DOCSLIB.ORG

Comprehensive Proteomic Study of Bacillus Amyloliquefaciens Strain FZB42 and Its Response to Plant Root Exudates

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Comprehensive Proteomic Study of Bacillus Amyloliquefaciens Strain FZB42 and Its Response to Plant Root Exudates Comprehensive proteomic study of Bacillus amyloliquefaciens strain FZB42 and its response to plant root exudates Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) im Fach Biologie eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät I der Humboldt-Univesität zu Berlin von M. Sc. Kinga Kierul, Geburtsname Szczypinska Präsident der Humboldt-Universität zu Berlin Prof. Dr. Jan-Hendrik Olbertz Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I Prof. Dr. Andreas Herrmann Gutachter: 1. Prof. Dr. Rainer Borriss 2. Prof. Dr. Thomas Schweder 3. Prof. Dr. Thomas Eitinger Tag der mündlichen Prüfung: 21.08.12 SUMMARY ...................................................................................................................................IX 1 INTRODUCTION ................................................................................................................... 1 1.1 PLANT-MICROBE INTERACTIONS ............................................................................................... 1 1.2 PLANT-ROOT COLONIZATION .................................................................................................... 2 1.3 PLANT-DERIVED COMPOUNDS IN THE SOIL AND THEIR FUNCTION ........................................... 2 1.4 MECHANISMS OF EXUDATION ................................................................................................... 3 1.5 PLANT ASSOCIATED BACTERIA AND THEIR USE IN AGRICULTURE ............................................ 4 1.6 WHAT MAKES B. AMYLOLIQUEFACIENS FZB42 A GOOD PGPR ................................................. 5 1.7 PROTEOME ANALYSIS ............................................................................................................... 6 1.8 GENE REGULATION IN BACILLUS .............................................................................................. 7 1.8.1 Transition state regulators ................................................................................................ 8 1.8.1.1 DegS-DegU two-component regulation system ........................................................................................... 8 1.8.1.2 AbrB global regulator ................................................................................................................................... 9 1.8.1.3 Hpr global regulator ................................................................................................................................... 10 1.8.2 Sigma factors ................................................................................................................... 10 1.8.2.1 Primary function sigma factors .................................................................................................................. 12 1.8.2.2 Alternative sigma factors ............................................................................................................................ 12 1.8.2.2.1 Flagellar sigma factors .......................................................................................................................... 12 1.8.2.2.2 Extracytoplasmic function (ECF) sigma factors ................................................................................... 13 1.8.2.2.3 Heat shock sigma factors ...................................................................................................................... 14 1.8.2.3 Sigma-factors involved in sporulation ........................................................................................................ 15 1.9 AIMS OF THE STUDY ................................................................................................................ 15 2 RESULTS .............................................................................................................................. 17 2.1 GROWTH CONDITIONS OF BACILLUS AMYLOLIQUEFACIENS FZB42 STRAIN FOR THE ANALYSIS OF THE EXTRACELLULAR PROTEOME ................................................................................................. 17 2.2 THE SECRETOME OF B. AMYLOLIQUEFACIENS FZB42 PREDICTED ACCORDING TO PRESENCE OF SIGNAL PEPTIDES ............................................................................................................................... 18 2.3 REFERENCE MAP OF THE SECRETOME OF B. AMYLOLIQUEFACIENS ......................................... 19 2.4 COMPARISON OF B. AMYLOLIQUEFACIENS EXTRACELLULAR PROTEOMES AT TRANSITION AND STATIONARY PHASES ......................................................................................................................... 24 2.5 ANALYSIS OF THE B. AMYLOLIQUEFACIENS SECRETOME IN RESPONSE TO MAIZE ROOT EXUDATES .......................................................................................................................................... 28 2.5.1 Differentially secreted proteins at transition phase ........................................................ 28 2.5.2 Differentially secreted proteins at stationary phase ........................................................ 32 2.6 DETERMINATION OF INVOLVEMENT OF SIGMA FACTORS AND GLOBAL REGULATORS IN RESPONSE TO ROOT EXUDATES – EXTRACELLULAR PROTEOME ANALYSIS ....................................... 