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Tungsten Biochemistry in Pyrococcus Furiosus

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Tungsten Biochemistry in Pyrococcus furiosus Tungsten Biochemistry in Pyrococcus furiosus Proefschrift ter verkrijging van de graad van doctor aan de Technische Universiteit Delft, op gezag van de Rector Magnificus Prof.dr.ir. J. T. Fokkema, voorzitter van het College voor Promoties, in het openbaar te verdedigen op maandag 10 maart 2008 om 15:00 uur door Loes Elizabeth BEVERS doctorandus in de scheikunde geboren te Nijmegen Dit proefschrift is goedgekeurd door de promotor: Prof.dr. W. R. Hagen Samenstelling promotie commissie: Rector Magnificus voorzitter Prof.dr. W. R. Hagen Technische Universiteit Delft, promotor Prof.dr. G. Schwarz Universität zu Köln Prof.dr. S. de Vries Technische Universiteit Delft Prof.dr. J. H. de Winde Technische Universiteit Delft Prof.dr. J. van der Oost Wageningen Universiteit Dr. A. Magalon Laboratoire de Chimie Bactérienne, IBSM, CNRS, Marseille Dr. P.L. Hagedoorn Technische Universiteit Delft This research has been financed by a grant from the Council for Chemical Sciences of the Netherlands Organization for Scientific research (NWO) (700.51.301). Cover design by Esengül Yildirim Contents Aim and outline of thesis 6 Chapter 1: Introduction: The bioinorganic chemistry of tungsten 9 Part I - Uptake of tungstate Chapter 2: Tungsten transport protein A (WtpA) in Pyrococcus furiosus : first member of a new class of molybdate and tungstate transporters 51 Part II - Tungsten cofactor synthesis Chapter 3: The function of MoaB proteins in the biosynthesis of the molybdenum 73 and tungsten cofactors Chapter 4: Metal incorporation into the molybdopterin cofactor by MoeA proteins 91 Chapter 5: Replacing tungsten by molybdenum in aldehyde oxidoreductases in Pyrococcus furiosus 105 Part III - Tungstoenzymes Chapter 6: WOR5: A novel tungsten containing aldehyde oxidoreductase from Pyrococcus furiosus with a broad substrate specificity 119 Chapter 7: Cellular localization and quaternary structure of WOR5 133 Chapter 8: Redox chemistry of tungsten and iron-sulfur prosthetic groups in Pyrococcus furiosus formaldehyde ferredoxin oxidoreductase 149 Chapter 9: Concluding remarks and outlook 165 Chapter 10: Summary 168 Samenvatting 170 Curriculum Vitae 172 List of Publicaties 173 Dankwoord 174 Aim and outline of thesis This thesis describes a study on the role of the metal tungsten in the hyperthermophilic archaeon Pyrococcus furiosus . P. furiosus can be considered as a model system for hyperthermophilic archaea; its genome has been sequenced [1], the organism can be cultivated easily in a batch or continuous culture [2,3], and many of its proteins have been the subject of studies in recent years. P. furiosus can also be considered as a model system with respect to its tungsten metabolism. Cultivation studies showed a strong tungsten- dependent growth [2], and in the last decades four tungsten containing aldehyde oxidoreductases (AORs) were purified from P. furiosus cell-free extract [4-7]. The aim of this thesis project was to extend the knowledge on different aspects of tungsten metabolism in P. furiosus by trying to answer some fundamental questions: how do the cells take up the tungstate from the media? Can they also take up molybdate and incorporate the molybdenum in the active site of the AOR enzymes? To what extent is tungsten-cofactor (Wco) synthesis similar to molybdenum–cofactor (Moco) synthesis? And can we identify new tungsten-containing enzymes in P. furiosus ? The results of this study are presented in this thesis in the following order: Chapter 1 provides a general introduction to the bioinorganic chemistry of tungsten. Repeatedly, reference is made to the homologous metal molybdenum, whose literature is generally more developed both for biological and model systems. The presentation of the experimental data is divided into three parts representing different aspects of tungsten metabolism: uptake, incorporation, and catalytic action. Part I starts with the uptake of the metal from the media by describing the identification and characterization of the