DOCSLIB.ORG

Uhm Phd 4561 R.Pdf

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Uhm Phd 4561 R.Pdf UNIVERSITY OF HAWAl'I LIBRARY DIVALENT CATION CHANNELS WITH INTRINSIC ALPHA-KINASE ACTIVITY DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI'I IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOMEDICAL SCIENCES (PHYSIOLOGY) MAY 2005 By Bret Fajans Bessac Dissertation Committee: Andrea Fleig, Chairperson John R. Claybaugh David A. Lally Martin D. Rayner John G. Starkus Reinhold Penner ACKNOWLEDGEMENTS The spirituality ofthis universe, thank you for the hope, mercy, love, passion,joy, grace, wonderment, perplexities, teachings, protection and companionship to my semi­ schizophrenic mind, dreams and seeking soul as my awareness, interpretation and enlightenment continues, as well as, the coral reefs, rain forests, beaches and sunshine, and their flora and fauna, ofthese islands. My mother, Susanne Leppmann Bessac, MA, and my father, Francis Bagnall Bessac, PhD, thank you for the support, love, guidance, nurturing and shaping me with a stubborn, argumentative, never-surrender attitude, to perceive in the intellectual, humanistic, artistic and spiritual aspects oflife. My love, Tricia Michelle Kratz, MSW, thank you for melting me with your heart, support, friendship and compassion, and for enduring and helping me through my studies and dissertation. Thank you for the friendship and ohana of your mother, Patricia Sonnenberg, and son, Matthew Kratz. My advisor, Andrea Fleig, PhD, thank you for the patience, help, instruction, friendship, support, guidance and direction of my distractible wanderings in gathering comprehension of electrophysiology methods and the concepts of channels, as well as, knowledgeable daily discourse, insights in the experimental procedures, and keeping me on the direction and scope of my project. This dissertation would not have existed without you. My committee member, Reinhold Penner, PhD/MD, thank you for your friendship and brilliant intellect, insights and discourse into science, electrophysiology, my project and hypothesis. My committee member and graduate student advisor, David A. Lally, PhD, thank you for your friendship, help and support in getting through this process. My committee member, John G. Starkus, PhD, thank you for your friendship, intrigue, insights and discourse in electrophysiology. My committee member, Martin D. Rayner, PhD, thank you for your friendship, guidance, advise, support and setting the foundation for all this to occur. My committee member, John R. Claybaugh, PhD, thank you for your friendship, intrigue, insight and discourse in physiology. My sisters, brothers-in-law, nieces and nephews; Barbara Tracy, MS, MLT, David Tracy, Josh Tracy, Eli Tracy, Willow Tracy, Andrea Maxeiner, PhD, James Maxeiner, PhD/JD, Cassey Maxeiner, Peter Maxeiner, Turan Albini, Martin Albini, BSE, Fiona Albini, III Jethro Albini, Ivan Albini, Joan Steelquist, Mark Steelquist, Rueben Steelquist and Frances Steelquist thank you for all the love and support. My editor and fellow laboratory mate, Andreas Beck, PhD, thank you for your friendship, humor, intellect, attention to detail and taking the time to read and re-read this dissertation and try to fonn it into something comprehensible. Fellow graduate student and laboratory mate, Mahealani Monteilh-Zoller, MS, thank