35 2.6.1 Transition state regulators .............................................................................................. 36 2.6.1.1 The ∆degU strain ........................................................................................................................................ 36 2.6.1.2 The ∆abrB strain......................................................................................................................................... 38 2.6.2 Heat shock sigma factor ∆sigB ........................................................................................ 40 2.6.3 ECF sigma factors SigM, SigV and SigX ........................................................................ 40 2.7 TWO DIMENSIONAL MAP OF CYTOSOLIC PROTEINS OF B. AMYLOLIQUEFACIENS FZB42 ......... 43 2.7.1 Map of the theoretical proteome of B. amyloliquefaciens ............................................... 43 2.7.2 Reference map of cytosolic proteins of cells grown in standard medium at transition phase 44 2.7.2.1 Functional classification and localization of the identified proteins ........................................................... 45 III 2.7.2.2 Codon adaptation index ............................................................................................................................. 46 2.7.2.3 Grand average of hydropathy ..................................................................................................................... 47 2.7.3 Reference map of cytosolic proteins of cells grown in minimal medium at transition phase 48 2.7.4 Metabolic pathways of the B. amyloliquefaciens FZB42 ................................................. 50 2.7.4.1 Glycolysis .................................................................................................................................................. 50 2.7.4.2 Nucleotide metabolism .............................................................................................................................. 50 2.7.4.3 Peptidoglycan precursor assembly ............................................................................................................. 50 2.7.4.4 Secondary metabolites synthesis ................................................................................................................ 51 2.7.4.5 Stress adaptation proteins .......................................................................................................................... 51 2.7.5 Differences in expression of proteins caused by growth of B. amyloliquefaciens FZB42 in different media at transition growth phase .................................................................................... 51 2.7.5.1 Carbohydrate degradation .......................................................................................................................... 52 2.7.5.2 Amino acid metabolism ............................................................................................................................. 52 2.7.5.3 Hypothetical and conserved hypothetical proteins ..................................................................................... 53 2.7.5.4 Coenzymes and prostetic groups metabolism ............................................................................................ 53 2.8 DIFFERENTIALLY EXPRESSED CYTOSOLIC PROTEINS OF B. AMYLOLIQUEFACIENS IN RESPONSE TO ROOT EXUDATES ........................................................................................................................... 56 2.9 THE ROLE OF SIGMA FACTORS AND GLOBAL REGULATORS IN RESPONSE TO ROOT EXUDATES 59 2.9.1 Transition state regulators............................................................................................... 60 2.9.1.1 The ∆degU strain ....................................................................................................................................... 60 2.9.1.2 The ∆abrB strain ........................................................................................................................................ 62 2.9.2 Heat shock sigma factor sigB .......................................................................................... 64 2.9.3 ECF sigma factors SigM, SigV and SigX ........................................................................
Load more

Recommended publications
  • ATP-Citrate Lyase Has an Essential Role in Cytosolic Acetyl-Coa Production in Arabidopsis Beth Leann Fatland Iowa State University
    Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2002 ATP-citrate lyase has an essential role in cytosolic acetyl-CoA production in Arabidopsis Beth LeAnn Fatland Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Molecular Biology Commons, and the Plant Sciences Commons Recommended Citation Fatland, Beth LeAnn, "ATP-citrate lyase has an essential role in cytosolic acetyl-CoA production in Arabidopsis " (2002). Retrospective Theses and Dissertations. 1218. https://lib.dr.iastate.edu/rtd/1218 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. ATP-citrate lyase has an essential role in cytosolic acetyl-CoA production in Arabidopsis by Beth LeAnn Fatland A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Plant Physiology Program of Study Committee: Eve Syrkin Wurtele (Major Professor) James Colbert Harry Homer Basil Nikolau Martin Spalding Iowa State University Ames, Iowa 2002 UMI Number: 3158393 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted.
