DOCSLIB.ORG

Advanced Studies of Blasticidin S Biosynthesis Redacted for Privacy Abstract Approved

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Advanced Studies of Blasticidin S Biosynthesis Redacted for Privacy Abstract Approved AN ABSTRACT OF THE THESIS OF Qibo Zhang for the degree of Doctor of Philosophy in Chemistry presented on November 3, 1998. Title: Advanced Studies of Blasticidin S Biosynthesis Redacted for Privacy Abstract approved: Blasticidin S (BS, 1) is a peptidyl nucleoside antibiotic produced by Streptomyces griseochromogenes. The biosynthesis of blasticidin S was investigated using a combined approach of whole-cell feeding and cell-free analysis. Fermentations of S. griseochromogenes supplemented with the arginine analogue argininic acid or the argininosuccinate synthase inhibitor 2-methylaspartic acid were found to produce a new metabolite, 58.Demethylblasticidin S (4) was produced and isolatedinlargequantitiesfromfermentationssupplementedwithmethionine hydroxamate, an inhibitor of methionine adenosyltransferase. When cytosinine and pyridoxal phosphate were incubated with cell-free extracts of S. griseochromogenes prepared in DA),2H NMR analysis of recovered cytosinine revealed deuterium enrichment at 1-1-4' and 11-2'. The results demonstrated the presence of a cytosinine:pyridoxal phosphate tautomerase activity that contributes to the net transamination at C-4' in the conversion of cytosylglucuronic acid to blasticidin 5, and its discovery supports the role of cytosinine as a biosynthetic intermediate. Cytosyl- 4'- ketoxylose (62). an analogue of 4'-keto CGA, was synthesized and labeled isotopically. Adding labeled 62 to S. griseochromogenes fermentationsresulted in labeled pentopyranine C (8), and this result supports the involvementof a 4'-keto intermediate in the conversion of CGA to cytosinine. Blasticidin S acetyltransferase activity was detected when BS and acetyl CoA were incubated with cell-free extracts of S. griseochromogenes. The enzyme was alsofound to acetylate demethylblasticidin S to produce acetyldemethylblasticidin S, which could then be converted to acetylblasticidin S when S-adenosyl-L-methioine (SAM) was added to cell-free extracts.These acetylated compounds are devoid of antibiotic activity and acetylation is proposed to be a detoxification mechanism. Additionally, cell-free extracts of S. griseochromogene.s, along with SAM, transformed leucyldemethylblasticidin S to leucylblasticidin S, which is then converted to I3S. Leucylblasticidin S also has very low antibiotic activity and these results suggested that formation of leucylblasticidin S is directly involved in the biosynthesis of BS as a self-resistance mechanism. A mechanism of resistance towards BS was also identified in Sfreptornyce,s lividans, a non-producer of BS. This organism produces a BS deaminase, converting BS to the inactive uracil analog. ©Copyright by Qibo Zhang November 3, 1998 All Rights Reserved ADVANCED STUDIES OF BLASTICIDIN S BIOSYNTHESIS by Qibo Zhang A Thesis Submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented November 3, 1998 Commencement June 1999 Doctor of Philosophy thesis of Qibo Zhang presented on November 3, 1998 APPROVED: Redacted for Privacy Major Professor, representing mistry Redacted for Privacy Chair of Dep ent of Chemistry Redacted for Privacy Dean of Gradto School I understand that my thesis will become part of the permanent collection of OregonState University libraries. My signature below authorizes release of my thesis to any reader upon request. Redacted for Privacy Qibo Zhang.uthor Acknowledgments I am forever indebted to Professors Steven J. Gould and T. Mark Zabriskie whose friendships, constant supports, encouragement and guidance ultimately made this work successful. Their