DOCSLIB.ORG

Enhancing H2O2 Resistance of an Esterase from Pyrobaculum Calidifontis by Structure-Guided Engineering of the Substrate Binding Site

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Enhancing H2O2 Resistance of an Esterase from Pyrobaculum Calidifontis by Structure-Guided Engineering of the Substrate Binding Site Appl Microbiol Biotechnol (2017) 101:5689–5697 DOI 10.1007/s00253-017-8299-0 BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Enhancing H2O2 resistance of an esterase from Pyrobaculum calidifontis by structure-guided engineering of the substrate binding site Pengfei Zhou1 & Dongming Lan2 & Grzegorz Maria Popowicz3 & Xuping Wang2 & Bo Yang1 & Yonghua Wang 2 Received: 19 January 2017 /Revised: 6 April 2017 /Accepted: 12 April 2017 /Published online: 17 May 2017 # Springer-Verlag Berlin Heidelberg 2017 Abstract Green technologies are attracting increasing at- Introduction tention in industrial chemistry where enzymatic reac- tions can replace dangerous and environmentally un- Oxidation reactions, such as epoxidation, hydroxylation, N- friendly chemical processes. In situ enzymatic synthesis oxidation, S-oxidation, and Baeyer-Villiger oxidation, are at- of peroxycarboxylic acid is an attractive alternative for tractive for organic synthesis of value compounds for science several industrial applications although concentrated and industry purposes (Hernandez et al. 2012). Although the H2O2 can denature the biocatalyst, limiting its useful- high catalytic efficiency of oxidation reactions can be ness. Herein, we report the structure-guided engineering achieved by using chemical methods, the toxicity and envi- of the Pyrobaculum calidifontis esterase (PestE) ronmental pollution arising from side products and reaction substrate binding site to increase its stability and residues deserve our attention. Alternatively, oxidases includ- perhydrolysis activity. The L89R/L40A PestE mutant ing peroxidase (Ullrich et al. 2008), peroxygenase (Kluge showed better tolerance toward concentrated H2O2 com- et al. 2009), P450 (Bernhardt 2006), chloroperoxidase pared with wild-type PestE, and retained over 72% of (Colonna et al. 1992), haloperoxidase (Dembitsky 2003), its initial activity after 24-h incubation with 2 M H2O2. and Baeyer-Villiger monooxygenase (Fraaije et al. 2005)are Surprisingly, the half-life (t1/2,80°C)ofPestEin- promising biocatalysts for these reactions because of their creasedfrom28to54h.Thekcat/Km values of the high catalytic efficiency under mild reaction conditions and mutant increased 21- and 3.4-fold toward pentanoic acid low toxicity to the environment. Additionally, esterases and and H2O2, respectively. This work shows how protein lipases can catalyze the reaction of carboxylic acids and H2O2 engineering can be used to enhance the H2O2 resistance to generate peroxycarboxylic acid, which can transfer its ox- and catalytic efficiency of an enzyme. ygen to olefins (Björkling et al. 1990; Björkling et al. 1992; Moreira et al. 2005; Ankudey et al. 2006;Wangetal.2015), cyclic ketones (Kotlewska et al. 2011; Drozdz et al. 2015), Keywords Pyrobaculum calidifontis . Esterase . Stability . amines (Irimescu and Kato 2004), and organic sulfur com- Structure-guided engineering . Perhydrolysis activity pounds (Nieuwenhuizen 1949; Lou et al. 2008). Thus, in situ enzymatic synthesis of peroxycarboxylic acid by esterases and lipases is an attractive alternative for a number of industrial * Yonghua Wang applications compared with the use of a chemical in situ gen- [email protected] eration method (Björkling et al. 1990; Rusch et al. 2002). Unfortunately, except for its role as a substrate, H O 1 School of Bioscience and Bioengineering, South China University of 2 2 Technology, Guangzhou, People’s Republic of China has a strong detrimental effect on the chemical structure of proteins because of its ability to oxidize amino acids. 