DOCSLIB.ORG

148240825.Pdf

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
148240825.Pdf University of Groningen Novel Dehalogenase Mechanism for 2,3-Dichloro-1-Propanol Utilization in Pseudomonas putida Strain MC4 Arif, Muhammad Ilan; Samin, Ghufrana; van Leeuwen, Jan G. E.; Oppentocht, Jantina; Janssen, Dick Published in: Applied and Environmental Microbiology DOI: 10.1128/AEM.00760-12 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2012 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Arif, M. I., Samin, G., van Leeuwen, J. G. E., Oppentocht, J., & Janssen, D. B. (2012). Novel Dehalogenase Mechanism for 2,3-Dichloro-1-Propanol Utilization in Pseudomonas putida Strain MC4. Applied and Environmental Microbiology, 78(17), 6128-6136. DOI: 10.1128/AEM.00760-12 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 11-02-2018 Novel Dehalogenase Mechanism for 2,3-Dichloro-1-Propanol Utilization in Pseudomonas putida Strain MC4 Muhammad Irfan Arif, Ghufrana Samin,* Jan G. E. van Leeuwen , Jantien Oppentocht, and Dick B. Janssen Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, Netherlands A Pseudomonas putida strain (MC4) that can utilize 2,3-dichloro-1-propanol (DCP) and several aliphatic haloacids and haloal- Downloaded from cohols as sole carbon and energy source for growth was isolated from contaminated soil. Degradation of DCP was found to start with oxidation and concomitant dehalogenation catalyzed by a 72-kDa monomeric protein (DppA) that was isolated from cell lysate. The dppA gene was cloned from a cosmid library and appeared to encode a protein equipped with a signal peptide and that possessed high similarity to quinohemoprotein alcohol dehydrogenases (ADHs), particularly ADH IIB and ADH IIG from Pseudomonas putida HK. This novel dehalogenating dehydrogenase has a broad substrate range, encompassing a number of ؊1 1 nonhalogenated alcohols and haloalcohols. With DCP, DppA exhibited a kcat of 17 s . H nuclear magnetic resonance experi- ments indicated that DCP oxidation by DppA in the presence of 2,6-dichlorophenolindophenol (DCPIP) and potassium ferri- http://aem.asm.org/ cyanide [K 3Fe(CN)6] yielded 2-chloroacrolein, which was oxidized to 2-chloroacrylic acid. ichloropropanols are widely used in the chemical industry, nicotinamide cofactor binding site. They catalyze the intramolec- Dparticularly as intermediates for epichlorohydrin produc- ular displacement of a halogen by the vicinal hydroxyl group, tion. The classical epichlorohydrin manufacturing process pro- yielding an epoxide, a halide ion, and a proton ( 52 ). At least six ceeds via hydrochlorination of allylchloride, which yields both different halohydrin dehalogenases have been found so far: two 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol (30 ). Be- enzymes from Corynebacterium sp. strain N-1074 (HheA and cause of the increasing availability of glycerol as a side product HheB) (57 ) and homologs in Arthrobacter sp. strain AD2 on September 27, 2012 by University of Groningen from biodiesel synthesis, this classical process is being replaced (HheBAD2) ( 52 ), Agrobacterium sp. strain NHG3 (DehB) (20 ), Ar- with the use of glycerol as a renewable feedstock for epichlorohy- throbacter erithii H10a (DehA) (5), and Agrobacterium radiobacter drin manufacture, again via chlorination to the same dichloropro- strain AD1 (HheC) (51 ). The structures are known, and the cata- panols or via 1-chloro-2,3-propanediol ( 6). Epichlorohydrin itself lytic mechanism is well understood ( 11 , 12 , 21 ). The halohydrin as well as the production intermediates 2,3-dichloropropanol dehalogenases have a preference for substrates with the halogen (DCP) and 1,3-dichloropropanol are mutagenic, genotoxic, and group on a terminal (primary) carbon atom, which can be ex- carcinogenic, and therefore their release and the possibility of hu- plained by analysis of X-ray structures ( 11 , 21 ). However, impor- man exposure are of significant concern ( 38 ). DCP also occurs as tant compounds, such as 2-chloro-1-propanol and DCP, are not a contaminant in cellulose and starch hydrolysates, soy sauce, and easily converted. The potential importance of DCP as an interme- baked foods (27 ). diate in the degradation of 1,2,3-trichloropropane prompted us to Microorganisms that metabolize dichloropropanols are of in- search for new pathways of DCP metabolism ( 9). terest in view of their role in the removal of these compounds from A well-established mechanism for the conversion of alcohols is waste streams and contaminated environments (14 ), from food oxidative conversion by alcohol dehydrogenases (ADHs). Many and pulp products, and also carbohydrate hydrolysates (55 ) and ADHs are NAD- or NADP-dependent