you for your friendship, humor, intellect and maintaining this laboratory. Ka'ohi Dang and Carolyn Oki, MS, thank you for your friendship, cell culture and maintance ofthe laboratoqr. My fellow laboratory mates, Phillip Demusse, PhD, Martin Kolisek, PhD, Henrique Chang, PhD/DVM thank you for your friendship, intellect and humor. Helen Turner, PhD and Alexander James Stokes, PhD, thank you for your friendship, support and critical idea to help make logical sense from my muddy writings, as well as, techniques and insights in biotechnology and molecular biology. The laboratory of Jean-Pierre Kinet in Boston, MA, thank you for the stably transfected mouse TRPM7 cells. The laboratory of Andrew Scharenberg in Seattle, WA, thank you for the stably transfected human TRPM7, mutated human TRPM7 with the kinase not expressed and single-point mutated human TRPM7 of a glycine (1599) to a glutamate or a lysine to an arginine (1648). The laboratory of Anne-Laure Perraud in Denver, CO, thank you for the stably transfected human TRPM6 or both TRPM6 and human TRPM7 or both TRPM6 and mutated kinase-deleted TRPM7. The laboratory of Alexey Ryazanov in Piscataway, NJ, thank you for the stably transfected human TRPM7 with or without human annexin I or mutated annexin I of a serine to alanine or to aspartate. The laboratory ofBernd Nilius in Brussels, Belgium, thank you for TRPM6 eDNA. The constitution of the United States and Hawai'i and the taxpayers within these governments, as well as, The late Queen Ema, who paid for my dissertation from their institutions of National Institute of Health, University of Hawai'i and The Queen's Center for Biomedical Research. IV ABSTRACT TRPM7and its homologue TRPM6 are members of the melastatin-related Transient­ Receptor-Potential (TRPM) family. TRPM6 and TRPM7 "chanzymes" form both transmembrane cation channels and cytosolic a -kinase enzymes. TRPM7 channel 2 2 specifically conducts Mg +, Ca + and trace divalent cations at resting potential, is 2 constitutively active and regulated by intracellular (IC) Mg + and Mg-ATP. TRPM7 a­ kinase phosphorylates TRPM7, annexin I and probably other proteins. TRPM7 is 2 2 ubiquitous, essential and implicated in cellular respiration and Mg + homeostasis, Ca +­ involvement in cell cycle and anoxic cell death. TRPM7 over-expression induces HEK-293 cells to change morphology, detach and aggregate, but not immediately die. The properties of TRPM7 to induce these effects were examined for clues to TRPM7 physiological functions. TRPM7 channel was not 2 directly involved, because over-expression effects kinetics was similar in high Ca +, high 2 Mg + or standard media. Over-expression of TRPM7 without the kinase did not change cells, unless co-expressed with TRPM6. TRPM6 co-expression maintained kinase­ 2 2 deleted TRPM7 hypersensitivity to Mg +. Ca + free media mimicked the affects of TRPM7 over-expression and increased the kinetics. TRPM7 kinase may bind and 2 inactivate a factor involved in cell morphology and adhesion, which involves Ca +. Evidence shows it is not annexin-I. TRPM7 osmosensitivity was examined, because osmotic stress signaling may involve membrane kinases and channels, and some TRP channels are osmosensitive. Hypertonic extracellular (EC) or hypo-osmotic IC media dose-dependently inhibits endogenous and v over-expressed TRPM7 currents in