2 School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, People’s Republic of China Aminoacids,suchasArg,Pro,Lys,Met,Cys,Tyr,and His, are sensitive to a high concentration of H O ,which 3 Institute of Structural Biology, Helmholtz ZentrumMünchen 2 2 Deutsches Forschungszentrum für Gesundheitund Umwelt (GmbH), destabilizes enzymes and limits their applications in oxi- Ingolstädter Landstrasse l, 85764 Oberschleißheim, Germany dation reactions (Villegas et al. 2000;Kroutiletal.2004; 5690 Appl Microbiol Biotechnol (2017) 101:5689–5697 Kitajima et al. 2008; Hernandez et al. 2012). To solve this and 2-chloro-5,5-dimethyl-1,3-cyclohexanedione problem, several strategies have been developed to (monochlorodimedone) was from Alfa Aesar (Shanghai, increase the tolerance of enzymes toward H2O2. China). All other regents were of analytical grade. Immobilization of enzymes on suitable supporting mate- rials can improve their resistance to extreme reaction con- Expression and purification of PestE and its mutants ditions, while chemical modification of enzymes is an alternative method to achieve similar effects. However, The gene-encoding PestE (GenBank accession number: in most cases, these strategies may sacrifice enzyme ac- AB078331.1) was chemically synthesized and cloned into the tivity for enhanced protein stability (Hernandez et al. pET23a vector as a cellulose-binding domain (CBD) fusion at 2012). Unlike physical and chemical methods, genetic en- the N terminus and His tag at the C terminus. The protein was gineering is a simple way to increase the protein stability expressed in Escherichia coli BL21(DE3). E. coli BL21(DE3) by substituting the H2O2-sensitive amino acid with an cells harboring the plasmid pET23a encoding the target genes insensitive one, and its feasibility has been confirmed by were grown at 37 °C for 8 h in Luria-Bertani (LB) medium many researchers (Villegas et al. 2000; Miyazaki and containing 100 μg ampicillin mL−1, and then the microorgan- Takahashi 2001;Kuoetal.2004;RyanandÓ’Fágáin isms were inoculated into auto-induction medium until the op- 2007; Kitajima et al. 2008; Ogola et al. 2010). The man- tical density (OD600) reached 0.8, and then the gene expression ganese peroxidase from Phanerochaete chrysosporium was induced by setting the temperature at 20 °C for 12 h at showed higher resistance to H2O2 inactivation when the 200 rpm as previously described (Studier 2005). H2O2-sensitive Met in the H2O2-binding pocket was re- Cells were collected, disrupted by sonication, and the pro- placed with Leu (Ryan and Ó’Fágáin 2007). Three mu- tein of interest was recovered by adsorption to cellulose. The tants of horseradish peroxidase, K232N, K241F and CBD tag was removed by proteolysis with 3C protease and T110V, showed higher H2O2 tolerances of 25-, 18-, and centrifugation and the protein was recovered in the superna- 12-fold compared with that of the parent enzyme tant. The recombinant enzyme was further purified by metal (Miyazaki and Takahashi 2001). ion affinity chromatography. The protein concentration in the PestE, an esterase from the hyperthermophilic archeon final preparation was determined using the Lowry Bradford Pyrobaculum calidifontis, has potential industrial applications method (Sedmak and Grossberg 1977) and bovine serum al- because of its extreme stability in the presence of heat and bumin was used as a standard. Mutants were obtained by site- organic solvents (Palm et al. 2011). However, it is not stable directed mutagenesis and expressed exactly as the wild type. in the presence of a high concentration of H2O2, limiting its All mutants were verified by sequencing of the relevant application in peroxycarboxylic acid production. In this study, constructs. we selected