timely advice and suggestions during my graduate study helped me in my professional development. I would like to express my gratitude to all members of the "Gould group" and Department of Chemistry at Oregon State University. In particular, Drs. Martha C. Cone and John R. Carney are highly appreciated for their helpful discussions and suggestions. Mr. Michael D. Jackson is also thanked for his suggestions and the critical reading of a draft of this thesis. Appreciation also goes to my current committee members for their help and suggestions. I regret that Professor James D. White resigned from my committee due to a time conflict for my defense; he is always thanked for his advice and encouragement over the past years. Finally, I gratefully acknowledge the undying patience and loving support of my wife. Her willingness to endure my lengthy stay in graduate school will not be forgotten. Table of Contents page Chapter 1. Introduction 1 Isolation and Structure 1 Biological Activity and Mechanism of Action of BS 2 Biological Transformation of BS and Applications in Molecular Biology 3 Related Nucleoside Antibiotics 4 Synthetic Efforts to BS 6 Previous Biosynthetic Studies on BS 7 Objectives and Significance of the Present Studies 14 References 18 Chapter 2. Stereochemistry of C-6' Decarboxylation 24 Introduction Results and Discussion 26 Synthesis of [5,6,6'-2H3]Glucose (22c) 26 Feeding [5.6,6'-2H3]Glucose 26 Summary 30 Experimental 31 General 31 2H NMR Conditions 32 HPLC Analysis 32 Maintenance of S. griseochromogenes 32 Seed and Production Medium 33 Synthesis of [5,6,6'-2H3]-D-Glucose (22c) 33 1 ,2-acetonide-a-D-glucurono-6.3-lactone (38) 33 1,2-acetonide-5 -keto-oc- D-glucurono-6,3 -lactone (39) 34 1,2-acetonide-a-D-[5.6,6'-2H3]glucofuranose (40) 34 [5,6,6'-2H3]-D-glucose (22c) 35 Feeding Protocol 35 Purification of PPNC (8c-d) 35 Purification of CGA (5b) 36 Table of Contents (Continued) page Formation of Triacetyl PPNC (41a-b) 36 References 37 Chapter 3. Feeding Metabolic Inhibitors and Purification of New Metabolites 38 Introduction 38 Results and Discussion 41 Feeding Inhibitors 41 Results of Inhibitor and Primary Precursor Feedings 43 Production and Purification of Large Quantities of DBS 46 Purification and Structure Elucidation of a New Cytosine Glycoside 47 Summary 52 Experimental 53 General 53 2-Fluorofumarate 53 HPLC Analysis 54 Culture Maintenance and Fermentation Conditions 54 Feeding Protocol 55 Isolation of New cytosine Glycoside 58 55 Purification of Demethylblasticidin S 56 References 58 Chapter IV. Enzymatic Studies Related to Cytosinine 60 Introduction 60 Results and Discussion 62 Coupling of Cytosinine and p-Arginine 62 Indentification of Cytosinine:Pyridoxal Phosphate Tautomerase 63 Summary 68 Experimental 69 General 69 Preparation of Cytosinine from Hydrolysis of Blasticidin S 69 Preparation of Cell-free Extracts 69 Table of Contents (Continued) page Incubation Conditions for the Coupling Reaction Assay 70 Standard Assay Work-up for Detection of the Tautomerase 70 Enzyme Incubation #1 71 Enzyme Incubation #2 71 Enzyme Incubation #3 72 References 73 Chapter V. Studies of 4'-Keto Intermediates 75 Introduction 75 Results and Discussion 77 Detection of CGA Oxidoreductase 77 Synthesis of 4'-Keto-CGA 77 Synthesis of Cytosyl- 4'- ketoxylose 81 Feeding Deuterium Labeled Cytosyl- 4'- ketoxylose 84 Summary 91 Experimental 92 General 92 Methyl Tetraacetyl Cytosylglucuronate(66) 92 Methyl 2',3',4' -O- Triacetyl Cytosylglucuronate(67) 93 Methyl N-B0C-2',3',4'-0-Triacetyl Cytosylglucuronate 93 Methyl N-BOC Cytosylglucuronate(68) 94 Methyl Acetonide N-BOC Cytosylglucuronate (69 &72) 94 Methyl 2',3'-Acetonide-N-B0C-4'-keto-cytosylglucuronate(70) 95 Methyl 4'-Keto-cytosylglucuronate(71) 95 Methyl 3',4'-Acetonide-2'-acetyl-N-B0C-cytosylglucuronate