HEK-293 cells. Inhibition is not from capacitance or 2 ionic strength changes, and is independent of f-actin, cAMP, pH, Mg-ATP or Mg +. Countering osmotic pressure with pressure through the patch-pipette cancels hypertonic inhibition of TRPM7. Hypotonic EC application rapidly increases over-expressed 2 TRPM7 currents suppressed with IC Mg + (3 mM), Mg-ATP (4 mM), hypo-osmolar IC media or a-kinase deletion, but does not affect endogenous TRPM7. Hyper-osmotic IC media also facilitates TRPM7. It was found TRPM7 is not involved in osmotic stress­ 2 induced volume regulation. Possibly IC Mg + fluctuations or mechanosensitivity explain TRPM7osmosensitivity. TRPM7 channel osmosensitivity and TRPM7 kinase apparent binding factors of morphology and adhesion suggest new possible physiological functions of TRPM7 of initiating signaling cascades and regulating proteins involved in adjusting cellular morphology, membrane dynamics or growth. vi TABLE OF CONTENTS ACKNOWLEDGMENTS .iii-iv ABSTRACT v-vi LIST OF TABLES x LIST OF FIGURES xi-xii LIST OF ABBREVIATIONS xiii-xvi CHAPTER 1: INTRODUCTION 1-18 1.1. TRPM6 & TRPM7: Divalent Cation Channels with a-Kinases 1-5 1.2. Glutamate Effect on TRPM7 Pore and Divalent Cation Permeation 5-7 1.3. The Channel and the Kinase Involvement in TRPM7 Over-Expression- Induced-Changes ofCell Morphology 8-14 1.3.1. TRPM6, TRPM7 and TRPM6/7: Physiological Roles ofChannels 8-10 1.3.2. TRPM6 and TRPM7: Physiological Roles ofthe a-kinase 10-14 104. TRPM6 and TRPM7 Osmosensitivity 14-18 1.5. Synopsis ofObjectives 18 CHAPTER 2: MOLECULAR CHARACTERISTICS 19-36 2.1. Transient Receptor Potential (TRP) Channels 19-25 2.1.1. Transient Receptor Potential (TRP) Channels 19-22 2.1.2. Melastatin-like Transient Receptor (TRPM) Channels 23-25 2.2. TRPM6 and TRPM7 Primary Structure 25-36 2.2.1. PROSCAN Putative Functional Motifs 25-27 2.2.2. Amino-terminal Region ofTRPM6 and TRPM7 28-29 Vll 2.2.3. Putative Transmembrane Region ofTRPM6 and TRPM7 .29-33 2.2.4. Carboxyl-terminal Region ofTRPM6 and TRPM7 .33-36 CHAPTER 3: MATERIALS AND METHODS 37-51 3.1. Cells 37-39 3.2. Solutions 39-40 3.3. Patch-Clamp Electrophysiology .41-44 3.3.1. Whole-cell Patch-clamp Method .41-43 3.3.2. Solution Application 44 3.3.3. "Balloon" Patch Volume Application .44 3.4. Fura-2: Fluorescent Indicator of Calcium and Cell Volume .45-48 3.5. Cell Vitality Staining and Microscopy .49 CHAPTER 4: RESULTS 50-78 4.1. TRPM7 Cell Volume-Membrane Strain Sensitivity 50-56 4.1.1. Hypertonic-induced Inhibition ofTRPM7 and MagNuM 50-56 4.1.2. Hypotonic-induced facilitation ofTRPM7 56-58 4.1.3. Balloon Patch Counteracts Hypertonic Inhibition ofTRPM7 59-60 4.1.4. Cytochalasin D, pH nor cAMP Alter Osmotic Effects on TRPM7 60-61 4.1.5. Intracellular Osmotic Stress Effects on TRPM7 Currents 61-63 4.] .6. Truncated TRPM7 without the Kinase Domain is Hyper-sensitive to Osmotic Stressors 64-65 4.1.7. TRPM6/7 Osmotic Sensitivities 65-67 Vlll 4.1.8. TRPM7 Over-Expression Does Not Alter Cell Volume-Responses to Osmolarity 67-68 4.2. TRPM7 and TRPM6/7 Over-Expression Effects 69-77 4.2.1. TRPM7 And TRPM6/7 Over-Expression Effects 69-71 2 2 4.2.2. Ca + and Mg + Media Levels on TRPM7 Over-Expression Effects 71-73 4.2.3. Kinase Mutations Effects on Cells Adhesion and Morphology 73-74 4.2.4. Annexin-I Co-Expression on TRPM7 Over-Expression Effects 74-76 4.2.5.