    [Show full text]
  • Table 2. Significant
    Table 2. Significant (Q < 0.05 and |d | > 0.5) transcripts from the meta-analysis Gene Chr Mb Gene Name Affy ProbeSet cDNA_IDs d HAP/LAP d HAP/LAP d d IS Average d Ztest P values Q-value Symbol ID (study #5) 1 2 STS B2m 2 122 beta-2 microglobulin 1452428_a_at AI848245 1.75334941 4 3.2 4 3.2316485 1.07398E-09 5.69E-08 Man2b1 8 84.4 mannosidase 2, alpha B1 1416340_a_at H4049B01 3.75722111 3.87309653 2.1 1.6 2.84852656 5.32443E-07 1.58E-05 1110032A03Rik 9 50.9 RIKEN cDNA 1110032A03 gene 1417211_a_at H4035E05 4 1.66015788 4 1.7 2.82772795 2.94266E-05 0.000527 NA 9 48.5 --- 1456111_at 3.43701477 1.85785922 4 2 2.8237185 9.97969E-08 3.48E-06 Scn4b 9 45.3 Sodium channel, type IV, beta 1434008_at AI844796 3.79536664 1.63774235 3.3 2.3 2.75319499 1.48057E-08 6.21E-07 polypeptide Gadd45gip1 8 84.1 RIKEN cDNA 2310040G17 gene 1417619_at 4 3.38875643 1.4 2 2.69163229 8.84279E-06 0.0001904 BC056474 15 12.1 Mus musculus cDNA clone 1424117_at H3030A06 3.95752801 2.42838452 1.9 2.2 2.62132809 1.3344E-08 5.66E-07 MGC:67360 IMAGE:6823629, complete cds NA 4 153 guanine nucleotide binding protein, 1454696_at -3.46081884 -4 -1.3 -1.6 -2.6026947 8.58458E-05 0.0012617 beta 1 Gnb1 4 153 guanine nucleotide binding protein, 1417432_a_at H3094D02 -3.13334396 -4 -1.6 -1.7 -2.5946297 1.04542E-05 0.0002202 beta 1 Gadd45gip1 8 84.1 RAD23a homolog (S.
    [Show full text]
  • Amino Acid Catabolism in Staphylococcus Aureus
    University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Fall 12-16-2016 Amino Acid Catabolism in Staphylococcus aureus Cortney Halsey University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Bacteriology Commons Recommended Citation Halsey, Cortney, "Amino Acid Catabolism in Staphylococcus aureus" (2016). Theses & Dissertations. 160. https://digitalcommons.unmc.edu/etd/160 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. Amino Acid Catabolism in Staphylococcus aureus By Cortney R. Halsey A DISSERTATION Presented to the Faculty of The Graduate College in the University of Nebraska In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy Pathology and Microbiology Under the Supervision of Dr. Paul D. Fey University of Nebraska Medical Center Omaha, Nebraska October 2016 Supervisory Committee: Kenneth Bayles, Ph.D. Steven Carson, Ph.D. Paul Dunman, Ph.D. Rakesh Singh, Ph.D. ii Acknowledgements First and foremost, I would like to thank my mentor, Dr. Paul Fey, whose patience and support over the past six years has been critical to my success as a graduate student. Paul has given me opportunities to grow as a scientist and person, for which I will be forever thankful. I would also like to thank Dr. Ken Bayles, Dr. Steven Carson, Dr. Paul Dunman, and Dr. Rakesh Singh for serving on my supervisory committee.