PestE as a model enzyme to increase its stability toward H2O2 by engineering its substrate binding pocket by General assays structure-guided mutagenesis. The H2O2-tolerance, thermo- stability, and biochemical characterization of the PestE mu- The stability of PestE and its mutants in the presence of H2O2 tants were investigated. We present a new approach for en- was determined by incubating the test enzyme (0.05 μM) in hancing the H2O2 stability of PestE esterase by introducing phosphate buffer containing different concentrations of H2O2 mutations that allow stabilization of H2O2 in the enzyme ac- (5 mM to 2 M) at room temperature. Samples were withdrawn tive site. Surprisingly, these modifications dramatically en- at different time points and the residual perhydrolysis activity hanced both enzymatic activity and H2O2 stability. was determined. The residual activity was expressed as a frac- tion of the residual activity of the reference sample treated in the same way except for the addition of H2O2. The thermal Materials and methods stability of PestE and its mutants was measured by using pNP butyrate as the substrate. Enzymes were incubated in phos- Chemicals and reagents phate buffer at pH 7.0 at 80 °C for 24 h. Samples were taken from the media at different time intervals. The optimal pH for The artificial substrates p-nitrophenyl (pNP) acetate (C2), the perhydrolysis reaction was determined by varying the pH pNP butyrate (C4), pNP caproate (C6), pNP caprylate (C8), of the pentanoic acid buffer. pNP caprate (C10), pNP laurate (C12), pNP myristate (C14), The temperature dependence of the perhydrolysis reaction pNP palmitate (C16), and oleic acid were purchased from was tested by varying the temperature from 40 to 55 °C. The Sigma-Aldrich (Shanghai, China). Formic acid, acetic acid, dependence of the hydrolysis rate on the length of the aliphatic propanoic acid, butyric acid, pentanoic acid, n-hexylic acid, chain of the carboxylic acid substrate was determined by ex- hydrogen peroxide (30% wt), and sodium bromide were pur- changing the pentanoic acid buffer into a buffer containing the chased from Aladdin Chemistry Co., Ltd. (Shanghai, China), tested acid. Appl Microbiol Biotechnol
Load more

Recommended publications
  • Ep 1734129 B1
    (19) TZZ_¥_ _T (11) EP 1 734 129 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12P 7/24 (2006.01) C12P 17/06 (2006.01) 24.02.2016 Bulletin 2016/08 (86) International application number: (21) Application number: 05727417.7 PCT/JP2005/005719 (22) Date of filing: 28.03.2005 (87) International publication number: WO 2005/098012 (20.10.2005 Gazette 2005/42) (54) PROCESS FOR PRODUCTION OF CHIRAL HYDROXYALDEHYDES VERFAHREN ZUR HERSTELLUNG CHIRALER HYDROXYALDEHYDE PROCÉDÉ DE FABRICATION D’HYDROXYALDÉHYDES CHIRALES (84) Designated Contracting States: (56) References cited: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR WO-A2-03/006656 HU IE IS IT LI LT LU MC NL PL PT RO SE SI SK TR • SAKURABA, H. ET AL.: "The First Crystal (30) Priority: 29.03.2004 JP 2004095263 Structure of Archaeal Aldolase. unique tetrameric structure of (43) Date of publication of application: 2-deoxy-D-ribose-5-phosphate aldolase from the 20.12.2006 Bulletin 2006/51 hyperthermophilic Archaea Aeropyrum pernix" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. (73) Proprietor: Mitsui Chemicals, Inc. 278, no. 12, 21 March 2003 (2003-03-21), pages Tokyo 105-7117 (JP) 10799-10806, XP002596667 • SAKURABA, H. ET AL.: "Sequential Aldol (72) Inventors: Condensation Catalyzed by Hyperthermophilic • MATSUMOTO, Kazuya, 2-Deoxy-D-Ribose-5-Phosphate Aldolase" c/o Mitsui Chemicals, Inc. APPLIED AND ENVIRONMENTAL Mobara-shi, Chiba 2970017 (JP) MICROBIOLOGY, vol. 73, no. 22, November 2007 • KAZUNO, Yasushi, (2007-11), pages 7427-7434, XP002596660 c/o Mitsui Chemicals, Inc.