Load more

Recommended publications
  • Genes in Eyecare Geneseyedoc 3 W.M
    Genes in Eyecare geneseyedoc 3 W.M. Lyle and T.D. Williams 15 Mar 04 This information has been gathered from several sources; however, the principal source is V. A. McKusick’s Mendelian Inheritance in Man on CD-ROM. Baltimore, Johns Hopkins University Press, 1998. Other sources include McKusick’s, Mendelian Inheritance in Man. Catalogs of Human Genes and Genetic Disorders. Baltimore. Johns Hopkins University Press 1998 (12th edition). http://www.ncbi.nlm.nih.gov/Omim See also S.P.Daiger, L.S. Sullivan, and B.J.F. Rossiter Ret Net http://www.sph.uth.tmc.edu/Retnet disease.htm/. Also E.I. Traboulsi’s, Genetic Diseases of the Eye, New York, Oxford University Press, 1998. And Genetics in Primary Eyecare and Clinical Medicine by M.R. Seashore and R.S.Wappner, Appleton and Lange 1996. M. Ridley’s book Genome published in 2000 by Perennial provides additional information. Ridley estimates that we have 60,000 to 80,000 genes. See also R.M. Henig’s book The Monk in the Garden: The Lost and Found Genius of Gregor Mendel, published by Houghton Mifflin in 2001 which tells about the Father of Genetics. The 3rd edition of F. H. Roy’s book Ocular Syndromes and Systemic Diseases published by Lippincott Williams & Wilkins in 2002 facilitates differential diagnosis. Additional information is provided in D. Pavan-Langston’s Manual of Ocular Diagnosis and Therapy (5th edition) published by Lippincott Williams & Wilkins in 2002. M.A. Foote wrote Basic Human Genetics for Medical Writers in the AMWA Journal 2002;17:7-17. A compilation such as this might suggest that one gene = one disease.
    [Show full text]
  • Teleomorph Hypocrea Jecorina)
    Downloaded from orbit.dtu.dk on: Dec 20, 2017 Unraveling the Secondary Metabolism of the Biotechnological Important Filamentous Fungus Trichoderma reesei ( Teleomorph Hypocrea jecorina) Jørgensen, Mikael Skaanning; Larsen, Thomas Ostenfeld; Mortensen, Uffe Hasbro; Aubert, Dominique Publication date: 2013 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Jørgensen, M. S., Larsen, T. O., Mortensen, U. H., & Aubert, D. (2013). Unraveling the Secondary Metabolism of the Biotechnological Important Filamentous Fungus Trichoderma reesei ( Teleomorph Hypocrea jecorina). Kgs. Lyngby: Technical University of Denmark (DTU). General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Unraveling the Secondary Metabolism of the Biotechnological Important Filamentous Fungus Trichoderma reesei (Teleomorph Hypocrea jecorina) MJ-T-030 Mikael Skaanning Jørgensen Ph.D. Thesis November 2012 MJ-T-020 MJ-T-032 28 8 20 27 5 28 1 30 17 8 24 25 9 25 31 13 15 26 16 4 24 12 32 18 11 3 14 13 19 10 5 7 2 9 2521 22 23 Unraveling the Secondary Metabolism of the Biotechnological Important Filamentous Fungus Trichoderma reesei (Teleomorph Hypocrea jecorina) Mikael Skaanning Jørgensen Ph.D.