P. furiosus periplasmic tungstate binding protein (WtpA) ( chapter 2). This protein, part of an ATP binding cassette (ABC) transport system, has an extremely low K D for tungstate (K D = 17 ± 7 pM) but is also able to bind molybdate with a relatively high affinity (K D = 11 ± 5 nM). Part II focuses on a subsequent step of tungsten metabolism; the incorporation of the metal into the pterin cofactor in order to tune its redox properties in a manner required for biological activity. In chapter 3 , the hexameric P. furiosus MoaB protein is shown to catalyse the adenylylation of metal-binding pterin (MPT) as activation step prior to metal insertion. This finding shows that adenylylation of MPT is a conserved step in Wco/Moco biosynthesis in both prokaryotes and eukaryotes. The subsequent step of metal insertion catalyzed by the P. furiosus MoeA proteins is investigated in chapter 4. Chapter 5 presents the in vivo incorporation of tungsten-homologous molybdenum into the cofactor of the P. furiosus AOR enzymes. So far, these enzymes had only been purified containing tungsten in their active site. Part III focuses on the tungstoenzymes. Chapter 6 describes the purification and biochemical characterization of a new AOR, tungsten oxidoreductase number five (WOR5), which completes the family of P. furiosus AORs. The gene adjacent to wor5 (PF1479) encodes a putative four [4Fe-4S] clusters binding protein, which distinguishes WOR5 from 6 the other (monocistronic) AORs. Chapter 7 provides a study on this PF1479 protein and proposes it to form a heterodimeric structure with WOR5, localized in the periplasmic space. Chapter 8 describes the redox chemistry of the tungsten and iron-sulfur prosthetic groups in P. furiosus formaldehyde oxidoreductase (FOR). In addition, the K M value for formaldehyde is suggested to be three orders of magnitude lower than previously reported, due to an unfavorable hydratation equilibrium which converts free formaldehyde into methylene glycol. Finally, chapter 9 contains the concluding remarks and perspectives for future research, and is followed by chapter 10 : a summary of all the results. 7 References [1] F.T. Robb, D.L. Maeder, J.R. Brown, J. DiRuggiero, M.D. Stump, R.K. Yeh, R.B. Weiss, D.M. Dunn, Meth. Enzym. 330 (2001) 134. [2] G. Fiala, K.O. Stetter, Arch. Microbiol. 145 (1986) 56. [3] N. Raven, N. Ladwa, D. Cossar, R. Sharp, Appl. Microbiol. Biotech. 38 (1992) 263. [4] S. Mukund, M.W. Adams, J. Biol. Chem. 270 (1995) 8389. [5] S. Mukund, M.W. Adams, J. Biol. Chem. 266 (1991) 14208. [6] R. Roy, S. Mukund, G.J. Schut, D.M. Dunn, R. Weiss, M.W.W. Adams, J. Bacteriol. 181 (1999) 1171. [7] R. Roy, M.W.W. Adams, J. Bacteriol. 184 (2002) 6952. 8 Chapter 1 The Bioinorganic Chemistry of Tungsten Loes E. Bevers Peter-Leon Hagedoorn Wilfred R. Hagen Adapted from: Coordination Chemistry Reviews (2008) in press 9 Chapter 1 Abstract Tungsten is widely distributed in biology, however, the majority of the tungsten-containing enzymes purified to date, originates from anaerobic archaea and bacteria. Tungsten coordination complexes incorporated in these enzymes can be studied with similar analytical and spectroscopic techniques as tungsten model compounds. The metal is taken up by cells in the form of tungstate, and subsequently it is processed into a sulfur-rich coordination as part of a metal-organic cofactor referred to as tungstopterin, which is equivalent to the molybdopterin forms found as active centers in several molybdenum- containing enzymes. For biology tungsten is significantly different from molybdenum and this review focuses on the (bio)molecular basis of this differential cellular use of W compared to Mo in terms of their active transport, cofactor synthesis, and functioning as catalytically active sites. 