enzymes ( 34 ). Oxidation of because dichloropropanols occur as intermediates in a catabolic alcohols by oxidases, which generates hydrogen peroxide, is also pathway for degradation of the emerging priority contaminant possible. A special class of alcohol dehydrogenases is formed by 1,2,3-trichloropropane (8). Of the dichloropropanols, 2,3-di- the periplasmic quinoprotein ADHs, which contain a quinoid co- chloro-1-propanol is chemically more stable and more difficult to factor, such as pyrroloquinoline quinone (PQQ), and Ca 2ϩ. A degrade than 1,3-dichloropropanol (14 ). Furthermore, microor- quinoprotein methanol dehydrogenase has been found to be re- ganisms that convert dichloropropanols can be used in the prep- sponsible for 2-chloroethanol oxidation in the 1,2-dichloroethane aration of enantiopure building blocks for the pharmaceutical in- catabolic pathway (26 ). PQQ-dependent ADHs have been discov- dustry (27 , 30 ). Several bacterial strains are known to grow on ered in a wide variety of bacteria, including Acetobacter, Glucono- dichloropropanols, such as Pseudomonas sp. strain OS-K-29 (30 ), Alcaligenes sp. strain DS-K-S38 (29 ), Mycobacterium sp. strain GP1 (40 ), Agrobacterium sp. strain NHG3 (14 , 20 ), and Arthrobac- Received 7 March 2012 Accepted 14 June 2012 ter sp. strain AD2 (51 ). Published ahead of print 29 June 2012 During the microbial conversion of vicinal haloalcohols, deha- Address correspondence to Dick B. Janssen, [email protected]. logenation is usually the first step, and this reaction can be cata- * Permanent address: Ghufrana Samin, Department of Chemistry, University of lyzed by haloalcohol dehalogenases ( 24 ). These enzymes, also Engineering and Technology Lahore, Faisalabad Campus, Faisalabad, Pakistan. called halohydrin dehalogenases, are composed of 2 to 4 subunits Supplemental material for this article may be found at http://aem.asm.org/. of molecular masses between 28 and 35 kDa and are phylogeneti- Copyright © 2012, American Society for Microbiology. All Rights Reserved. cally related to the short-chain dehydrogenase reductase super- doi:10.1128/AEM.00760-12 family (SDR proteins) (54 ), even though they do not possess a 6128 aem.asm.org Applied and Environmental Microbiology p. 6128–6136 September 2012 Volume 78 Number 17 Quinohemoprotein Dehydrogenase/Dehalogenase bacter, Pseudomonas, and Comamonas strains (1, 2, 4, 19 , 47 ). Enzyme characterization. For molecular weight determination of na- Some of these enzymes contain heme as a secondary prosthetic tive enzyme, purified dehydrogenase (DppA) was analyzed by gel filtra- group and are known as quinohemoproteins ( 4). The periplasmic tion (see the supplemental material). The purity and molecular weight of quinohemoproteins transfer electrons to the membrane-bound the protein were determined by SDS-PAGE (12% gel) analysis. Heme bacterial respiratory chain (4, 34 ). staining of the SDS-PAGE gels was done by using the method of Francis In this paper we show that such a quinohemoprotein alcohol and Becker (15 ). To determine heme c concentrations, we measured difference spectra dehydrogenase may act as DCP dehalogenase. We started with the of reduced and oxidized pyridine hemochrome according to a published isolation of a DCP-degrading organism from a site polluted with protocol (53a). chlorinated compounds. We report the properties of this new Enzyme assays. All enzyme assays were performed at 25°C. The dehy- DCP-utilizing bacterium, analyze the gene encoding the quinohe- drogenase (DppA) activity toward DCP in cell extracts was measured by Downloaded from moprotein alcohol dehydrogenase, and propose a pathway for following the reduction of the electron acceptor DCPIP with PMS as an DCP metabolism. intermediate electron carrier. The reaction mixture (1 ml) contained 50 mM potassium phosphate, pH 7.4, 35 ␮M 2,6-DCPIP, 5 mM DCP, and MATERIALS AND METHODS cell extract. After addition of 1.6 mM PMS, the absorbance was monitored at 600 nm. The absorption coefficient of DCPIP at 600 nm is 21.0 mM Ϫ1 Chemicals, reagents and enzymes. All chemicals were obtained from Alfa cm Ϫ1 (16 ). Aesar, Sigma-Aldrich, and Acros Organics. Oxidase test discs were ob- For routine measurements, the enzyme activity was measured by fol- tained from Fluka. Plasmid DNA was isolated with the Qiagen plasmid lowing the reduction of potassium ferricyanide, K 3Fe(CN)6, at 420 nm in isolation kit. Enzymes used for cloning were either from Roche or New http://aem.asm.org/ a 1-ml reaction mixture consisting of 50 mM Tris-SO , pH 8.0, 5 mM England BioLabs. The PCR master mix for screening was purchased from 4 DCP, and 1 mM potassium ferricyanide (extinction coefficient, 1 mM Ϫ1 Promega. cm Ϫ1 at 420 nm [ 22 ]).