    [Show full text]
  • The Inducers 1,3-Diaminopropane and Spermidine Cause The
    JOURNAL OF PROTEOMICS 85 (2013) 129– 159 Available online at www.sciencedirect.com www.elsevier.com/locate/jprot The inducers 1,3-diaminopropane and spermidine cause the reprogramming of metabolism in Penicillium chrysogenum, leading to multiple vesicles and penicillin overproduction Carlos García-Estradaa,⁎, Carlos Barreiroa, Mohammad-Saeid Jamia, Jorge Martín-Gonzáleza, Juan-Francisco Martínb,⁎⁎ aINBIOTEC, Instituto de Biotecnología de León, Avda. Real no. 1, Parque Científico de León, 24006 León, Spain bÁrea de Microbiología, Departamento de Biología Molecular, Universidad de León, Campus de Vegazana s/n; 24071 León, Spain ARTICLE INFO ABSTRACT Article history: In this article we studied the differential protein abundance of Penicillium chrysogenum in Received 21 February 2013 response to either 1,3-diaminopropane (1,3-DAP) or spermidine, which behave as inducers Accepted 15 April 2013 of the penicillin production process. Proteins were resolved in 2-DE gels and identified by Available online 30 April 2013 tandem MS spectrometry. Both inducers produced largely identical changes in the proteome, suggesting that they may be interconverted and act by the same mechanism. Keywords: The addition of either 1,3-DAP or spermidine led to the overrepresentation of the last 1,3-diaminopropane enzyme of the penicillin pathway, isopenicillin N acyltransferase (IAT). A modified form of Spermidine the IAT protein was newly detected in the polyamine-supplemented cultures. Both Penicillin inducers produced a rearrangement of the proteome resulting in an overrepresentation of Penicillium chrysogenum enzymes involved in the biosynthesis of valine and other precursors (e.g. coenzyme A) of Vesicles penicillin. Interestingly, two enzymes of the homogentisate pathway involved in the degradation of phenylacetic acid (a well-known precursor of benzylpenicillin) were reduced following the addition of either of these two inducers, allowing an increase of the phenylacetic acid availability.
    [Show full text]
  • Structural Basis for the Activation of Phenylalanine in the Non-Ribosomal Biosynthesis of Gramicidin S
    The EMBO Journal Vol.16 No.14 pp.4174–4183, 1997 Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S Elena Conti1,2, Torsten Stachelhaus3, the peptide product. Each amino acid is activated by Mohamed A.Marahiel3 and Peter Brick1,4 adenylation of its carboxylate group with ATP and then transferred to the thiol group of an enzyme-bound phospho- 1Biophysics Section, Blackett Laboratory, Imperial College, pantetheine cofactor for possible modification and the 3 London SW7 2BZ, UK and Biochemie/Fachbereich Chemie, elongation reaction (Stachelhaus and Marahiel, 1995a; Philipps-Universita¨t Marburg, D-35032 Marburg, Germany Kleinkauf and von Do¨hren, 1996). 2 Present address: Laboratory of Molecular Biophysics, The cloning and sequencing of several peptide synthe- Rockefeller University, New York, NY10021, USA tase genes have revealed a conserved and ordered modular 4Corresponding author organization. Each module encodes a functional building unit containing ~1000 amino acids, which specifically The non-ribosomal synthesis of the cyclic peptide recognizes a single amino acid. Within such a protein antibiotic gramicidin S is accomplished by two large template-directed peptide biosynthesis, the occurrence and multifunctional enzymes, the peptide synthetases 1 and specific order of the modules in the genomic DNA dictate 2. The enzyme complex contains five conserved subunits the number and sequence of the amino acids to be of ~60 kDa which carry out ATP-dependent activation incorporated into the resulting oligopeptide. The modular of specific amino acids and share extensive regions of arrangement of peptide synthetases closely parallels the sequence similarity with adenylating enzymes such multienzyme complexes responsible for the biogenesis of as firefly luciferases and acyl-CoA ligases.
    [Show full text]
  • Complete Thesis
    University of Groningen Nonribosomal peptide synthetases Zwahlen, Reto Daniel IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2018 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Zwahlen, R. D. (2018). Nonribosomal peptide synthetases: Engineering, characterization and biotechnological potential. University of Groningen. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 04-10-2021 Nonribosomal peptide synthetases: Engineering, characterization and biotechnological potential Academic Thesis, University of Groningen, the Netherlands ISBN: 978-94-034-0674-9 978-94-034-0673-2 (e-book) Printing: Eikon + Cover: Reto D.