CGA (74) 96 Methyl 2',3'-Acetonide-4'-acetyl-N-B0C-cytosylglucuronate(73) 96 Alkylation by-product75 97 2',3'-Acetonide-N-B0C-Cytosylglucuronate(76) 97 Tetraacetyl D-Xylose(78) 98 Tetraacetyl Cytosylxylose(79) 98 2',3',4' -O- Triacetyl Cytosylxylose (80) 99 N -BOC- 2',3',4' -O- Triacetyl Cytosylxylose 100 N-BOC Cytosylxylose (81) 100 2',3'-Acetonide-N-B0C-cytosylxylose(82) 101 Isomerized Product from the TPAP Oxidation(85) 102 Table of Contents (Continued) page 2' .3 '-Acetonide-4'-acetyl -N-BOC -cytosylxylose(86) 102 4'- Acetyl- N- BOC- cytosylxylose(87) 102 4'-Acetyl-2',3'-0-4'-N-triB0C-cytosylxylose(88) 103 2',3'-0-4'-N-TriB0C-cytosylxylose(89) 103 a,13-Unsaturated ketone 90 104 2',3'-Acetonide-N-BOC cytosy1-4'-ketoxylose(83) 104 Cytosyl- 4'- ketoxylose(62)and its Dimer84 105 Deuterium Exchange 105 Feeding Deuterated Cytosy1-4'-keto-xylose(62) 106 experiment 1 106 diacetyl PPNC 106 experiment 2 106 References 108 Chapter VI. Self-resistance Mechanism in S. griseochromogenes 109 Introduction 109 Results and Discussion 113 Synthesis of N-Acetylblasticidin S and its Identification in the Fermentation Broth 113 Detection of N-Acetyltransferase Activity 114 N-Acetylation of Demethylblasticidin S 114 Determination of V,aI K,,, of the N-Acetyltransferase 117 N-Acetyltransferase Substrate Competition Experiment 118 Methylation of N-Acetyldemethylblasticidin S 119 Acetylation as a Detoxification process 120 The Biosynthetic Role of Leucylblasticidin S 121 Conclusion 124 Summary 126 Experimental 127 General 127 HPLC Analysis 127 Preparation of Cell-free Extracts 127 Bioassay 128 Acetyltransferase Assay Conditions 128 Kinetic Data Measurement 128 N-Acetyltransferase Substrate Competition Experiment 129 Table of Contents (Continued) page Assay for Methyltransferase Activity Using AcDBS 129 Diacetylblasticidin S (92) 129 N-Acetylblasticidin S (93) 130 N-Acetyldemethylblasticidin S 130 Leucyldemethylblasticidin S (95) 131 Isolation of Leucylblasticidin S 131 Enzymatic Conversion of LBS to BS 132 Assay for Methyltransferase Activity Using LDBS 132 References 134 Chapter VII. BS Resistance in S. lividans 136 Introduction 136 Results
Load more

Recommended publications
  • Identification of Antibiotics for Use in Selection of the Chytrid Fungi Batrachochytrium
    bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.184721; this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Identification of antibiotics for use in selection of the chytrid fungi Batrachochytrium 2 dendrobatidis and Batrachochytrium salamandrivorans 3 4 5 Kristyn A. Robinson, Mallory Dunn, Shane P. Hussey, Lillian K. Fritz-Laylin* 6 7 8 The University of Massachusetts Amherst, Department of Biology, Amherst, MA 01003 9 *Correspondence: [email protected] 10 11 12 ABSTRACT 13 14 Global amphibian populations are being decimated by chytridiomycosis, a deadly skin infection 15 caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans 16 (Bsal). Although ongoing efforts are attempting to limit the spread of these infections, targeted 17 treatments are necessary to manage the disease. Currently, no tools for genetic manipulation 18 are available to identify and test specific drug targets in these fungi. To facilitate the 19 development of genetic tools in Bd and Bsal, we have tested five commonly used antibiotics 20 with available resistance genes: Hygromycin, Blasticidin, Puromycin, Zeocin, and Neomycin. 21 We have identified effective concentrations of each for selection in both liquid culture and on 22 solid media. These concentrations are within the range of concentrations used for selecting 23 genetically modified cells from a variety of other eukaryotic species. 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.184721; this version posted July 2, 2020.