Load more

Recommended publications
  • Conformational Landscapes of Halohydrin Dehalogenases and Their Accessible Active Site Tunnels
    catalysts Article Conformational Landscapes of Halohydrin Dehalogenases and Their Accessible Active Site Tunnels 1 1, , 1,2, Miquel Estévez-Gay , Javier Iglesias-Fernández * y and Sílvia Osuna * 1 CompBioLab Group, Institut de Química Computacional i Catàlisi (IQCC) and Departament de Química, Universitat de Girona, c/Maria Aurèlia Capmany 69, 17003 Girona, Catalonia, Spain; [email protected] 2 ICREA, Passeig Lluís Companys 23, 08010 Barcelona, Catalonia, Spain * Correspondence: [email protected] (J.I.-F.); [email protected] (S.O.) Present address: Nostrum Biodiscovery, Carrer de Baldiri Reixac, 10–12, 08028 Barcelona, Catalonia, Spain. y Received: 12 November 2020; Accepted: 28 November 2020; Published: 1 December 2020 Abstract: Halohydrin dehalogenases (HHDH) are industrially relevant biocatalysts exhibiting a promiscuous epoxide-ring opening reactivity in the presence of small nucleophiles, thus giving access to novel carbon–carbon, carbon–oxygen, carbon–nitrogen, and carbon–sulfur bonds. Recently, the repertoire of HHDH has been expanded, providing access to some novel HHDH subclasses exhibiting a broader epoxide substrate scope. In this work, we develop a computational approach based on the application of linear and non-linear dimensionality reduction techniques to long time-scale Molecular Dynamics (MD) simulations to study the HHDH conformational landscapes. We couple the analysis of the conformational landscapes to CAVER calculations to assess their impact on the active site tunnels and potential ability towards bulky epoxide ring opening reaction. Our study indicates that the analyzed HHDHs subclasses share a common breathing motion of the halide binding pocket, but present large deviations in the loops adjacent to the active site pocket and N-terminal regions.