    [Show full text]
  • Three Conserved Glycine Residues in Valine Activation of Gramicidin S Synthetase 2 from Bacillus Brevis1
    J. Biochem. 117, 276-282 (1995) Three Conserved Glycine Residues in Valine Activation of Gramicidin S Synthetase 2 from Bacillus brevis1 Masaki Saito, Kazuko Hori, Toshitsugu Kurotsu, Masayuki Kanda, and Yoshitaka Saito Department of Biochemistry, Hyogo College of Medicine, Mukogawa-cho, Nishinomiya, Hyogo 663 Received for publication, July 27, 1994 The translated product from the gene fragment containing the second and third domains of gramicidin S synthetase 2 was purified to an essentially homogeneous state. It showed valine and ornithine-activating activity and the second domain was proved to be the valine-activating domain. Three mutant genes from Bacillus brevis Nagano, BI-3, E-4, and E-5 strains, which encode defective valine-activating domains of gramicidin S synthetase 2, were sequenced. By comparison with the wild-type gene, single point mutations of guanine to adenine were found at the three conserved glycine codons; the 5303rd guanine in BI-3, the 5378th guanine in E-4, and the 4967th guanine in E-5, which corresponded to codon changes of the 1768th glycine to glutamic acid and the 1793rd and the 1656th glycine to aspartic acid. Loss of valine-adenylation activity by mutation at the 1656th glycine proved the direct participation of the TSGT/STGXPKG motif in the adenylation reaction, and suggests that this glycine residue with the conserved lysine residue of the motif forms the phosphate-binding loop for ATP-binding. The 1793rd glycine is a member of the YGXTE motif which was also conserved among adenylate-forming enzymes except acetyl-CoA synthetases. The 1768th glycine residue appears to maintain the conformation of the active site for aminoacyl adenylation since this residue is retained among the adenylate-forming enzymes, though flanking regions are not conserved.
    [Show full text]
  • Omics Approaches Applied to Penicillium Chrysogenum and Penicillin Production: Revealing the Secrets of Improved Productivity
    G C A T T A C G G C A T genes Review Omics Approaches Applied to Penicillium chrysogenum and Penicillin Production: Revealing the Secrets of Improved Productivity Carlos García-Estrada 1,2,*, Juan F. Martín 3, Laura Cueto 1 and Carlos Barreiro 1,4 1 INBIOTEC (Instituto de Biotecnología de León). Avda. Real 1—Parque Científico de León, 24006 León, Spain; [email protected] (L.C.); [email protected] (C.B.) 2 Departamento de Ciencias Biomédicas, Universidad de León, Campus de Vegazana s/n, 24071 León, Spain 3 Área de Microbiología, Departamento de Biología Molecular, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain; [email protected] 4 Departamento de Biología Molecular, Universidad de León, Campus de Ponferrada, Avda. Astorga s/n, 24401 Ponferrada, Spain * Correspondence: [email protected] or [email protected]; Tel.: +34-987210308 Received: 22 April 2020; Accepted: 24 June 2020; Published: 26 June 2020 Abstract: Penicillin biosynthesis by Penicillium chrysogenum is one of the best-characterized biological processes from the genetic, molecular, biochemical, and subcellular points of view. Several omics studies have been carried out in this filamentous fungus during the last decade, which have contributed to gathering a deep knowledge about the molecular mechanisms underlying improved productivity in industrial strains. The information provided by these studies is extremely useful for enhancing the production of penicillin or other bioactive secondary metabolites by means of Biotechnology or Synthetic Biology. Keywords: penicillin; Penicillium chrysogenum; omics; beta-lactam antibiotics 1. Introduction There are few examples of industrial microbial processes as deeply characterized as penicillin production by the filamentous fungus Penicillium chrysogenum.