    [Show full text]
  • Pyrobaculum Igneiluti Sp. Nov., a Novel Anaerobic Hyperthermophilic Archaeon That Reduces Thiosulfate and Ferric Iron
    TAXONOMIC DESCRIPTION Lee et al., Int J Syst Evol Microbiol 2017;67:1714–1719 DOI 10.1099/ijsem.0.001850 Pyrobaculum igneiluti sp. nov., a novel anaerobic hyperthermophilic archaeon that reduces thiosulfate and ferric iron Jerry Y. Lee, Brenda Iglesias, Caleb E. Chu, Daniel J. P. Lawrence and Edward Jerome Crane III* Abstract A novel anaerobic, hyperthermophilic archaeon was isolated from a mud volcano in the Salton Sea geothermal system in southern California, USA. The isolate, named strain 521T, grew optimally at 90 C, at pH 5.5–7.3 and with 0–2.0 % (w/v) NaCl, with a generation time of 10 h under optimal conditions. Cells were rod-shaped and non-motile, ranging from 2 to 7 μm in length. Strain 521T grew only in the presence of thiosulfate and/or Fe(III) (ferrihydrite) as terminal electron acceptors under strictly anaerobic conditions, and preferred protein-rich compounds as energy sources, although the isolate was capable of chemolithoautotrophic growth. 16S rRNA gene sequence analysis places this isolate within the crenarchaeal genus Pyrobaculum. To our knowledge, this is the first Pyrobaculum strain to be isolated from an anaerobic mud volcano and to reduce only either thiosulfate or ferric iron. An in silico genome-to-genome distance calculator reported <25 % DNA–DNA hybridization between strain 521T and eight other Pyrobaculum species. Due to its genotypic and phenotypic differences, we conclude that strain 521T represents a novel species, for which the name Pyrobaculum igneiluti sp. nov. is proposed. The type strain is 521T (=DSM 103086T=ATCC TSD-56T). Anaerobic respiratory processes based on the reduction of recently revealed by the receding of the Salton Sea, ejects sulfur compounds or Fe(III) have been proposed to be fluid of a similar composition at 90–95 C.
    [Show full text]
  • A Virus of Hyperthermophilic Archaea with a Unique Architecture Among DNA Viruses
    A virus of hyperthermophilic archaea with a unique architecture among DNA viruses Elena Ilka Rensena,1, Tomohiro Mochizukia,b,1, Emmanuelle Quemina, Stefan Schoutenc, Mart Krupovica,2, and David Prangishvilia,2 aDepartment of Microbiology, Institut Pasteur, 75015 Paris, France; bEarth-Life Science Institute, Tokyo Institute of Technology, Tokyo 152-8550, Japan; and cDepartment of Marine Organic Biogeochemistry, Royal Netherlands Institute for Sea Research, 1790 AB Den Burg, The Netherlands Edited by James L. Van Etten, University of Nebraska-Lincoln, Lincoln, NE, and approved January 19, 2016 (received for review September 23, 2015) Viruses package their genetic material in diverse ways. Most known shell consisting of two protein layers and an external envelope. strategies include encapsulation of nucleic acids into spherical or Our results provide new insights into the diversity of architec- filamentous virions with icosahedral or helical symmetry, respec- tural solutions used by filamentous viruses. tively. Filamentous viruses with dsDNA genomes are currently as- sociated exclusively with Archaea. Here, we describe a filamentous Results hyperthermophilic archaeal virus, Pyrobaculum filamentous virus 1 Virus and Host Isolation. From the environmental sample collected (PFV1), with a type of virion organization not previously observed at the Pozzuoli Solfatara, Italy, enrichment cultures were estab- in DNA viruses. The PFV1 virion, 400 ± 20 × 32 ± 3 nm, contains an lished in conditions known to favor the growth of aerobic mem- envelope and an inner core consisting of two structural units: a rod- bers of the archaeal genus Pyrobaculum (14). The virus-like particles shaped helical nucleocapsid formed of two 14-kDa major virion pro- (VLPs) were detected in the enrichment culture by transmission teins and a nucleocapsid-encompassing protein sheath composed electron microscopy (TEM).