    [Show full text]
  • Molecular Basis of Two Novel Dehalogenating Activities in Bacteria
    Molecular basis of two novel dehalogenating activities in bacteria Structure/function relationships in an evolutionary context 1 Foto’s omslag: Dinanda Lutikhedde, MFA Druk: Digital Printing Partners Utrecht bv The research presented in this work was performed in the protein crystallography group at the Laboratory of Biophysical Chemistry of the University of Groningen. This work was supported by The Netherlands Foundation of Chemical Research (CW) with financial aid from The Netherlands Foundation for Scientific Research (NWO). 2 RIJKSUNIVERSITEIT GRONINGEN Molecular basis of two novel dehalogenating activities in bacteria Structure/function relationships in an evolutionary context Proefschrift ter verkrijging van het doctoraat in de Wiskunde en Natuurwetenschappen aan de Rijksuniversiteit Groningen op gezag van de Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op vrijdag 30 januari 2004 om 14.15 uur door René Marcel de Jong geboren op 22 maart 1976 te Heerenveen 3 Promotor: Prof. dr. B. W. Dijkstra Beoordelingscommissie: Prof. dr. D. B. Janssen Prof. dr. A. E. Mark Prof. dr. J.B.F.N. Engberts 4 CONTENTS CHAPTER 1: GENERAL INTRODUCTION ..........................................................9 EVOLUTION IN THE MICROBIAL WORLD.................................................................................. 10 EVOLUTION AT THE MOLECULAR LEVEL................................................................................. 12 BACTERIAL DEHALOGENATING ACTIVITY TOWARDS ANTHROPOGENIC COMPOUNDS ................... 14 SCOPE
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 7,381,538 B2 Reardon Et Al
    US007381538B2 (12) United States Patent (10) Patent No.: US 7,381,538 B2 Reardon et al. (45) Date of Patent: Jun. 3, 2008 (54) OPTICAL BIOSENSOR WITH ENHANCED 6,060,327 A * 5/2000 Keen ..................... 204,403.14 ACTIVITY RETENTION FOR DETECTION 6,159,681 A 12/2000 Zebala .......................... 435/4 OF HALOGENATED ORGANIC COMPOUNDS OTHER PUBLICATIONS Derek W. Campbell, entitled “The Development of Biosensors for (75) Inventors: Kenneth F. Reardon, Fort Collins, CO the Detection of Halogenated Groundwater Contaminants,” avail (US); Neema Das, Murgeshpalya (IN) able from Morgan Library at the CSU in Fort Collins, Colorado, having been submitted by Derek W. Campbell in fulfillment of the (73) Assignee: Colorado State University Research requirements for the Degree of Master of Science at Colorado State Foundation (CSURF), Fort Collins, University, Spring 1998 (labeled “Attachment B” of applicants' CO (US) provisional app. filed Jun. 1, 2001 as containing general background technical information}. (*) Notice: Subject to any disclaimer, the term of this inst k Eyle A. PS SEE s past l S.s I listed under 35 12,alogenated 1998 by Reardonflydrocarbons, and Campbell, U.S. Appl. 7 pages No. (labeled,89U, “Attachment Illed Sep. M YW- (b) by ayS. C” of applicants’pp pprovisional app.pp filed Jun. 1, 2001 as containin9. general background technical information}. (21) Appl. No.: 10/478,822 Cord Müller. F. Schubert. T. Scheper,p "Multicomponentp fiberopticalp biosensor for use in hemodialysis monitoring.” Proc. SPIE vol. (22) PCT Filed: Jun. 1, 2002 2131, 555-562 Biomedical Fiber Optic Instrumentation(Jul 1994) {referenced as “Cord Müller, et al.” in applicants’ provisional app.
    [Show full text]
  • Wo 2008/127291 A2
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (43) International Publication Date PCT (10) International Publication Number 23 October 2008 (23.10.2008) WO 2008/127291 A2 (51) International Patent Classification: Jeffrey, J. [US/US]; 106 Glenview Drive, Los Alamos, GOlN 33/53 (2006.01) GOlN 33/68 (2006.01) NM 87544 (US). HARRIS, Michael, N. [US/US]; 295 GOlN 21/76 (2006.01) GOlN 23/223 (2006.01) Kilby Avenue, Los Alamos, NM 87544 (US). BURRELL, Anthony, K. [NZ/US]; 2431 Canyon Glen, Los Alamos, (21) International Application Number: NM 87544 (US). PCT/US2007/021888 (74) Agents: COTTRELL, Bruce, H. et al.; Los Alamos (22) International Filing Date: 10 October 2007 (10.10.2007) National Laboratory, LGTP, MS A187, Los Alamos, NM 87545 (US). (25) Filing Language: English (81) Designated States (unless otherwise indicated, for every (26) Publication Language: English kind of national protection available): AE, AG, AL, AM, AT,AU, AZ, BA, BB, BG, BH, BR, BW, BY,BZ, CA, CH, (30) Priority Data: CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, 60/850,594 10 October 2006 (10.10.2006) US ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, (71) Applicants (for all designated States except US): LOS LR, LS, LT, LU, LY,MA, MD, ME, MG, MK, MN, MW, ALAMOS NATIONAL SECURITY,LLC [US/US]; Los MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, Alamos National Laboratory, Lc/ip, Ms A187, Los Alamos, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, NM 87545 (US).