    [Show full text]
  • Structural Studies of Natural Product Biosynthetic Proteins Craig a Townsend
    Review 721 Structural studies of natural product biosynthetic proteins Craig A Townsend The first high-resolution structures of key proteins involved in the Introduction biosynthesis of several natural product classes are now appear- X-ray qstallography has dramatically advanced our under- ing. In some cases, they have resulted in a significantly improved standing of protein structure and the macromolecular asso- mechanistic understanding of the often complex processes ciation of proteins with IlKA, RNA, drugs, cofactors and catalyzed by these enzymes, and they have also opened the way other proteins. Progress has, however, been considerably for more rational efforts to modify the products made. slower in obtaining detailed insights into the enzymes that catalyze natural product biosynthesis. ‘I’he reasons for this Address: Department of Chemistry, The Johns Hopkins University, Baltimore, MD 21218, USA. are several; in particular the enzymes concerned have modest, even poor, kinetic properties, they are available in Correspondence: Craig A Townsend only minute amounts from wild-type organisms and they E-mail: [email protected] are often not very stable. As a consequence, they have proved difficult to detect, isolate and purify. Efforts towards Chemistry & Biology October 1997, 4:721-730 http://biomednet.com/elecref/1074552100400721 this end have been successful in the last decade, ho\vever, and now a number of genes encoding synthetic enzymes for C Current Biology Ltd ISSN 1074-5521 natural products of interest have been cloned and recombi- nant enzymes have been made available for thorough char- acterization. Given the commercial importance of natural products, the associated enzymes hai,e become targets for manipulation by chemical modification, muragenesis and combinatorial methods, not onI\; to improve the production of these high-value materials but also, through deeper mechanistic understanding, to achieve the efficient synthe- sis of new chemical entities.
    [Show full text]
  • University of Groningen Genome Sequencing and Analysis Of
    University of Groningen Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum van den Berg, Marco A.; Albang, Richard; Albermann, Kaj; Badger, Jonathan H.; Daran, Jean-Marc; Driessen, Arnold; Garcia-Estrada, Carlos; Fedorova, Natalie D.; Harris, Diana M.; Heijne, Wilbert H. M. Published in: Nature Biotechnology DOI: 10.1038/nbt.1498 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2008 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): van den Berg, M. A., Albang, R., Albermann, K., Badger, J. H., Daran, J-M., Driessen, A. J. M., ... Bovenberg, R. A. L. (2008). Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum. Nature Biotechnology, 26(10), 1161-1168. DOI: 10.1038/nbt.1498 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
    [Show full text]
  • Saccharomyces Cerevisiae As Host for the Recombinant Production of Polyketides and Nonribosomal Peptides Anna Tippelt and Markus Nett*
    Tippelt and Nett Microb Cell Fact (2021) 20:161 https://doi.org/10.1186/s12934-021-01650-y Microbial Cell Factories REVIEW Open Access Saccharomyces cerevisiae as host for the recombinant production of polyketides and nonribosomal peptides Anna Tippelt and Markus Nett* Abstract As a robust, fast growing and genetically tractable organism, the budding yeast Saccharomyces cerevisiae is one of the most widely used hosts in biotechnology. Its applications range from the manufacturing of vaccines and hor- mones to bulk chemicals and biofuels. In recent years, major eforts have been undertaken to expand this portfolio to include structurally complex natural products, such as polyketides and nonribosomally synthesized peptides. These compounds often have useful pharmacological properties, which make them valuable drugs for the treatment of infectious diseases, cancer, or autoimmune disorders. In nature, polyketides and nonribosomal peptides are gener- ated by consecutive condensation reactions of short chain acyl-CoAs or amino acids, respectively, with the substrates and reaction intermediates being bound to large, multidomain enzymes. For the reconstitution of these multistep catalytic processes, the enzymatic assembly lines need to be functionally expressed and the required substrates must be supplied in reasonable quantities. Furthermore, the production hosts need to be protected from the toxicity of the biosynthetic products. In this review, we will summarize and evaluate the status quo regarding the heterologous production of polyketides
