DOCSLIB.ORG

An Enzyme-Based Process for the Production of Long-Chain Α,Ω-Dicarboxylic Acids

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
An Enzyme-Based Process for the Production of Long-Chain Α,Ω-Dicarboxylic Acids A CYP solution: an enzyme-based process for the production of long-chain α,ω-dicarboxylic acids Ir. Delphine Devriese Supervisor: Prof. Dr. Bart Devreese Academic year 2020–2021 A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Doctor of Sciences: Biochemistry and Biotechnology Supervisor Prof. Dr. Bart Devreese Laboratory of Microbiology – Protein Research Unit, Ghent University Examination committee Prof. Dr. Savvas Savvides (Chair) VIB-UGent Center for Inflammation Research Dr. Kenneth Verstraete (Secretary) VIB-UGent Center for Inflammation Research Prof. Dr. ir. Inge Van Bogaert Centre for Synthetic Biology (CSB), Ghent University Prof. Dr. Serge Tavernier Intelligence in Processes, Advanced Catalysts & Solvents (iPRACS), University of Antwerp Dr. Lina De Smet Laboratory of Molecular Entomology and Honeybee Pathology (L-MEB), Ghent University This dissertation was part of the project Enzymes for Added Sustainability and Efficiency (EnzymASE). The project was funded by The Flemish Agency for Innovation and Entrepreneurship (VLAIO) and supported by Catalisti. A CYP solution: an enzyme-based process for the production of long-chain α,ω-dicarboxylic acids Ir. Delphine Devriese Supervisor: Prof. Dr. Bart Devreese Academic year 2020–2021 A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Doctor of Sciences: Biochemistry and Biotechnology The author and the supervisors give the authorization to consult and copy parts of this manuscript for personal use only. Any other use is limited by the laws of copyright, especially the obligation to refer to the source whenever results from this manuscript are cited. Ghent Author Supervisor Ir. Delphine Devriese Prof. Dr. Bart Devreese II Dankwoord De afgelopen vier jaar kreeg ik de kans om een doctoraatsonderzoek uit te voeren in de PRU onderzoeksgroep van het labo microbiologie, voormalig het L-PROBE labo. Ik heb mezelf hier kunnen ontwikkelen als onderzoeker maar ook daarbuiten leerde ik veel bij. Dit alles was niet mogelijk geweest zonder de volgende mensen en bij deze wil ik hen ook bedanken. Allereerst wil ik mijn promotor professor Bart Devreese bedanken. Iets meer dan vier jaar geleden botste ik op een vacature bij het toenmalige L-PROBE labo betreffende het onderzoek van enzymen voor de chemische industrie. Vanwege mijn interesse in de eiwitchemie, solliciteerde ik voor dit doctoraat en werd ik tot mijn vreugde aangenomen. Niet alleen wil ik mijn promotor bedanken om mij de kans te geven voor het uitvoeren van dit doctoraat, ook kon ik afgelopen vier jaar rekenen op een aangename samenwerking waarbij de deur ook vaak letterlijk openstond voor ons. In het labo kon ik altijd terugvallen op mijn collega’s. Stijn wil ik bedanken om mij bij de start van mijn doctoraat te begeleiden en mij te introduceren in het labo alsook in het EnzymASE project. Isabel verdient hier ook zeker een dankjewel. Als lab manager kon ik steeds op haar rekenen voor haar raad en daad met betrekking tot veiligheid, producten, toestellen en meer. Daarnaast was ze mee betrokken in EnzymASE en kon ik bij haar terecht voor het bediscussiëren van dit project. Steeds als ik iets meer wilde weten of hulp nodig had bij massa spectrometrie, kon ik aankloppen bij Sören. Voor extra informatie in verband met de membraanvesikels kon ik dan weer rekenen op Jolien Vitse. Ook mijn andere collega’s wil ik graag bedanken, namelijk Ingrid, Zhoujian en Gonzalez, alsook ex-collega’s Jolien Claeys, Sofie en Kasia en nieuwe collega’s Amir, Amaury en Eva. Iedereen droeg en draagt bij aan een aangename sfeer in het labo, van een babbeltje tussendoor en tijdens de lunch, tot de jaarlijkse teambuilding, waardoor ik steeds met plezier naar het labo kwam. Doorheen mijn doctoraat kon ik ook rekenen op mensen buiten het PRU labo. Zo wil ik Jordy Bauwelinck en Pieter Surmont bedanken voor het uitvoeren van de GC-MS analyses. Om mijn membraanvesikels te analyseren, kon ik gebruik maken van de NanoSight toestellen, beschikbaar in het labo experimenteel kankeronderzoek in het UZ Gent en in het labo algemene biochemie en fysische farmacie aan de faculteit farmaceutische wetenschappen. Daarbij wil ik Lien Lippens en Herlinde De Keersmaecker, respectievelijk, in het bijzonder bedanken voor de introductie van het NanoSight toestel. Ik wil van de gelegenheid gebruik maken om mijn examen commissie te bedanken voor de kritische analyse van mijn doctoraat en de behulpzame suggesties die gegeven werden om deze dissertatie te maken tot wat ik hier nu mag afleveren. III Afgelopen vier jaar kon ik rekenen op mijn familie en vrienden en kon ik er ook steeds terecht voor een ontspannend momentje. Ik zou hierbij graag mijn ouders bedanken voor hun steun doorheen de jaren. Daarnaast verdient mijn zus Joke hier zeker een grote dankjewel. Ook zij legde een doctoraat af. Ze wist me zo steeds bij te staan met raad en daad en als er