10 The Bioinorganic Chemistry of Tungsten Introduction Tungsten is the bioelement with the highest atomic number, 74, and the only bioelement in the third transition row of the periodic table. Tungsten is widely distributed in biology, however, it is not a universal bioelement. For some species tungsten is essential: their life depends on the presence of the element; for other species tungsten is a facultative bioelement: they choose to make biological use of the element when they experience specific environmental constraints; for the remaining species tungsten is biochemically indifferent or possibly xenobiotic: they have not developed a functional use of the element, although, upon its inadvertent intake, their physiology may well be affected. Present knowledge places all eukaryotes, including man, in the last category. Two fundamental questions form the Leitmotif of this review; the first one is the ‘why’-question: why do some cells go for tungsten chemistry and others not? The second is the ‘how’-question: how do cells discriminate between tungsten and molybdenum. Molybdenum is in many ways the twin element of tungsten. Also in biology the coordination chemistries of W and Mo are similar in structural and functional aspects. Molybdenum is the only bioelement in the second transition row. Like tungsten it is widely, though possibly not universally, distributed in biology. Its usage appears to be to a considerable extent the mirror image of that of tungsten. Some forms of life, e.g. humans, are strictly dependent on the availability of Mo while they are independent of W; other species, e.g. the archaeon Pyrococcus furiosus , have no apparent use for Mo, while they are strictly dependent on W; yet other species, e.g. the archaeon Methanobacterium thermoautotrophicum , appear to be able to choose between W and Mo as a function of a variable environment. And yet other species, e.g. the archaeon Pyrobaculum aerophilum , may have learned to employ the chemistries of Mo and W simultaneously for distinct functions. Mo-biochemistry and W-biochemistry are presently both very active areas of research, the latter in particular in relation to the fundamental why and how questions formulated above. Mo has been known to be a biological trace element for a long time, and the development of its biochemistry has commonly been taken to be an endeavor in its own right.
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  • UNIVERSITY of EMBU EDWARD NDERITU KARANJA Phd 2020

    UNIVERSITY of EMBU EDWARD NDERITU KARANJA Phd 2020

    UNIVERSITY OF EMBU EDWARD NDERITU KARANJA PhD 2020 MICROBIAL COMMUNITY DIVERSITY AND STRUCTURE WITHIN ORGANIC AND CONVENTIONAL FARMING SYSTEMS IN CENTRAL HIGHLANDS OF KENYA EDWARD NDERITU KARANJA (MSc) A THESIS SUBMITTED IN PARTIAL FULFILLMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN APPLIED MICROBIOLOGY IN THE UNIVERSITY OF EMBU NOVEMBER, 2020 DECLARATION This thesis is my original work and has not been presented for a degree in any other University Signature……………………………. Date………….……….. Edward Nderitu Karanja Department of Biological Science B801/147/2015 This thesis has been submitted for examination with our approval as the University Supervisors Signature……………………………. Date………….………. Prof. Romano Mwirichia Department of Biological science University of Embu (UoEm), Kenya Signature………. …………………………. Date………….…………. Dr. Andreas Fliessbach Department of Soil Science Research Institute of Organic Agriculture - FIBL, Switzerland i DEDICATION I dedicated to my family; my wife Anne Kelly Kambura, my children; Shawn Karanja, Melissa Wangithi, Joseph Munyuithia, Shayne Koome and Ann Wanjiku, my parents; Mr. Samuel Karanja and Mrs. Agnes Wangithi, my siblings, Ruth Wairimu, Juliet Muthoni, Alex Ngochi, James Karuma and Nelly Njoki. I appreciate the support you have accorded me during my studies. Your inspiration and backing in this journey made it easier to manage all challenges encountered. ii ACKNOWLEDGEMENT I express gratitude toward Almighty God for his mercies from the beginning of this long and thought-provoking journey. This was conducted in the framework of long-term systems comparison program, with financial support from Biovision Foundation, Coop Sustainability Fund, Liechtenstein Development Service (LED) and the Swiss Agency for Development and Cooperation (SDC). I acknowledge icipe core funding for the kind contribution provided by UK-Aid from UK Government, Swedish International Development Cooperation Agency, Swiss Agency for Development and Cooperation, Federal Democratic Republic of Ethiopia and the Kenyan Government.