    [Show full text]
  • Discovery and Characterization of Blse, a Radical S- Adenosyl-L-Methionine Decarboxylase Involved in the Blasticidin S Biosynthetic Pathway
    Discovery and Characterization of BlsE, a Radical S- Adenosyl-L-methionine Decarboxylase Involved in the Blasticidin S Biosynthetic Pathway Jun Feng1, Jun Wu1, Nan Dai2, Shuangjun Lin1, H. Howard Xu3, Zixin Deng1, Xinyi He1* 1 State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China, 2 New England Biolabs, Inc., Research Department, Ipswich, Massachusetts, United States of America, 3 Department of Biological Sciences, California State University Los Angeles, Los Angeles, California, United States of America Abstract BlsE, a predicted radical S-adenosyl-L-methionine (SAM) protein, was anaerobically purified and reconstituted in vitro to study its function in the blasticidin S biosynthetic pathway. The putative role of BlsE was elucidated based on bioinformatics analysis, genetic inactivation and biochemical characterization. Biochemical results showed that BlsE is a SAM-dependent radical enzyme that utilizes cytosylglucuronic acid, the accumulated intermediate metabolite in blsE mutant, as substrate and catalyzes decarboxylation at the C5 position of the glucoside residue to yield cytosylarabinopyranose. Additionally, we report the purification and reconstitution of BlsE, characterization of its [4Fe–4S] cluster using UV-vis and electron paramagnetic resonance (EPR) spectroscopic analysis, and investigation of the ability of flavodoxin (Fld), flavodoxin 2+ reductase (Fpr) and NADPH to reduce the [4Fe–4S] cluster. Mutagenesis studies demonstrated that Cys31, Cys35, Cys38 in the C666C6MC motif and Gly73, Gly74, Glu75, Pro76 in the GGEP motif were crucial amino acids for BlsE activity while mutation of Met37 had little effect on its function. Our results indicate that BlsE represents a typical [4Fe–4S]-containing radical SAM enzyme and it catalyzes decarboxylation in blasticidin S biosynthesis.
    [Show full text]
  • Nucleoside Intermediates in Blasticidin S Biosynthesis Identified by the in Vivo Use of Enzyme Inhibitors
    Nucleoside intermediates in blasticidin S biosynthesis identified by the in vivo use of enzyme inhibitors STEVENJ. GOULD' Departments of Chemistry and of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331-4003, U.S.A. JINCAN GUOAND ANJAGEITMANN Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331-4003, U.S.A. KARL DFXESUS Department of Chemistry, Oregon State University, Corvallis, OR 97331-4003, U.S.A. Received March 9, 1993 This paper is dedicated to Professors Ian Sperzser and David B. MacLean STEVENJ. GOULD, JINCAN GUO,ANJA GEITMANN,and KARLDmus. Can. J. Chem. 72,6 (1994). Intermediates in the biosynthesis of blasticidin S and its nucleoside co-metabolites were detected by altering fermentation con- ditions. Inhibitors of specific types of biochemical reactions that were expected to be involved in blasticidin biosynthesis were fed to Streptomyces griseochromogenes, in some cases with the inclusion of large quantities of the primary precursors of blas- ticidin S. The types of reactions and inhibitors used were (1) transaminase (aminooxyacetic acid and 2-methylglutamate), (2) amidotransferase (azaserine and 6-diazo-5-0x0-L-norleucine), (3) arginine biosynthesis (arginine hydroxamate), and (4) meth- yltransferase (ethionine). These manipulations apparently distorted the pools of precursors and (or) intermediates, and led to sub- stantial accumulations of three known, previously minor, metabolites of S. griseochromogenes, cytosylglucuronic acid, pentopyranine C, and demethylblasticidin S, and of two new ones, pentopyranone and isoblasticidin S. New cytosyl metabolites were detected by HPLC with photodiode array detection. Fermentations to which arginine hydroxamate and cytosine had been added also produced three aberrant metabolites that were derived from pentopyranone and arginine hydroxamate.