    [Show full text]
  • Different Proteins Mediate Step-Wise Chromosome Architectures in 2 Thermoplasma Acidophilum and Pyrobaculum Calidifontis
    bioRxiv preprint doi: https://doi.org/10.1101/2020.03.13.982959; this version posted May 4, 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 4.0 International license. 1 Different Proteins Mediate Step-wise Chromosome Architectures in 2 Thermoplasma acidophilum and Pyrobaculum calidifontis 3 4 5 Hugo Maruyama1†*, Eloise I. Prieto2†, Takayuki Nambu1, Chiho Mashimo1, Kosuke 6 Kashiwagi3, Toshinori Okinaga1, Haruyuki Atomi4, Kunio Takeyasu5 7 8 1 Department of Bacteriology, Osaka Dental University, Hirakata, Japan 9 2 National Institute of Molecular Biology and Biotechnology, University of the Philippines 10 Diliman, Quezon City, Philippines 11 3 Department of Fixed Prosthodontics, Osaka Dental University, Hirakata, Japan 12 4 Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, 13 Kyoto University, Kyoto, Japan 14 5 Graduate School of Biostudies, Kyoto University, Kyoto, Japan 15 † These authors have contributed equally to this work 16 17 * Correspondence: 18 Hugo Maruyama 19 [email protected]; [email protected] 20 21 Keywords: archaea, higher-order chromosome structure, nucleoid, chromatin, HTa, histone, 22 transcriptional regulator, horizontal gene transfer 23 Running Title: Step-wise chromosome architecture in Archaea 24 Manuscript length: 6955 words 25 Number of Figures: 7 26 Number of Tables: 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.13.982959; this version posted May 4, 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.
    [Show full text]
  • A Method for Achieving Complete Microbial Genomes and Improving Bins from Metagenomics Data
    bioRxiv preprint doi: https://doi.org/10.1101/2020.03.05.979740; this version posted July 18, 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-ND 4.0 International license. 1 A method for achieving complete microbial genomes and improving 2 bins from metagenomics data 3 4 Authors: 5 Lauren M. Lui1, Torben N. Nielsen1, Adam P. Arkin1,2,3* 6 7 Affiliations: 8 1Environmental Genomics and Systems Biology Division, Lawrence Berkeley National 9 Laboratory, Berkeley, CA, USA. 10 2Department of Bioengineering, University of California, Berkeley, CA, USA 11 3Innovative Genomics Institute, Berkeley, CA, USA 12 13 *Correspondence: [email protected] 14 15 16 17 18 19 20 21 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.05.979740; this version posted July 18, 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-ND 4.0 International license. 22 Abstract 23 Metagenomics facilitates the study of the genetic information from uncultured microbes 24 and complex microbial communities. Assembling complete microbial genomes (i.e., 25 circular with no misassemblies) from metagenomics data is difficult because most 26 samples have high organismal complexity and strain diversity. Less than 100 27 circularized bacterial and archaeal genomes have been assembled from metagenomics 28 data despite the thousands of datasets that are available.
    [Show full text]
  • Discovery of Pyrobaculum Small RNA Families with Atypical Pseudouridine Guide RNA Features
    Downloaded from rnajournal.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press REPORT Discovery of Pyrobaculum small RNA families with atypical pseudouridine guide RNA features DAVID L. BERNICK,1 PATRICK P. DENNIS,2 MATTHIAS HO¨ CHSMANN,1 and TODD M. LOWE1,3 1Department of Biomolecular Engineering, University of California, Santa Cruz, California 95064, USA 2Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA ABSTRACT In the Eukarya and Archaea, small RNA-guided pseudouridine modification is believed to be an essential step in ribosomal RNA maturation. While readily modeled and identified by computational methods in eukaryotic species, these guide RNAs have not been found in most archaeal genomes. Using high-throughput transcriptome sequencing and comparative genomics, we have identified ten novel small RNA families that appear to function as H/ACA pseudouridylation guide sRNAs, yet surprisingly lack several expected canonical features. The new RNA genes are transcribed and highly conserved across at least six species in the archaeal hyperthermophilic genus Pyrobaculum. The sRNAs exhibit a single hairpin structure interrupted by a conserved kink- turn motif, yet only two of ten families contain the complete canonical structure found in all other H/ACA sRNAs. Half of the sRNAs lack the conserved 39-terminal ACA sequence, and many contain only a single 39 guide region rather than the canonical 59 and 39 bipartite guides. The predicted sRNA structures contain guide sequences that exhibit strong complementarity to ribosomal RNA or transfer RNA. Most of the predicted targets of pseudouridine modification are structurally equivalent to those known in other species.