nood was aan ontspanning, stond ze alvast klaar met haar gezelschapsspelen. Als laatste had ik mijn vriend Toon willen bedanken. Doorheen de afgelopen jaren heeft hij altijd een luisterend oor geboden en klaargestaan voor mij. Hij wist me steeds te motiveren en kon mij ook tot rust brengen bij stress momenten. Dankjewel iedereen! Delphine IV Summary α,ω-Dicarboxylic acids (DCAs) are valuable chemical compounds and are used in a wide variety of applications. They serve as monomers for polyamides such as nylons and polyesters. Moreover, these compounds are used as precursors for pharmaceuticals, perfumes, lubricants, adhesives, plasticizers, powder coatings and corrosion inhibitors. Today, mainly short- and medium-chain DCAs are used. The application of DCAs with longer chain lengths (C > 14) is less widespread due their lower availability and elevated costs. This is the result of their challenging production process. On the one hand, long-chain α,ω-DCAs are produced through chemical conversion of fatty acids by means of metathesis. On the other hand, a fermentation process with the industrial DCA producer Candida viswanathii is established. Unfortunately, both processes come with a number of disadvantages and there is a need for an alternative long- chain α,ω-DCA production process for increased availability and decreased prices. Cytochrome P450 monooxygenases (CYP) constitute a large superfamily of enzymes, performing a wide variety of reactions and accepting a diverse range of substrates. These enzymes have the ability to introduce one oxygen atom from molecular oxygen in a non- activated C-H bond, while reducing the other oxygen atom to water. This is a reaction difficult to achieve by conventional chemical routes. Therefore, CYPs are considered of great value in synthetic biology applications. Unfortunately, disadvantages such as low specific activity, the need for a redox partner and the cofactor NAD(P)H limits their use, and current application is restricted to whole-cell bioconversions. A number of CYP families have been reported to hydroxylate fatty acid substrates at the ω-position to their corresponding ω-hydroxy fatty acids. In some cases, overoxidation to the α,ω-DCAs was observed. One of these enzymes was selected for the development of an in vitro biocatalytic process, producing α,ω-DCAs from fatty acid substrates, i.e. CYP52A13 from the industrial DCA producer C. viswanathii. An in vitro biocatalytic process was pursued due to its many advantages over the currently used processes. No rare metal catalysts are needed, the enzyme performs its conversion at ambient temperature in mild reaction conditions, no side reactions occur due to absence of other enzymes and no cell membrane needs to be crossed, especially important with respect to the desired long-chain DCAs. Moreover, a biocatalytic process is easier to implement in the chemical industry as fermentation requires specific know-how and specialized equipment. In the first experimental chapter (chapter 3) it was investigated whether it would be feasible to create a soluble self-sufficient enzyme for the implementation in an enzyme reactor. CYP52A13 belongs to the class II CYP enzymes, meaning it is a membrane-bound protein, requiring the redox partner CYP reductase (CPR), which is also membrane-bound. As a soluble system was of interest due to its ease of implementation in a reactor system, both enzymes were truncated in order to remove their membrane anchor. Pichia pastoris was chosen as this fungal host offers many advantages towards industrial protein production. For example, little endogenous protein is secreted by this recombinant host. Producing CYP and CPR in a secreted V form was therefore pursued as this should greatly facilitate downstream processing. Furthermore, it was of interest to create a chimeric construct, where both redox partners were present in the same polypeptide chain. In this way, only one enzyme instead of two needs to be produced and fusion of the redox partners was reported to enhance coupling efficiency. For the optimization of this construct, a nontruncated variant was produced in the same recombinant host. Both truncated CYP and CPR were successfully produced using an artificial N-terminal tag, replacing the N-terminal membrane anchor. Even more, this N-tag enabled secretion to the medium for both enzymes. Unfortunately, activity could only be proven for CPR. A chimer was successfully produced but not secreted in P. pastoris and the chimer was retained in the microsomal membrane. However, the artificial N-tag proved to be valuable still. The chimer could be isolated in a soluble form in absence of detergents by washing the microsome with a high salt buffer. Furthermore, the N-tag improved the production yield of this class II CYP compared to the nontruncated form, which was only produced to a low level. In the following chapter (chapter 4), an in vivo immobilization strategy was considered in order to enhance stability of the CYP by introduction in a membrane environment, while still retaining the advantages of an in vitro approach.