    [Show full text]
  • Purification and Characterization of a Puromycin-Hydrolyzing Enzyme from Blasticidin S-Producing Streptomyces Morookaensis
    J. Biochem. 123, 247-252 (1998) Purification and Characterization of a Puromycin-Hydrolyzing Enzyme from Blasticidin S-Producing Streptomyces morookaensis Motohiro Nishimura,*,' Hiroaki Matsuo ,t Akiko Nakamura,* and Masanori Sugiyama•õ *Department of Chemical and Biological Engineering , Ube National College of Technology Tokiwadai, Ube, Y amaguchi 755; and •õInstitute of Pharmaceutical Sciences , Hiroshima University School of Medicine, Kasumi 1 -2-3 , Minami-ku, Hiroshima, Hiroshima 734 Received for publication, August 4, 1997 Blasticidin S-producing Streptomyces morookaensis JCM4673 produces an enzyme which inactivates puromycin (PM) by hydrolyzing an amide linkage between its aminonucleoside and O-methyl-L-tyrosine moieties [Nishimura et al. (1995) FEMS Microbiol . Lett. 132,95- 100]. In this study, we purified to homogeneity the enzyme from the cell-free extracts of S . morookaensis. The molecular weight of PM-hydrolyzing enzyme , estimated by SDS-PAGE and gel filtration, was 68 and 66 kDa, respectively, suggesting that this protein is monomeric. The PM-hydrolyzing activity was strongly inhibited by Zn2+ , Fe2+, Cu2+, Hg2+, and N-bromosuccinimide, but was stimulated by DTT. The optimum pH and temperature for PM-hydrolyzing activity were 8.0 and 45•Ž, respectively. Several L-aminoacyl-ƒÀ-naph thylamides were good substrates for the enzyme, suggesting that the PM-inactivating enzyme has an aminopeptidase activity. The N-terminal sequence of the first 14 amino acids (Val-Ser-Thr-Ala-Pro-Tyr-Gly-Ala-Trp-Gln-Ser-Pro-Ile-Asp) of the enzyme showed no significant homology with any published hydrolase sequences. Key words: aminoacyl-ƒÀ-naphthylamide, aminopeptidase, inactivating enzyme, puro mycin, Streptomyces morookaensis. The antibiotic-resistance mechanisms in actinomycetes of enzymatic inactivations of the drug in the resistant have been studied with respect to enzymatic inactivation microorganisms, Streptomyces morookaensis (20) and (1-4), resistance of target site (5-7), and membrane Aspergillus fumigatus (21), respectively.
    [Show full text]
  • Antibiotics That Bind to the a Site of the Large Ribosomal Subunit Can Induce Mrna Translocation
    Downloaded from rnajournal.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation DMITRI N. ERMOLENKO,1,5 PETER V. CORNISH,2 TAEKJIP HA,3 and HARRY F. NOLLER4 1Department of Biochemistry and Biophysics and Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642, USA 2Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA 3Department of Physics and Howard Hughes Medical Institute, University of Illinois, Urbana, Illinois 61801, USA 4Department of Molecular, Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064, USA ABSTRACT In the absence of elongation factor EF-G, ribosomes undergo spontaneous, thermally driven fluctuation between the pre- translocation (classical) and intermediate (hybrid) states of translocation. These fluctuations do not result in productive mRNA translocation. Extending previous findings that the antibiotic sparsomycin induces translocation, we identify additional peptidyl transferase inhibitors that trigger productive mRNA translocation. We find that antibiotics that bind the peptidyl transferase A site induce mRNA translocation, whereas those that do not occupy the A site fail to induce translocation. Using single-molecule FRET, we show that translocation-inducing antibiotics do not accelerate intersubunit rotation, but act solely by converting the intrinsic, thermally driven dynamics of the ribosome into translocation. Our results support the idea that the ribosome is a Brownian ratchet machine, whose intrinsic dynamics can be rectified into unidirectional translocation by ligand binding. Keywords: antibiotics; Brownian ratchet mechanism; mRNA translocation; ribosome INTRODUCTION kle et al.