    [Show full text]
  • Two Pectate Lyases from Caldicellulosiruptor Bescii with the Same CALG Domain Had
    bioRxiv preprint doi: https://doi.org/10.1101/2020.01.16.910000; this version posted January 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Two pectate lyases from Caldicellulosiruptor bescii with the same CALG domain had 2 distinct properties on plant biomass degradation 3 Hamed I. Hamoudaa,b,c, Nasir Alia, Hang Sua,b, Jie Fenga, Ming Lua,†and Fu-Li Li a,† 4 a Shandong Provincial Key Laboratory of Energy Genetics, Key Laboratory of Biofuel, 5 Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 6 Qingdao 266101, China 7 b University of Chinese Academy of Sciences, Beijing 100039, China. 8 c Egyptian Petroleum Research Institute, Nasr City 11727, Cairo, Egypt. 9 †Corresponding authors: Dr. Ming Lu (E-mail: [email protected]) and Dr. Fu-Li Li 10 (E-mail: [email protected]), Qingdao Institute of Bioenergy and Bioprocess Technology, 11 Chinese Academy of Sciences, Qingdao 266101, China 12 13 Keywords: Caldicellulosiruptor, Pectin, Pectate lyase, Polysaccharide lyase, Concanavalin 14 A-like lectin/glucanase (CALG) 15 16 17 18 19 20 21 22 23 24 25 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.16.910000; this version posted January 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 26 Abstract 27 Pectin deconstruction is the initial step in breaking the recalcitrance of plant biomass by using 28 selected microorganisms that carry pectinolytic enzymes.
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • Transcriptomic and Proteomic Profiling Provides Insight Into
    BASIC RESEARCH www.jasn.org Transcriptomic and Proteomic Profiling Provides Insight into Mesangial Cell Function in IgA Nephropathy † † ‡ Peidi Liu,* Emelie Lassén,* Viji Nair, Celine C. Berthier, Miyuki Suguro, Carina Sihlbom,§ † | † Matthias Kretzler, Christer Betsholtz, ¶ Börje Haraldsson,* Wenjun Ju, Kerstin Ebefors,* and Jenny Nyström* *Department of Physiology, Institute of Neuroscience and Physiology, §Proteomics Core Facility at University of Gothenburg, University of Gothenburg, Gothenburg, Sweden; †Division of Nephrology, Department of Internal Medicine and Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan; ‡Division of Molecular Medicine, Aichi Cancer Center Research Institute, Nagoya, Japan; |Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden; and ¶Integrated Cardio Metabolic Centre, Karolinska Institutet Novum, Huddinge, Sweden ABSTRACT IgA nephropathy (IgAN), the most common GN worldwide, is characterized by circulating galactose-deficient IgA (gd-IgA) that forms immune complexes. The immune complexes are deposited in the glomerular mesangium, leading to inflammation and loss of renal function, but the complete pathophysiology of the disease is not understood. Using an integrated global transcriptomic and proteomic profiling approach, we investigated the role of the mesangium in the onset and progression of IgAN. Global gene expression was investigated by microarray analysis of the glomerular compartment of renal biopsy specimens from patients with IgAN (n=19) and controls (n=22). Using curated glomerular cell type–specific genes from the published literature, we found differential expression of a much higher percentage of mesangial cell–positive standard genes than podocyte-positive standard genes in IgAN. Principal coordinate analysis of expression data revealed clear separation of patient and control samples on the basis of mesangial but not podocyte cell–positive standard genes.