    [Show full text]
  • Biotechnology of Archaea- Costanzo Bertoldo and Garabed Antranikian
    BIOTECHNOLOGY– Vol. IX – Biotechnology Of Archaea- Costanzo Bertoldo and Garabed Antranikian BIOTECHNOLOGY OF ARCHAEA Costanzo Bertoldo and Garabed Antranikian Technical University Hamburg-Harburg, Germany Keywords: Archaea, extremophiles, enzymes Contents 1. Introduction 2. Cultivation of Extremophilic Archaea 3. Molecular Basis of Heat Resistance 4. Screening Strategies for the Detection of Novel Enzymes from Archaea 5. Starch Processing Enzymes 6. Cellulose and Hemicellulose Hydrolyzing Enzymes 7. Chitin Degradation 8. Proteolytic Enzymes 9. Alcohol Dehydrogenases and Esterases 10. DNA Processing Enzymes 11. Archaeal Inteins 12. Conclusions Glossary Bibliography Biographical Sketches Summary Archaea are unique microorganisms that are adapted to survive in ecological niches such as high temperatures, extremes of pH, high salt concentrations and high pressure. They produce novel organic compounds and stable biocatalysts that function under extreme conditions comparable to those prevailing in various industrial processes. Some of the enzymes from Archaea have already been purified and their genes successfully cloned in mesophilic hosts. Enzymes such as amylases, pullulanases, cyclodextrin glycosyltransferases, cellulases, xylanases, chitinases, proteases, alcohol dehydrogenase,UNESCO esterases, and DNA-modifying – enzymesEOLSS are of potential use in various biotechnological processes including in the food, chemical and pharmaceutical industries. 1. Introduction SAMPLE CHAPTERS The industrial application of biocatalysts began in 1915 with the introduction of the first detergent enzyme by Dr. Röhm. Since that time enzymes have found wider application in various industrial processes and production (see Enzyme Production). The most important fields of enzyme application are nutrition, pharmaceuticals, diagnostics, detergents, textile and leather industries. There are more than 3000 enzymes known to date that catalyze different biochemical reactions among the estimated total of 7000; only 100 enzymes are being used industrially.
    [Show full text]
  • Maturation of Trna in Haloferax Volcanii. Dissertation Presented In
    Maturation of tRNA in Haloferax volcanii. Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Richard Neil Nist BA, MS Graduate Program in Microbiology The Ohio State University 2011 Dissertation Committee: Professor Charles J. Daniels, Advisor Associate Professor Juan Alfonzo Professor Venkat Gopalan Copyright by Richard Neil Nist 2011 Abstract Transfer RNA maturation in Archaea is a multi-faceted process requiring the activities of numerous enzymes in order to generate functional tRNAs from primary transcripts. Upon transcription, ribonuclease P cleaves the 5’ leader, tRNase Z cleaves the 3’ trailer, RNA-dependent and RNA-independent enzymes introduce modifications to bases and sugars, 3’ maturation is completed by the CCA-adding enzyme, and introns are removed. We examined the modification and intron removal stages of maturation for the Haloferax volcanii intron-containing pre-tRNA. H. volcanii tRNAMet possesses a 2’-O-methyl modification at position C34. A similar modification was characterized in pre-tRNATrp as being guided by a box C/D sRNP complex. Furthermore, just as sequence analysis and in vitro experiments have shown the intron-containing pre-tRNATrp to be a substrate for box C/D-guided modification, sequence analysis of intron-containing pre-tRNAMet suggests it is a substrate for box C/D-guided modification. We have determined not only that the C34 modification of tRNAMet is guided by a box C/D sRNP, but also that the intron- containing pre-tRNAMet appears to be a modification substrate in vivo. By the action of intron endonuclease, tRNA introns are removed in Archaea.