    [Show full text]
  • Inhibition of Translation Termination by Small Molecules
    www.nature.com/scientificreports OPEN Inhibition of translation termination by small molecules targeting ribosomal release factors Xueliang Ge 1, Ana Oliveira1, Karin Hjort 2, Terese Bergfors 1, Hugo Gutierrez de Terán 1, Dan I. Andersson2, Suparna Sanyal 1 & Johan Åqvist 1* The bacterial ribosome is an important drug target for antibiotics that can inhibit diferent stages of protein synthesis. Among the various classes of compounds that impair translation there are, however, no known small-molecule inhibitors that specifcally target ribosomal release factors (RFs). The class I RFs are essential for correct termination of translation and they difer considerably between bacteria and eukaryotes, making them potential targets for inhibiting bacterial protein synthesis. We carried out virtual screening of a large compound library against 3D structures of free and ribosome-bound RFs in order to search for small molecules that could potentially inhibit termination by binding to the RFs. Here, we report identifcation of two such compounds which are found both to bind free RFs in solution and to inhibit peptide release on the ribosome, without afecting peptide bond formation. Bacterial ribosomes with their auxiliary translation factors are major targets for drug intervention by a wide range of diferent antibiotics. Such compounds are ofen derived from natural products and inhibit diferent phases of protein synthesis, including initiation, peptide chain elongation and mRNA-tRNA translocation, or severely increase the error rate of the translation process1. Many of these antibiotics bind directly to the ribosome and thereby interfere with specifc stages of protein synthesis. Tere are also examples, such as kirromycin and fusidic acid, which act by binding to elongation factors (EF-Tu and EF-G, respectively) and thereby blocking their release from the ribosome2.
    [Show full text]
  • Blasticidin-S Deaminase, a New Selection Marker for Genetic Transformation of the Diatom Phaeodactylum Tricornutum
    Blasticidin-S deaminase, a new selection marker for genetic transformation of the diatom Phaeodactylum tricornutum Jochen M. Buck1, Carolina Río Bártulos1, Ansgar Gruber1,2 and Peter G. Kroth1 1 Department of Biology, University of Konstanz, Konstanz, Germany 2 Institute of Parasitology, Biology Centre, Czech Academy of Sciences, České Budějovice, Czech Republic ABSTRACT Most genetic transformation protocols for the model diatom Phaeodactylum tricornutum rely on one of two available antibiotics as selection markers: Zeocin (a formulation of phleomycin D1) or nourseothricin. This limits the number of possible consecutive genetic transformations that can be performed. In order to expand the biotechnological possibilities for P. tricornutum, we searched for additional antibiotics and corresponding resistance genes that might be suitable for use with this diatom. Among the three different antibiotics tested in this study, blasticidin-S and tunicamycin turned out to be lethal to wild-type cells at low concentrations, while voriconazole had no detectable effect on P. tricornutum. Testing the respective resistance genes, we found that the blasticidin-S deaminase gene (bsr) effectively conferred resistance against blasticidin-S to P. tricornutum. Furthermore, we could show that expression of bsr did not lead to cross-resistances against Zeocin or nourseothricin, and that genetically transformed cell lines with resistance against Zeocin or nourseothricin were not resistant against blasticidin-S. In a proof of concept, we also successfully generated double resistant (against blasticidin-S and nourseothricin) P. tricornutum cell lines by co-delivering the bsr vector with a vector conferring nourseothricin resistance to wild-type cells. Submitted 3 July 2018 Accepted 8 October 2018 Published 14 November 2018 Subjects Genetics, Marine Biology, Microbiology, Molecular Biology, Plant Science Corresponding author Keywords Phaeodactylum tricornutum, Voriconazole, Genetic transformation, Genome editing, Jochen M.
