TY - GEN A1 - Yan, Robert A1 - Friemel, Martin A1 - Aloisi, Claudia A1 - Huynen, Martijn A1 - Taylor, Ian A. A1 - Leimkühler, Silke A1 - Pastore, Annalisa T1 - The eukaryotic-specific Isd11 is a complex- orphan protein with ability to bind the prokaryotic IscS T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The eukaryotic protein Isd11 is a chaperone that binds and stabilizes the central component of the essential metabolic pathway responsible for formation of iron-sulfur clusters in mitochondria, the desulfurase Nfs1. Little is known about the exact role of Isd11. Here, we show that human Isd11 (ISD11) is a helical protein which exists in solution as an equilibrium between monomer, dimeric and tetrameric species when in the absence of human Nfs1 (NFS1). We also show that, surprisingly, recombinant ISD11 expressed in E. coli co-purifies with the bacterial orthologue of NFS1, IscS. Binding is weak but specific suggesting that, despite the absence of Isd11 sequences in bacteria, there is enough conservation between the two desulfurases to retain a similar mode of interaction. This knowledge may inform us on the conservation of the mode of binding of Isd11 to the desulfurase. We used evolutionary evidence to suggest Isd11 residues involved in the interaction. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 551 KW - sulfur cluster formation KW - Escherichia coli KW - cysteine desulfurase KW - interacting protein KW - bacterial frataxin KW - statistical-model KW - biogenesis KW - biosynthesis KW - NFS1 KW - deficiency Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-411906 SN - 1866-8372 IS - 551 ER - TY - GEN A1 - Spricigo, Roberto A1 - Dronov, Roman A1 - Lisdat, Fred A1 - Leimkühler, Silke A1 - Scheller, Frieder W. A1 - Wollenberger, Ursula T1 - Electrocatalytic sulfite biosensor with human sulfite oxidase co-immobilized with cytochrome c in a polyelectrolyte-containing multilayer T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - An efficient electrocatalytic biosensor for sulfite detection was developed by co-immobilizing sulfite oxidase and cytochrome c with polyaniline sulfonic acid in a layer-by-layer assembly. QCM, UV-Vis spectroscopy and cyclic voltammetry revealed increasing loading of electrochemically active protein with the formation of multilayers. The sensor operates reagentless at low working potential. A catalytic oxidation current was detected in the presence of sulfite at the modified gold electrode, polarized at +0.1 V ( vs. Ag/AgCl 1 M KCl). The stability of the biosensor performance was characterized and optimized. A 17-bilayer electrode has a linear range between 1 and 60 mu M sulfite with a sensitivity of 2.19 mA M-1 sulfite and a response time of 2 min. The electrode retained a stable response for 3 days with a serial reproducibility of 3.8% and lost 20% of sensitivity after 5 days of operation. It is possible to store the sensor in a dry state for more than 2 months. The multilayer electrode was used for determination of sulfite in unspiked and spiked samples of red and white wine. The recovery and the specificity of the signals were evaluated for each sample. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 945 KW - bioelectrocatalysis KW - sulfite KW - sulfite oxidase KW - cytochrome c KW - multilayer Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-431176 SN - 1866-8372 IS - 945 SP - 225 EP - 233 ER - TY - JOUR A1 - Marelja, Zvonimir A1 - Leimkühler, Silke A1 - Missirlis, Fanis T1 - Iron sulfur and molybdenum cofactor enzymes regulate the drosophila life cycle by controlling cell metabolism JF - Frontiers in physiology N2 - Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which may function as a mitochondrial iron sensor since it is inactivated by iron; (iii) with the Krebs cycle thus disrupted, citrate is exported to the cytosol for fatty acid synthesis, while succinyl-CoA and the iron are used for heme biosynthesis; (iv) as iron is used for heme biosynthesis its concentration in the matrix drops allowing for manganese to reactivate superoxide dismutase and Fe-S cluster biosynthesis to reestablish the Krebs cycle. KW - aldehyde oxidase KW - DNA polymerase KW - electron transport chain KW - ecdysone KW - iron regulatory protein KW - quiescent mitochondria KW - magnetoreceptor KW - mitoflashes Y1 - 2018 U6 - https://doi.org/10.3389/fphys.2018.00050 SN - 1664-042X VL - 9 PB - Frontiers Research Foundation CY - Lausanne ER - TY - GEN A1 - Marelja, Zvonimir A1 - Leimkühler, Silke A1 - Missirlis, Fanis T1 - Iron sulfur and molybdenum cofactor enzymes regulate the Drosophila life cycle by controlling cell metabolism T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which may function as a mitochondrial iron sensor since it is inactivated by iron; (iii) with the Krebs cycle thus disrupted, citrate is exported to the cytosol for fatty acid synthesis, while succinyl-CoA and the iron are used for heme biosynthesis; (iv) as iron is used for heme biosynthesis its concentration in the matrix drops allowing for manganese to reactivate superoxide dismutase and Fe-S cluster biosynthesis to reestablish the Krebs cycle. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 925 KW - aldehyde oxidase KW - DNA polymerase KW - electron transport chain KW - ecdysone KW - iron regulatory protein KW - quiescent mitochondria KW - magnetoreceptor KW - mitoflashes Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-445670 SN - 1866-8372 IS - 925 ER - TY - GEN A1 - Lemaire, Olivier N. A1 - Infossi, Pascale A1 - Chaouche, Amine Ali A1 - Espinosa, Leon A1 - Leimkühler, Silke A1 - Giudici-Orticoni, Marie-Thérèse A1 - Méjean, Vincent A1 - Iobbi-Nivol, Chantal T1 - Small membranous proteins of the TorE/NapE family, crutches for cognate respiratory systems in Proteobacteria T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - In this report, we investigate small proteins involved in bacterial alternative respiratory systems that improve the enzymatic efficiency through better anchorage and multimerization of membrane components. Using the small protein TorE of the respiratory TMAO reductase system as a model, we discovered that TorE is part of a subfamily of small proteins that are present in proteobacteria in which they play a similar role for bacterial respiratory systems. We reveal by microscopy that, in Shewanella oneidensis MR1, alternative respiratory systems are evenly distributed in the membrane contrary to what has been described for Escherichia coli. Thus, the better efficiency of the respiratory systems observed in the presence of the small proteins is not due to a specific localization in the membrane, but rather to the formation of membranous complexes formed by TorE homologs with their c-type cytochrome partner protein. By an in vivo approach combining Clear Native electrophoresis and fluorescent translational fusions, we determined the 4: 4 stoichiometry of the complexes. In addition, mild solubilization of the cytochrome indicates that the presence of the small protein reinforces its anchoring to the membrane. Therefore, assembly of the complex induced by this small protein improves the efficiency of the respiratory system. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 933 KW - trimethylamine n-oxide KW - molybdenum cofactor biosynthesis KW - cytochrome bd oxidase KW - c-type cytochromes KW - escherichia-coli KW - swiss-model KW - native electrophoresis KW - mutational analysis KW - reductase KW - nitrate KW - microbiology KW - microbiology techniques Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-459208 SN - 1866-8372 IS - 933 ER -