    [Show full text]
  • University of Groningen Biocatalytic Conversion of Epoxides De Vries, Erik
    University of Groningen Biocatalytic conversion of epoxides de Vries, Erik; Janssen, DB Published in: Current Opinion in Biotechnology DOI: 10.1016/S0958-1669(03)00102-2 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2003 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): de Vries, E., & Janssen, DB. (2003). Biocatalytic conversion of epoxides. Current Opinion in Biotechnology, 14(4), 414-420. https://doi.org/10.1016/S0958-1669(03)00102-2 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
    [Show full text]
  • Aquaculture Journals – Table of Contents April 2012
    Aquaculture Journals – Table of Contents With the financial support of Flemish Interuniversity Councel Aquaculture Journals – Table of Contents April 2012 Information of interest !! Animal Feed Science and Technology * Antimicrobial Agents and Chemotherapy Applied and Environmental Microbiology Applied Microbiology and Biotechnology Aqua Aquaculture * Aquaculture Economics & Management Aquacultural Engineering * Aquaculture International * Aquaculture Nutrition * Aquaculture Research * Current Opinion in Microbiology * Diseases of Aquatic Organisms * Fish & Shellfish Immunology * Fisheries Science * Hydrobiologia * Indian Journal of Fisheries International Journal of Aquatic Science Journal of Applied Ichthyology * Journal of Applied Microbiology * Journal of Applied Phycology Journal of Aquaculture Research and Development Journal of Experimental Marine Biology and Ecology * Journal of Fish Biology Journal of Fish Diseases * Journal of Invertebrate Pathology* Journal of Microbial Ecology* Aquaculture Journals Page: 1 of 108 Aquaculture Journals – Table of Contents Journal of Microbiological Methods Journal of Shellfish Research Journal of the World Aquaculture Society Letters in Applied Microbiology * Marine Biology * Marine Biotechnology * Nippon Suisan Gakkaishi Reviews in Aquaculture Trends in Biotechnology * Trends in Microbiology * * full text available Aquaculture Journals Page: 2 of 108 Aquaculture Journals – Table of Contents BibMail Information of Interest - April, 2012 FAO COFI Subcommittee Aquaculture, Cape Town (South Africa),
    [Show full text]
  • Bioremediation 3.0: Engineering Pollutant-Removing Bacteria in The
    1 Bioremediation 3.0: Engineering pollutant-removing bacteria 2 in the times of systemic biology 3 4 by 5 6 Pavel Dvořák1, Pablo I. Nikel2, Jiří Damborský3,4, Víctor de Lorenzo1# 7 8 1 Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), 9 28049 Madrid, Spain 10 2 The Novo Nordisk Foundation Center for Biosustainability, 2800 Lyngby, Denmark 11 3 Loschmidt Laboratories, Centre for Toxic Compounds in the Environment RECETOX 12 and Department of Experimental Biology, Faculty of Science, Masaryk University, 62500 13 Brno, Czech Republic 14 4 International Clinical Research Center, St. Anne's University Hospital, Pekarska 53, 656 15 91 Brno, Czech Republic 16 17 # E-mail: [email protected], Tel.: +34 91-585 4536 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 1 1 2 3 4 Table of Contents 5 6 1. Introduction 7 2. Approaches and tools of systems biology and metabolic engineering for tailoring 8 biodegradation pathways 9 2.1 Step 1: Get to know the contaminant and find a suitable catabolic pathway 10 2.1.1 Databases 11 2.1.2 Pathway prediction systems and toxicity prediction algorithms 12 2.1.3 Detection and quantification of pathway building blocks 13 2.2 Step2: Select and get to know a suitable microbial host 14 2.2.1 Omic techniques in studies of bacterial degraders 15 2.3 Step 3: Build the pathway and optimize its performance in the context of host 16 metabolism 17 2.3.1 Computational tools for pathway and strain optimization 18 2.3.2 Experimental tools for pathway and strain optimization 19 2.3.3 Protein engineering to eliminate bottlenecks of biodegradation pathways 20 3.