Load more

Recommended publications
  • Title Total Biosynthesis of Opiates by Stepwise Fermentation Using
    Total biosynthesis of opiates by stepwise fermentation using Title engineered Escherichia coli. Nakagawa, Akira; Matsumura, Eitaro; Koyanagi, Takashi; Katayama, Takane; Kawano, Noriaki; Yoshimatsu, Kayo; Author(s) Yamamoto, Kenji; Kumagai, Hidehiko; Sato, Fumihiko; Minami, Hiromichi Citation Nature communications (2016), 7 Issue Date 2016-02-05 URL http://hdl.handle.net/2433/204378 This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; Right if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Type Journal Article Textversion publisher Kyoto University ARTICLE Received 30 Oct 2015 | Accepted 7 Dec 2015 | Published 5 Feb 2016 DOI: 10.1038/ncomms10390 OPEN Total biosynthesis of opiates by stepwise fermentation using engineered Escherichia coli Akira Nakagawa1, Eitaro Matsumura1, Takashi Koyanagi1, Takane Katayama2, Noriaki Kawano3, Kayo Yoshimatsu3, Kenji Yamamoto1, Hidehiko Kumagai1, Fumihiko Sato4 & Hiromichi Minami1 Opiates such as morphine and codeine are mainly obtained by extraction from opium poppies. Fermentative opiate production in microbes has also been investigated, and complete biosynthesis of opiates from a simple carbon source has recently been accom- plished in yeast. Here we demonstrate that Escherichia coli serves as an efficient, robust and flexible platform for total opiate synthesis. Thebaine, the most important raw material in opioid preparations, is produced by stepwise culture of four engineered strains at yields of 2.1 mg l À 1 from glycerol, corresponding to a 300-fold increase from recently developed yeast systems.
    [Show full text]
  • “Biosynthesis of Morphine in Mammals”
    “Biosynthesis of Morphine in Mammals” D i s s e r t a t i o n zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Naturwissenschaftlichen Fakultät I Biowissenschaften der Martin-Luther-Universität Halle-Wittenberg von Frau Nadja Grobe geb. am 21.08.1981 in Querfurt Gutachter /in 1. 2. 3. Halle (Saale), Table of Contents I INTRODUCTION ........................................................................................................1 II MATERIAL & METHODS ........................................................................................ 10 1 Animal Tissue ....................................................................................................... 10 2 Chemicals and Enzymes ....................................................................................... 10 3 Bacteria and Vectors ............................................................................................ 10 4 Instruments ........................................................................................................... 11 5 Synthesis ................................................................................................................ 12 5.1 Preparation of DOPAL from Epinephrine (according to DUNCAN 1975) ................. 12 5.2 Synthesis of (R)-Norlaudanosoline*HBr ................................................................. 12 5.3 Synthesis of [7D]-Salutaridinol and [7D]-epi-Salutaridinol ..................................... 13 6 Application Experiments .....................................................................................
    [Show full text]
  • © Central University of Technology, Free State DECLARATION
    © Central University of Technology, Free State DECLARATION DECLARATION I, MOHAMMAD PARVEZ (INDIAN PASSPORT NUMBER ), hereby certify that the dissertation submitted by me for the degree DOCTOR OF HEALTH SCIENCES IN BIOMEDICAL TECHNOLOGY, is my own independent work; and complies with the Code of Academic Integrity, as well as other relevant policies, procedures, rules and regulations of the Central University of Technology (Free State). I hereby declare, that this research project has not been previously submitted before to any university or faculty for the attainment of any qualification. I further waive copyright of the dissertation in favour of the Central University of Technology (Free State). I also state that expression vector modified/generated in this study is in collaboration with my colleagues, Mr HANS DENIS BAMAL (student number: ) and Ms IPELENG KOPANO ROSINAH KGOSIEMANG (student number: ). MOHAMMAD PARVEZ DATE II | P a g e © Central University of Technology, Free State © Central University of Technology, Free State ACKNOWLEDGEMENT ACKNOWLEDGEMENTS This thesis would have remained a dream, had it not been for my supervisor, Prof Khajamohiddin Syed, who continuously supported me during my doctoral study. I would like to extend my gratitude to him, for his patience, motivation, enthusiasm, and immense knowledge in P450 research. I would also like to thank him for giving me the opportunity to attend national and international scientific conferences. He has shown great faith and trust in me throughout my studies and without such attributes I may not be where I am now. I would also like to thank Prof Samson Sitheni Mashele for his support, and always listening to our concerns.