    [Show full text]
  • Characterization of the Metal Ion Binding Site in the Anti-Terminator Protein, Hutp, of Bacillus Subtilis Thirumananseri Kumarevel, Hiroshi Mizuno1 and Penmetcha K
    Published online September 28, 2005 5494–5502 Nucleic Acids Research, 2005, Vol. 33, No. 17 doi:10.1093/nar/gki868 Characterization of the metal ion binding site in the anti-terminator protein, HutP, of Bacillus subtilis Thirumananseri Kumarevel, Hiroshi Mizuno1 and Penmetcha K. R. Kumar* Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, Tsukuba, Ibaraki 305-8566, Japan and 1NEC Soft Ltd, 1-18-6, Shinkiba, Koto-ku, Tokyo 106-8608, Japan Downloaded from https://academic.oup.com/nar/article-abstract/33/17/5494/1067798 by guest on 06 March 2019 Received July 10, 2005; Revised August 22, 2005; Accepted September 9, 2005 PDB ID: 1WRN, 1WRO and 1WPT ABSTRACT terminator structure (+459 to +571 nt, Figure 1) in between the hutP and hutH genes, and regulates the transcription of the HutP is an RNA-binding protein that regulates the hut operon by an anti-termination mechanism (1–3). Several expression of the histidine utilization (hut) operon bacterial proteins that regulate anti-terminator/terminator in Bacillus subtilis, by binding to cis-acting regulatory structures have been described, including TRAP, PyrR, sequences on hut mRNA. It requires L-histidine and LacT, BglG, SacT and GlpP (4–10). One such protein is an Mg21 ion for binding to the specific sequence the hutP gene product, HutP, of B.subtilis. HutP is a 16.2 within the hut mRNA. In the present study, we show kDa protein that shares 60% sequence identity with that several divalent cations can mediate the HutP– the HutP proteins found in other Bacillus species, such as RNA interactions.
    [Show full text]
  • The Genome of an Industrial Workhorse
    NEWS AND VIEWS The genome of an industrial workhorse Dan Cullen Sequencing of the filamentous fungus Aspergillus niger offers new opportunities for the production of specialty chemicals and enzymes. Few microbes compare with the filamentous fungus Aspergillus niger in its ability to pro­ Environment CAT (2) H O H O + O duce prodigious amounts of useful chemicals 2 2 2 2 and enzymes. This fungus is the principal GOX (3) GLN (1) source of citric acid for food, beverages and D-glucono­ Glucose Gluconate Oxalate pharmaceuticals1 and of several important 1,5-lactone Citrate http://www.nature.com/naturebiotechnology http://www.nature.com/naturebiotechnology commercial enzymes, including glucoamy­ lase, which is widely used for the conversion of starch to food syrups and to fermentative Oxalate + acetate PEP feedstocks for ethanol production. Although OAH most of these fermentation processes are well cMDH (3) established, the underlying genetics are still cPYC (1) cACO (2) cIDH (1) poorly understood. In this issue, Pel et al.2 Pyruvate OAA MAL Citrate Isocitrate 2-ketoglutarate report the genome sequence of A. niger strain Cytosol CBS 513.88. The availability of this sequence OAT (1) CMC (2) mPYC (1) should provide invaluable aid toward improv­ Pyruvate OAA MAL Citrate Isocitrate 2-ketoglutarate ing the production of chemicals and enzymes PDH Nature Publishing Group Group Nature Publishing 7 in this organism. mMDH (1) mACO (2) mIDH (3) Pel et al. sequenced tiled bacterial artificial CS (3) 200 Acetyl-CoA TCA cycle © chromosomes representing the entire A. niger genome to produce a high-quality assembly of Mitochondrion 19 supercontigs with a combined length of 33.9 Mb.