    [Show full text]
  • Engineering of the Synthetic Metabolic Pathway for Biodegradation of Environmental Pollutant
    MASARYK UNIVERSITY FACULTY OF SCIENCE LOSCHMIDT LABORATORIES DEPARTMENT OF EXPERIMENTAL BIOLOGY Engineering of the synthetic metabolic pathway for biodegradation of environmental pollutant Doctoral dissertation Pavel Dvořák Supervisors: Prof. Mgr. Jiří Damborský, Dr. Doc. RNDr. Zbyněk Prokop, Ph.D. BRNO 2014 Poděkování Na tomto místě bych chtěl poděkovat svému školiteli Jiřímu Damborskému za profesionální vedení, schopnost a odhodlání dělat kvalitní, nepodlézavou vědu, ochotu setkávat se, diskutovat a radit, která není ani mezi školiteli postgraduálních studentů zcela automatická, a konečně za jeho víru ve zdárné konce, která mě dovedla až k sepsání této práce. Velmi děkuji i Zbyňku Prokopovi, mému školiteli specialistovi, za jeho optimismus a cenné rady a všem svým současným i minulým kolegům, kteří přispívali a přispívají k tomu, že Loschmidtovy laboratoře jsou nejenom špičkovým vědeckým týmem, ale i příjemným místem pro práci, kvůli kterému se člověk každé pondělní ráno rád přiměje vstát z postele. Největší dík ale patří mé ženě Monice za její lásku, přátelství, toleranci a schopnost vracet mě z badatelských výšin zpátky nohama na zem. Bibliographic entry Author: Mgr. Pavel Dvořák Loschmidt Laboratories Department of Experimental Biology Faculty of Science Masaryk University Title of dissertation: Engineering of the synthetic metabolic pathway for biodegradation of environmental pollutant Study Programme: Biology Field of study: Molecular and Cellular Biology Supervisor: Prof. Mgr. Jiří Damborský, Dr. Supervisor-specialist: doc. RNDr.
    [Show full text]
  • Enantioselective Formation and Ring-Opening of Epoxides Catalysed by Halohydrin Dehalogenases
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by UniversityBiocatalysis of Groningen Digital 291 Archive Enantioselective formation and ring-opening of epoxides catalysed by halohydrin dehalogenases D.B. Janssen*1, M. Majeric-Elenkov*´ 2, G. Hasnaoui*, B. Hauer† and J.H. Lutje Spelberg*3 *Biochemical Laboratory, Groningen Biomolecular Sciences and Biotechnology Institute, Groningen, The Netherlands, and †BASF AG, Fine Chemicals Research, Ludwigshafen, Germany Abstract Halohydrin dehalogenases catalyse the conversion of vicinal halohydrins into their corresponding epoxides, while releasing halide ions. They can be found in several bacteria that use halogenated alcohols or com- pounds that are degraded via halohydrins as a carbon source for growth. Biochemical and structural studies have shown that halohydrin dehalogenases are evolutionarily and mechanistically related to enzymes of the SDR (short-chain dehydrogenase/reductase) superfamily. In the reverse reaction, which is epoxide-ring opening, different nucleophiles can be accepted, including azide, nitrite and cyanide. This remarkable catalytic promiscuity allows the enzymatic production of a broad range of β-substituted alcohols from epoxides. In these oxirane-ring-opening reactions, the halohydrin dehalogenase from Agrobacterium radiobacter displays high enantioselectivity, making it possible to use the enzyme for the preparation of enantiopure building blocks for fine chemicals. Introduction and stopped flow fluorescence measurements have indicated The bacterial degradation of epichlorohydrin, 2,3-dibromo- that the release of the halide ion from the active site is the 1-propanol, 1,2-dibromoethane and some related compounds slowest step in the catalytic cycle [4]. Based on phylogenetic involves a step in which a vicinal halohydrin is converted relationships, the known halohydrin dehalogenases can be into the corresponding epoxide and free halide.