    [Show full text]
  • Www .Alfa.Com
    Bio 2013-14 Alfa Aesar North America Alfa Aesar Korea Uni-Onward (International Sales Headquarters) 101-3701, Lotte Castle President 3F-2 93 Wenhau 1st Rd, Sec 1, 26 Parkridge Road O-Dong Linkou Shiang 244, Taipei County Ward Hill, MA 01835 USA 467, Gongduk-Dong, Mapo-Gu Taiwan Tel: 1-800-343-0660 or 1-978-521-6300 Seoul, 121-805, Korea Tel: 886-2-2600-0611 Fax: 1-978-521-6350 Tel: +82-2-3140-6000 Fax: 886-2-2600-0654 Email: [email protected] Fax: +82-2-3140-6002 Email: [email protected] Email: [email protected] Alfa Aesar United Kingdom Echo Chemical Co. Ltd Shore Road Alfa Aesar India 16, Gongyeh Rd, Lu-Chu Li Port of Heysham Industrial Park (Johnson Matthey Chemicals India Toufen, 351, Miaoli Heysham LA3 2XY Pvt. Ltd.) Taiwan England Kandlakoya Village Tel: 866-37-629988 Bio Chemicals for Life Tel: 0800-801812 or +44 (0)1524 850506 Medchal Mandal Email: [email protected] www.alfa.com Fax: +44 (0)1524 850608 R R District Email: [email protected] Hyderabad - 501401 Andhra Pradesh, India Including: Alfa Aesar Germany Tel: +91 40 6730 1234 Postbox 11 07 65 Fax: +91 40 6730 1230 Amino Acids and Derivatives 76057 Karlsruhe Email: [email protected] Buffers Germany Tel: 800 4566 4566 or Distributed By: Click Chemistry Reagents +49 (0)721 84007 280 Electrophoresis Reagents Fax: +49 (0)721 84007 300 Hydrus Chemical Inc. Email: [email protected] Uchikanda 3-Chome, Chiyoda-Ku Signal Transduction Reagents Tokyo 101-0047 Western Blot and ELISA Reagents Alfa Aesar France Japan 2 allée d’Oslo Tel: 03(3258)5031 ...and much more 67300 Schiltigheim Fax: 03(3258)6535 France Email: [email protected] Tel: 0800 03 51 47 or +33 (0)3 8862 2690 Fax: 0800 10 20 67 or OOO “REAKOR” +33 (0)3 8862 6864 Nagorny Proezd, 7 Email: [email protected] 117 105 Moscow Russia Alfa Aesar China Tel: +7 495 640 3427 Room 1509 Fax: +7 495 640 3427 ext 6 CBD International Building Email: [email protected] No.
    [Show full text]
  • Antibiotics and Translation
    Dissertation zur Erlangung des Doktorgrades der Fakultät fur Chemie und Pharmazie der Ludwig-Maximilians-Universität München Antibiotics and translation Overcoming emerging bacterial resistance to old and new antimicrobials Agata Lucyna Starosta aus Rzeszow, Polen 2011 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 (in der Fassung der Sechsten Änderungssatzung von 16. August 2010) von Herrn Prof. Dr. Roland Beckmann betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet. München, am 24.11.2011 Agata Lucyna Starosta Dissertation eingereicht am 24.11.2011 1. Gutachter: Herr Prof. Dr. Roland Beckmann 2. Gutachter: Herr Prof. Dr. Klaus Förstemann Mündliche Prüfung am 31.01.2012 2 Agata L. Starosta List of contents List of contents Acknowledgements .................................................................................................................................. 5 List of original publications .................................................................................................................... 6 Contribution report ................................................................................................................................. 8 Abbreviations ........................................................................................................................................ 10 Summary ...............................................................................................................................................
    [Show full text]
  • In the Course of an Investigation on the Fate of Blasticidin S,1) An
    VOL. XXVIII NO. 1 THE JOURNAL OF ANTIBIOTICS 7 ISOLATION AND PURIFICATION OF BLASTICIDIN S DEAMINASE FROM ASPERGILLUS TERREUS ISAMU YAMAGUCHI, HIROKO SHIBATA, HARUO SETO* and TOMOMASA MISATO The Institute of Physical and Chemical Research, Wako-shi, Saitama Pref., Japan `Institute of Applied Microbiology , University of Tokyo, Tokyo (Received for publication September 6, 1974) An enzyme catalyzing the deamination of the cytosine moiety of blasticidin S was extracted from a fungal strain that belongs to Aspergillus terreus. The enzyme was purified with ammonium sulfate fractionation, Sephadex G-100 column and DEAE cellulose column chromatography, followed by preparative polyacrylamide gel electro- phoresis. Blasticidin S deaminase could be separated easily from co-existing cytidine deaminase by DEAE column chromatography or gel electrophoresis, and preliminary study on the substrate specificity showed that this enzyme acts on blasticidin S derivatives, such as cytomycin and acetylblasticidin S, but not on cytosine, cytidine, purine bases or their nucleosides. Blasticidin S deaminase could be induced by the addition of blasticidin S to the culture, and sulfhydryl compounds, such as mercapto- ethanol, were effective in protecting the enzyme from inactivation. The homogeneity of the enzyme was examined by both sedimentation analysis and polyacrylamide gel electrophoresis. The molecular weight and isoelectric point were found to be around 30,000 and 4.35, respectively. Some other properties were also examined. In the course of an investigation on the fate of blasticidin S,1) an agricultural antibiotic, in the environment, we found that a fungal strain, isolated from a soil of paddy field, was able to decrease significantly the bioactivity of the antibiotic.2) The structure of blasticidin S was determined by OTAKE et al.3); it consists of cytosinine, a new nucleoside, and a n-amino acid designated as blastidic acid.