    [Show full text]
  • A Mathematical Model of Glutathione Metabolism Michael C Reed*1, Rachel L Thomas1, Jovana Pavisic1,2, S Jill James3, Cornelia M Ulrich4 and H Frederik Nijhout2
    Theoretical Biology and Medical Modelling BioMed Central Research Open Access A mathematical model of glutathione metabolism Michael C Reed*1, Rachel L Thomas1, Jovana Pavisic1,2, S Jill James3, Cornelia M Ulrich4 and H Frederik Nijhout2 Address: 1Department of Mathematics, Duke University, Durham, NC 27708, USA, 2Department of Biology, Duke University, Durham, NC 27708, USA, 3Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AK 72205, USA and 4Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA Email: Michael C Reed* - [email protected]; Rachel L Thomas - [email protected]; Jovana Pavisic - [email protected]; S Jill James - [email protected]; Cornelia M Ulrich - [email protected]; H Frederik Nijhout - [email protected] * Corresponding author Published: 28 April 2008 Received: 27 November 2007 Accepted: 28 April 2008 Theoretical Biology and Medical Modelling 2008, 5:8 doi:10.1186/1742-4682-5-8 This article is available from: http://www.tbiomed.com/content/5/1/8 © 2008 Reed et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Glutathione (GSH) plays an important role in anti-oxidant defense and detoxification reactions. It is primarily synthesized in the liver by the transsulfuration pathway and exported to provide precursors for in situ GSH synthesis by other tissues. Deficits in glutathione have been implicated in aging and a host of diseases including Alzheimer's disease, Parkinson's disease, cardiovascular disease, cancer, Down syndrome and autism.
    [Show full text]
  • Structural Studies of Three Enzymes: Telomerase, the Methyltransferase Cobj and Pectate Lyase
    Structural studies of three enzymes: Telomerase, the methyltransferase CobJ and Pectate lyase Teng Teng To Thesis submitted to the University of London for the Degree of Doctor of Philosophy 1 Abstract This thesis investigates the structure and function of three enzymes of biotechnological and biomedical interest: telomerase from Caenorhabtidis elegans , pectate lyase from Bacillus subtilis and the methyltransferase CobJ from Rhodobacter capsulatus . Telomerase is a ribonucleoprotein found in all eukaryotes and its function is to maintain telomere length, sustain chromosome integrity and circumvent the end-replication problem. The protein requires two subunits to function, telomerase reverse transcriptase (TERT), the catalytic component, and an intrinsic RNA template (TR). The TR makes telomerase a unique reverse transcriptase using the template in the synthesis of short iterative sequences which cap the ends of telomeres. This work reports the successful cloning of a small and therefore potentially crystallisable TERT from C. elegans and expression trials of this catalytic component. Cobalamin (vitamin B 12 ) is an intricate small molecule belonging to a group of compounds called cyclic tetrapyrroles. Its biosynthesis is achieved through a complex pathway encompassing over thirty different enzyme-mediated reactions. Within this pathway there are seven methyltransferases which add eight S-adenosyl-methionine (SAM) derived methyl groups to the macrocycle. CobJ catalyses the methylation of C17 and ring contraction at C20, this reaction which exudes C20 from the tetrapyrrole ring is unprecedented in nature. In this thesis I report the crystallisation of native CobJ and refinement and validation of a high resolution structure along side co-crystallisation and soaking experiments aimed at capturing an enzyme-tetrapyrrole complex.