    [Show full text]
  • Enhancing the Biocatalytic Manufacture of the Key Intermediate
    www.nature.com/scientificreports OPEN Enhancing the biocatalytic manufacture of the key intermediate of atorvastatin by Received: 04 August 2016 Accepted: 05 January 2017 focused directed evolution of Published: 06 February 2017 halohydrin dehalogenase Yu Luo1,2, Yangzi Chen1, Hongmin Ma3, ZhenHua Tian2, Yeqi Zhang3 & Jian Zhang1 Halohydrin dehalogenases (HHDHs) are biocatalytically interesting enzymes due to their ability to form C-C, C-N, C-O, and C-S bonds. One of most important application of HHDH was the protein engineering of HheC (halohydrin dehalogenase from Agrobacterium radiobacter AD1) for the industrial manufacturing of ethyl (R)-4-cyano-3-hydroxybutanoate (HN), a key chiral synthon of a cholesterol-lowering drug of atorvastatin. During our development of an alternative, more efficient and economic route for chemo-enzymatic preparation of the intermediate of atorvastatin, we found that the HheC2360 previously reported for HN manufacture, had insufficient activity for the cyanolysis production of tert-butyl (3 R,5 S)-6-cyano-3,5-dihydroxyhexanoate (A7). Herein, we present the focused directed evolution of HheC2360 with higher activity and enhanced biocatalytic performance using active site mutagenesis. Through docking of the product, A7, into the crystal structure of HheC2360, 6 residues was selected for combined active sites testing (CASTing). After library screening, the variant V84G/W86F was identified to have a 15- fold increase in activity. Time course analysis of the cyanolysis reaction catalyzed by this variant, showed 2- fold increase in space time productivity compared with HheC2360. These results demonstrate the applicability of the variant V84G/W86F as a biocatalyst for the efficient and practical production of atorvastatin intermediate.
    [Show full text]
  • The Role of Allosteric Scaffolds and Protein Dynamics in Enzyme Function – Lessons Learned by NMR
    The Role of Allosteric Scaffolds and Protein Dynamics in Enzyme Function – Lessons Learned by NMR By Keith Thomas Taverner A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Chemistry University of Toronto University of Toronto Department of Chemistry April 2020 © Copyright by Keith Taverner, 2020 The Role of Allosteric Scaffolds and Protein Dynamics in Enzyme Function – Lessons Learned by NMR Keith Thomas Taverner Master of Science Department of Chemistry University of Toronto 2020 Abstract Most proteins undergo significant cooperative processes over timescales spanning microseconds to many milliseconds. These dynamic processes allow the protein to span the entire reaction coordinate and accomplish its function. Thus, protein function is generally not achieved by a single conformer but rather through a functional ensemble of conformers. Here, we focus on a homodimeric enzyme, fluoroacetate dehalogenase (FAcD) which cleaves the CF bond - one of the strongest covalent bonds in nature - generating fluoride, water, and glycolate from fluoroacetate. The enzyme functions through half-of-sites reactivity where only one protomer is catalytically active. The empty protomer releases ~30 bound water molecules during the catalytic step, thereby entropically favouring the forward reaction. This thesis focuses on using NMR, crystallography and computational studies to study protein dynamics. Specifically, evaluating substrate and product-based regulation, asymmetry in a homodimeric enzyme, disruptions to the allosteric network, and inhibited dynamics due to induced stiffness. ii Acknowledgements I would like to start by thanking my supervisor Robert Scott Prosser for giving me the opportunity to do research in his lab and for all the help/advice along the way.
    [Show full text]
  • Approaching Boiling Point Stability of an Alcohol Dehydrogenase Through
    RESEARCH ARTICLE Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering Friso S Aalbers1,2, Maximilian JLJ Fu¨ rst1,3, Stefano Rovida2, Milos Trajkovic1, J Rube´ n Go´ mez Castellanos2, Sebastian Bartsch4, Andreas Vogel4, Andrea Mattevi2, Marco W Fraaije1* 1Molecular Enzymology Group, University of Groningen, Groningen, Netherlands; 2Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, Pavia, Italy; 3MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, United Kingdom; 4c-LEcta GmbH, Leipzig, Germany Abstract Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase. Of the 177 selected mutations, 25 mutations brought about a T significant increase in apparent melting temperature (D m +3 ˚C). By combining mutations, a 10- T fold mutant was generated with a m of 94 ˚C (+51 ˚C relative to wild type), almost reaching water’s boiling point, and the highest increase with FRESCO to date. The 10-fold mutant’s structure was elucidated, which enabled the identification of an activity-impairing mutation. After reverting this T mutation, the enzyme showed no loss in activity compared to wild type, while displaying a m of 88 ˚C (+45 ˚C relative to wild type). This work demonstrates the value of enzyme stabilization through computational library design. *For correspondence: [email protected] Competing interest: See Introduction page 15 The oxidation of alcohols and reduction of ketones in nature is primarily governed by alcohol dehy- Funding: See page 15 drogenases (ADHs) (EC 1.1.1.1) (Kavanagh et al., 2008; Oppermann et al., 2003).
    [Show full text]