    [Show full text]
  • Inhibition of Protein Synthesis by Blasticidin S I. Studies with Cell-Free
    The Journal of Biochemistry Vol. 57, No. 5, 1965 Inhibition of Protein Synthesis by Blasticidin S I. Studies with Cell-free Systems from Bacterial and Mammalian Cells By HIDEYO YAMAGUCHI, CHIEKO YAMAMOTO and NOBuo TANAKA (From the Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo) (Received for publication, December 14, 1964) It has been demonstrated that blasticidin S, a useful fungicidal antibiotics, also exhibits MATERIALS AND METHODS antibacterial activity (1), toxicity to mam Chemicals-Purified blasticidin S in a crystal form malians and tumor-inhibitory activity against was kindly supplied by Prof. Hiroshi Yonehara, transplantable animal tumors (2). Although Institute of Applied Microbiology, University of the structure of blasticidin S is not finally Tokyo. L-Leucine-C14 (U) and L-phenylalanine-C14(U) determined, the antibiotic is proved to consist were purchased from the Radiochemical Centre, of cytosine and a pentose linked with a sort Amersham, England, and poly U from CALBIOCHEM, Los Angeles, Colifornia. of amino acid (3). In this respect, blasticidin Preparation of Extracts of Bacterial Cells-E. coli S belongs to the group of nucleoside anti strain B was grown at 37°C in a nutrient broth sup- biotics, of which puromycin is most extensively plimented with 0.5% glucose with reciprocal shaking. investigated concerning the mechanism of The cells were harvested in logarithmic growth phase, action. It is established that puromycin washed twice with standard buffer containing 10-IM inhibits protein synthesis by acting on the potassium chloride, 10-2M magnesium chloride, process following aminoacyl-sRNA formation •~10-2M Tris buffer, pH 7.8, and 6•~10-1M ƒÀ- (4, 5), and by stimulating the release of mercaptoethanolamine, and were stocked in a frozen incomplete peptide chains from ribosomal state until used.
    [Show full text]
  • Characterization of Nonr, an Esterase That Confers Nonactin Resistance
    CHARACTERIZATION OF NONR, AN ESTERASE THAT CONFERS NONACTIN RESISTANCE A Thesis Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By James E. Cox, B.S., B.A. ***** The Ohio State University 2004 Dissertation Committee: Approved by Professor Robert W. Brueggemeier, Adviser Professor Larry W. Robertson Professor Lane Wallace _ _______________________ Adviser Professor Nigel D. Priestley Dean, College of Pharmacy ABSTRACT Nonactin is the parent compound of the macrotetrolide class of antibiotics, atypical cyclic polyethers, consisting of both enantiomers of nonactic acid subunits linked together via four ester linkages in a (+)(-)(+)(-) manner. The higher macrotetrolide homologs of nonactin are made up of successive substitutions of nonactic acid with homononactate or bishomononactate. The macrotetrolides act as ionophores, forming complexes with both monovalent and divalent ions. Organisms that produce antibiotics as a defense mechanism need to protect themselves from their own biosynthesis products. Self-protection is achieved using many methods such as modification of the antibiotic itself, modification of the cellular target, removal of the produced antibiotic to specific binding proteins, or changes in the cell wall. Many species employ several of these methods simultaneously, incorporating antibiotic modification steps into the biosynthetic pathway and using the other methods to offer layers of resistance. A genetic resistance element conferred nonactin resistance to a macrotetrolide sensitive host. The genetic element was cloned from the producer, Streptomyces griseus subsp. griseus. By sequence analysis the protein product, NonR, appeared to be an esterase. This work describes the subcloning of the gene nonR, its expression in a heterologous host, and examination of the activity of the expressed protein.
    [Show full text]