    [Show full text]
  • An Intron Within the 16S Ribosomal RNA Gene of the Archaeon Pyrobaculum Aerophilum (Phylogeny/Crenarchaeota/Pyrobaculum Islanicum/Open Reading Frame) S
    Proc. Natl. Acad. Sci. USA Vol. 90, pp. 2547-2550, March 1993 Evolution An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum (phylogeny/Crenarchaeota/Pyrobaculum islanicum/open reading frame) S. BURGGRAF*t, N. LARSENt, C. R. WOESEt, AND K. 0. STETTER* *Lehrstuhl fOr Mikrobiologie, Universitat Regensburg, 8400 Regensburg, Germany; and tDepartment of Microbiology, University of Illinois, 131 Burrill Hall, Urbana, IL 61801 Contributed by C. R. Woese, December Is, 1992 ABSTRACT The 16S rRNA genes of Pyrobaculum aero- Isolation of Nucleic Acids and Gene Amplification Proce- philum and Pyrobaculum islandicum were amplified by the dures. DNA and RNA were extracted and purified by stan- polymerase chain reaction, and the resulting products were dard procedures (14-16). The 16S rRNA gene was amplified sequenced directly. The two organisms are closely related by by the polymerase chain reaction (PCR) (17, 18). Amplifica- this measure (over 98% similar). However, they differ in that tion and purification of PCR products were performed as the (lone) 16S rRNA gene ofPyrobaculum aerophilum contains described earlier (19). a 713-bp intron not seen in the corresponding gene of Pyro- Sequencing. The dideoxynucleotide chain-termination baculum islandicum. To our knowledge, this is the only intron method was used to sequence both PCR-amplified DNA and so far reported in the small subunit rRNA gene ofa prokaryote. RNA (20-22). A set of standard archaeal "forward" and Upon excision the intron is circularized. A secondary structure "reverse" primers was used to sequence the 16S rRNAs (or model of the intron-containing rRNA suggests a splicing mech- their genes).
    [Show full text]
  • Mechanistic Studies on Pyrobaculum Calidifontis Porphobilinogen Synthase
    Bioorganic Chemistry 91 (2019) 103117 Contents lists available at ScienceDirect Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg Mechanistic studies on Pyrobaculum calidifontis porphobilinogen synthase T (5-aminolevulinic acid dehydratase) ⁎ Naseema Azima, Qurratulann Afza Gardnera, Naeem Rashida, Muhammad Akhtara,b, a School of Biological Sciences, University of the Punjab, New Campus, Lahore 54590, Pakistan b Biological Sciences, University of Southampton SO17 1BJ, UK ARTICLE INFO ABSTRACT Keywords: Porphobilinogen synthase (PBG synthase) gene from Pyrobaculum calidifontis was cloned and expressed in E. coli. Heme The recombinant enzyme was purified as an octamer and was found by mass spectrometry to have asubunit Mr Tetrapyrroles of 37676.59 (calculated, 37676.3). The enzyme showed high thermal stability and retained almost all of its Catalytic zinc activity after incubation at 70 °C for 16 h in the presence of β-mercaptoethanol (β-ME) and zinc chloride. Schiff base However, in the absence of the latter the enzyme was inactivated after 16 h although it regained full activity in MALDI and ESI mass spectrometry the presence of β-ME and zinc chloride. The protein contained 4 mol of tightly bound zinc per octamer. Further, Affinity labeling Active site residues 4 mol of low affinity zinc could be incorporated following incubation with exogenous zinc salts. The enzymewas inactivated by incubation with levulinic acid followed by treatment with sodium borohydride. Tryptic digest of the modified enzyme and mass spectrometric analysis showed that Lys257 was the site of modification, which has previously been shown to be the site for the binding of 5-aminolevulinic acid giving rise to the propionate-half of porphobilinogen.