    [Show full text]
  • Supplementary Table 1. the List of 1,675 All Stages of AD-Related Upregulated Genes
    Supplementary Table 1. The list of 1,675 all stages of AD-related upregulated genes Gene ID Gene symbol Gene name 51146 A4GNT ALPHA-1,4-N-ACETYLGLUCOSAMINYLTRANSFERASE 9625 AATK APOPTOSIS-ASSOCIATED TYROSINE KINASE 19 ABCA1 ATP-BINDING CASSETTE, SUB-FAMILY A (ABC1), MEMBER 1 20 ABCA2 ATP-BINDING CASSETTE, SUB-FAMILY A (ABC1), MEMBER 2 10058 ABCB6 ATP-BINDING CASSETTE, SUB-FAMILY B (MDR/TAP), MEMBER 6 89845 ABCC10 ATP-BINDING CASSETTE, SUB-FAMILY C (CFTR/MRP), MEMBER 10 5826 ABCD4 ATP-BINDING CASSETTE, SUB-FAMILY D (ALD), MEMBER 4 25 ABL1 V-ABL ABELSON MURINE LEUKEMIA VIRAL ONCOGENE HOMOLOG 1 3983 ABLIM1 ACTIN BINDING LIM PROTEIN 1 30 ACAA1 ACETYL-COENZYME A ACYLTRANSFERASE 1 (PEROXISOMAL 3-OXOACYL-COENZYME A THIOLASE) 35 ACADS ACYL-COENZYME A DEHYDROGENASE, C-2 TO C-3 SHORT CHAIN 8310 ACOX3 ACYL-COENZYME A OXIDASE 3, PRISTANOYL 56 ACRV1 ACROSOMAL VESICLE PROTEIN 1 2180 ACSL1 ACYL-COA SYNTHETASE LONG-CHAIN FAMILY MEMBER 1 86 ACTL6A ACTIN-LIKE 6A 93973 ACTR8 ACTIN-RELATED PROTEIN 8 91 ACVR1B ACTIVIN A RECEPTOR, TYPE IB 8747 ADAM21 ADAM METALLOPEPTIDASE DOMAIN 21 27299 ADAMDEC1 ADAM-LIKE, DECYSIN 1 56999 ADAMTS9 ADAM METALLOPEPTIDASE WITH THROMBOSPONDIN TYPE 1 MOTIF, 9 105 ADARB2 ADENOSINE DEAMINASE, RNA-SPECIFIC, B2 (RED2 HOMOLOG RAT) 109 ADCY3 ADENYLATE CYCLASE 3 113 ADCY7 ADENYLATE CYCLASE 7 120 ADD3 ADDUCIN 3 (GAMMA) 125 ADH1C ALCOHOL DEHYDROGENASE 1A (CLASS I), ALPHA POLYPEPTIDE 9370 ADIPOQ ADIPONECTIN, C1Q AND COLLAGEN DOMAIN CONTAINING 165 AEBP1 AE BINDING PROTEIN 1 4299 AFF1 AF4/FMR2 FAMILY, MEMBER 1 173 AFM AFAMIN 79814 AGMAT AGMATINE
    [Show full text]
  • Chapter 2: Literature Review
    Chapter 2: Literature Review 2.1 A historical perspective on the study of glycine conjugation The urinary excretion of hippurate after ingestion of benzoate was first observed by Alexander Ure in 1841 (Ure, 1841). This credits Ure with the first discovery of a biotransformation reaction, a finding that started the whole field of drug metabolism research. However, interest in glycine conjugation faded significantly after this great discovery, probably because very few pharmaceuticals are metabolised by conjugation to glycine (Badenhorst et al., 2013, Knights et al., 2007). This explains why, now more than 170 years later, the significance of glycine conjugation in metabolism is still not clearly understood. As mentioned in Chapter 1, GLYAT conjugates several endogenous and xenobiotic organic acids to glycine. Acylglycines from endogenous sources include butyrylglycine, hexanoylglycine, and isovalerylglycine. The xenobiotic acylglycines include hippurate, salicylurate, and methylhippurate (Bartlett and Gompertz, 1974, Nandi et al., 1979, Schachter and Taggart, 1954, Mawal and Qureshi, 1994). It seems as though this unusual range of metabolites formed by GLYAT has contributed to the lack of understanding of the glycine conjugation pathway. Some historical perspective sheds light on this situation. Initial studies of glycine conjugation were similar to Ure’s original experiments. Benzoic acid was ingested by human or animal test subjects, followed by detection and quantification of hippurate in the urine. This led to several interesting observations such as the decreased synthesis of hippurate in individuals with schizophrenia and in hepatitis patients (Quastel and Wales, 1938, Probstein and Londe, 1940, Wong, 1945, Saltzman and Caraway, 1953). In 1953 Schachter and Taggart showed that the synthesis of hippurate from benzoate and glycine is dependent on benzoyl-CoA, a high-energy form of benzoate (Schachter and Taggart, 1953).
    [Show full text]