    [Show full text]
  • EUROPEAN PATENT OFFICE, VIENNA Thousand Oaks, CA 91320 (US) SUB-OFFICE
    Europäisches Patentamt *EP001033405A2* (19) European Patent Office Office européen des brevets (11) EP 1 033 405 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.7: C12N 15/29, C12N 15/82, 06.09.2000 Bulletin 2000/36 C07K 14/415, C12Q 1/68, A01H 5/00 (21) Application number: 00301439.6 (22) Date of filing: 25.02.2000 (84) Designated Contracting States: • Brover, Vyacheslav AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU Calabasas, CA 91302 (US) MC NL PT SE • Chen, Xianfeng Designated Extension States: Los Angeles, CA 90025 (US) AL LT LV MK RO SI • Subramanian, Gopalakrishnan Moorpark, CA 93021 (US) (30) Priority: 25.02.1999 US 121825 P • Troukhan, Maxim E. 27.07.1999 US 145918 P South Pasadena, CA 91030 (US) 28.07.1999 US 145951 P • Zheng, Liansheng 02.08.1999 US 146388 P Creve Coeur, MO 63141 (US) 02.08.1999 US 146389 P • Dumas, J. 02.08.1999 US 146386 P , (US) 03.08.1999 US 147038 P 04.08.1999 US 147302 P (74) Representative: 04.08.1999 US 147204 P Bannerman, David Gardner et al More priorities on the following pages Withers & Rogers, Goldings House, (83) Declaration under Rule 28(4) EPC (expert 2 Hays Lane solution) London SE1 2HW (GB) (71) Applicant: Ceres Incorporated Remarks: Malibu, CA 90265 (US) THE COMPLETE DOCUMENT INCLUDING REFERENCE TABLES AND THE SEQUENCE (72) Inventors: LISTING IS AVAILABLE ON CD-ROM FROM THE • Alexandrov, Nickolai EUROPEAN PATENT OFFICE, VIENNA Thousand Oaks, CA 91320 (US) SUB-OFFICE.
    [Show full text]
  • A-Aminoadipyl-Cysteinyl-Valine Synthetases in J8-Lactam Producing Organisms
    VOL. 53 NO. 10, OCT.2000 THE JOURNAL OF ANTIBIOTICS pp.1008 - 1021 REVIEW ARTICLE a-Aminoadipyl-cysteinyl-valine Synthetases in j8-Lactam Producing Organisms From Abraham's Discoveries to Novel Concepts of Non-Ribosomal Peptide Synthesis Juan F. Martin Area of Microbiology, Faculty of Biology, University of Leon, 2407 1 Leon, and Institute of Biotechnology, INBIOTEC, Science Park of Leon, Avda. del Real, n° 1, 24006 Leon, Spain (Received for publication July 27, 2000) The tripeptide 5-(L-a-aminoadipyl)-L-cysteinyl-D-valine (ACV) was discovered by Arnstein and Morris in Penicillium chrysogenum and Abraham and coworkers in Acremoniumchrysogenum.Other analogous tripeptides and tetrapeptides werelater reported in these and other /3-lactam producing fungi and actinomycetes. The ACVtripeptide is synthesized by a large non-ribosomal peptide synthetase named ACVsynthetase encoded by the 1 1 kb pcbAB gene. This gene has been cloned from the DNAof four different filamentous fungi and two actinomycetes. Detailed analysis of the multifunctional ACVsynthetases reveals that they consist of three repeated modules (initially named domains) involved in activation of the corresponding amino acids L-a-aminoadipic acid, L-cysteine and L-valine. Each module consists of functional domains for amino acid activation (A), condensation (C) and thiolation (T). In addition the last module of the ACVsynthetase contains an epimerization domain (E) involved in conversion of the L-valine to its D-isomer when the tripeptide is still enzyme linked. There are seven epimerization motifs conserved in the third module of all ACVsynthetases. In addition, there is an integrated thioesterase domain in the C-terminal region of the ACV synthetases that appears to be involved in the selective release of the tripeptide with the correct lld configuration.
    [Show full text]