler - One of the best experts on this subject based on the ideXlab platform.
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the structure of the bacterial Oxidoreductase Enzyme dsba in complex with a peptide reveals a basis for substrate specificity in the catalytic cycle of dsba Enzymes
Journal of Biological Chemistry, 2009Co-Authors: Jason J Paxman, Natalie A Borg, James Horne, Philip E Thompson, Yanni K Y Chin, Pooja Sharma, Jamie S Simpson, Jerome Wielens, Susannah Piek, Charlene M KahlerAbstract:Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide Oxidoreductase Enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA Enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA Enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA Enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.
Stefan Nebl - One of the best experts on this subject based on the ideXlab platform.
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NMR fragment screening reveals a novel small molecule binding site near the catalytic surface of the disulfide–dithiol Oxidoreductase Enzyme DsbA from Burkholderia pseudomallei
Journal of Biomolecular NMR, 2020Co-Authors: Stefan Nebl, Wesam S. Alwan, Martin L. Williams, Gaurav Sharma, Ashley Taylor, Bradley C. Doak, Karyn L. Wilde, Róisín M. Mcmahon, Maria A. Halili, Jennifer L. MartinAbstract:The presence of suitable cavities or pockets on protein structures is a general criterion for a therapeutic target protein to be classified as ‘druggable’. Many disease-related proteins that function solely through protein–protein interactions lack such pockets, making development of inhibitors by traditional small-molecule structure-based design methods much more challenging. The 22 kDa bacterial thiol Oxidoreductase Enzyme, DsbA, from the gram-negative bacterium Burkholderia pseudomallei (BpsDsbA) is an example of one such target. The crystal structure of oxidized BpsDsbA lacks well-defined surface pockets. BpsDsbA is required for the correct folding of numerous virulence factors in B. pseudomallei , and genetic deletion of dsbA significantly attenuates B. pseudomallei virulence in murine infection models. Therefore, BpsDsbA is potentially an attractive drug target. Herein we report the identification of a small molecule binding site adjacent to the catalytic site of oxidized BpsDsbA. ^1H^N CPMG relaxation dispersion NMR measurements suggest that the binding site is formed transiently through protein dynamics. Using fragment-based screening, we identified a small molecule that binds at this site with an estimated affinity of K _D ~ 500 µM. This fragment inhibits BpsDsbA enzymatic activity in vitro. The binding mode of this molecule has been characterized by NMR data-driven docking using HADDOCK. These data provide a starting point towards the design of more potent small molecule inhibitors of BpsDsbA.