    [Show full text]
  • Vulcanisaeta Distributa Type Strain (IC-017)
    Lawrence Berkeley National Laboratory Recent Work Title Complete genome sequence of Vulcanisaeta distributa type strain (IC-017). Permalink https://escholarship.org/uc/item/1kv1s9fh Journal Standards in genomic sciences, 3(2) ISSN 1944-3277 Authors Mavromatis, Konstantinos Sikorski, Johannes Pabst, Elke et al. Publication Date 2010 DOI 10.4056/sigs.1113067 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Standards in Genomic Sciences (2010) 3:117-125 DOI:10.4056/sigs.1113067 Complete genome sequence of Vulcanisaeta distributa type strain (IC-017T) Konstantinos Mavromatis1, Johannes Sikorski2, Elke Pabst3, Hazuki Teshima1,4, Alla Lapidus1, Susan Lucas1, Matt Nolan1, Tijana Glavina Del Rio1, Jan-Fang Cheng1, David Bruce1,4, Lynne Goodwin1,4, Sam Pitluck1, Konstantinos Liolios1, Natalia Ivanova1, Natalia Mikhailova1, Amrita Pati1, Amy Chen5, Krishna Palaniappan5, Miriam Land1,6, Loren Hauser1,6, Yun-Juan Chang1,6, Cynthia D. Jeffries1,6, Manfred Rohde7, Stefan Spring2, Markus Göker2, Reinhard Wirth3, Tanja Woyke1, James Bristow1, Jonathan A. Eisen1,8, Victor Markowitz5, Philip Hugenholtz1, Hans-Peter Klenk2, and Nikos C. Kyrpides1* 1 DOE Joint Genome Institute, Walnut Creek, California, USA 2 DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany 3 University of Regensburg, Microbiology – Archaeenzentrum. Regensburg, Germany 4 Los Alamos National Laboratory, Bioscience Division, Los Alamos, New Mexico, USA 5 Biological Data Management and Technology Center, Lawrence Berkeley National Laboratory, Berkeley, California, USA 6 Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA 7 HZI – Helmholtz Centre for Infection Research, Braunschweig, Germany 8 University of California Davis Genome Center, Davis, California, USA *Corresponding author: Nikos C. Kyrpides Keywords: hyperthermophilic, acidophilic, non-motile, microaerotolerant anaerobe, Ther- moproteaceae, Crenarchaeota, GEBA Vulcanisaeta distributa Itoh et al.
    [Show full text]
  • A Genus Definition for Bacteria and Archaea Based on a Standard
    RESEARCH ARTICLE Ecological and Evolutionary Science A Genus Definition for Bacteria and Archaea Based on a Standard Genome Relatedness Index R. A. Barco,a,b,e G. M. Garrity,c J. J. Scott,d J. P. Amend,a,b K. H. Nealson,a,b D. Emersone aDepartment of Earth Sciences, University of Southern California, Los Angeles, California, USA Downloaded from bDepartment of Biological Sciences, University of Southern California, Los Angeles, California, USA cDepartment of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA dSmithsonian Tropical Research Institute, Panama, Republic of Panama eBigelow Laboratory for Ocean Sciences, East Boothbay, Maine, USA ABSTRACT Genus assignment is fundamental in the characterization of microbes, yet there is currently no unambiguous way to demarcate genera solely using stan- dard genomic relatedness indices. Here, we propose an approach to demarcate gen- http://mbio.asm.org/ era that relies on the combined use of the average nucleotide identity, genome alignment fraction, and the distinction between type- and non-type species. More than 3,500 genomes representing type strains of species from Ͼ850 genera of either bacterial or archaeal lineages were tested. Over 140 genera were analyzed in detail within the taxonomic context of order/family. Significant genomic differences be- tween members of a genus and type species of other genera in the same order/ family were conserved in 94% of the cases. Nearly 90% (92% if polyphyletic genera are excluded) of the type strains were classified in agreement with current taxon- omy. The 448 type strains that need reclassification directly impact 33% of the gen- on March 23, 2020 by guest era analyzed in detail.
    [Show full text]