    [Show full text]
  • Electron Transfer Partners of Cytochrome P450
    4 Electron Transfer Partners of Cytochrome P450 Mark J.l. Paine, Nigel S. Scrutton, Andrew W. Munro, Aldo Gutierrez, Gordon C.K. Roberts, and C. Roland Wolf 1. Introduction Although P450 redox partners are usually expressed independently, "self-sufficient" P450 monooxygenase systems have also evolved through Cytochromes P450 contain a heme center the fusion of P450 and CPR genes. These fusion where the activation of molecular oxygen occurs, molecules are found in bacteria and fungi, the best- resulting in the insertion of a single atom of known example being P450 BM3, a fatty acid oxygen into an organic substrate with the con­ (0-2 hydroxylase from Bacillus megaterium, which comitant reduction of the other atom to water. The comprises a soluble P450 with a fiised carboxyl- monooxygenation reaction requires a coupled and terminal CPR module (recently reviewed by stepwise supply of electrons, which are derived Munro^). BM3 has the highest catalytic activity from NAD(P)H and supplied via a redox partner. known for a P450 monooxygenase^ and was for P450s are generally divided into two major classes many years the only naturally occurring ftised sys­ (Class I and Class II) according to the different tem known until the identification of a eukaryotic types of electron transfer systems they use. P450s membrane-bound equivalent fatty acid hydroxy­ in the Class I family include bacterial and mito­ lase, CYP505A1, from the phytopathogenic fungus chondrial P450s, which use a two-component Fusarium oxysporurrP. A number of novel P450 sys­ shuttle system consisting of an iron-sulfur protein tems are starting to emerge from the large numbers (ferredoxin) and ferredoxin reductase (Figure 4.1).
    [Show full text]
  • UC Berkeley UC Berkeley Electronic Theses and Dissertations
    UC Berkeley UC Berkeley Electronic Theses and Dissertations Title Oriented Attachment of Cytochrome P450 2C9 to a Self-Assembled Monolayer on a Gold Electrode as a Biosensor Design Permalink https://escholarship.org/uc/item/1m67k8mm Author Schneider, Elizabeth Publication Date 2011 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California Oriented Attachment of Cytochrome P450 2C9 to a Self-Assembled Monolayer on a Gold Electrode as a Biosensor Design by Elizabeth Ann Schneider A dissertation submitted in partial satisfaction of the requirements for the degree of Joint Doctor of Philosophy with the University of California, San Francisco in Bioengineering in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Douglas S. Clark, Chair Associate Professor Shuvo Roy Professor Liwei Lin Dr. Robert Kostecki Fall 2011 Abstract Oriented Attachment of Cytochrome P450 2C9 to a Self-Assembled Monolayer on a Gold Electrode as a Biosensor Design by Elizabeth Ann Schneider Doctor of Philosophy in Bioengineering University of California, Berkeley Professor Douglas S. Clark, Chair Cytochrome P450s (CYPs) are a family of enzymes implicated in the metabolism of drugs in the body. Consequently, P450 reactions are of high interest to the pharmaceutical industry, where lead compounds in drug development are screened as potential substrates of CYPs. The P450 reaction involves electron transfer to an iron heme via NADPH and the electron transfer partner enzyme P450 reductase (CPR). By immobilizing CYPs on an electrode however, NADPH and CPR are potentially no longer needed and the immobilized CYP can act as a biosensor by accepting electrons directly from the electrode.
    [Show full text]
  • Evaluation of Yarrowia Lipolytica As a Host For
    EVALUATION OF YARROWIA LIPOLYTICA AS A HOST FOR CYTOCHROME P450 MONOOXYGENASE EXPRESSION By GEORGE OGELLO OBIERO B.Sc. (U.O.N); M.Sc. (U.B) Submitted in fulfilment of the requirements for the degree PHILOSOPHIAE DOCTOR In the Faculty of Natural and Agricultural Sciences, Department of Microbial, Biochemical and Food Biotechnology at the University of the Free State, Bloemfontein, South Africa June, 2006 Promoter: Prof. M.S. Smit "It's not that I'm so smart, it's just that I stay with problems longer." Albert Einstein (1879-1955) I This thesis is dedicated to my late mother, Mrs. Beldine Obiero and my dad, Mr. Gabriel Obiero whose unwavering support and encouragement has been my pillar of strength. II ACKNOWLEDGEMENTS I would like to express my sincere gratitude toward the following people: Prof. M.S. Smit for her invaluable guidance, patience and constructive criticism during the course of the study. Thanks for the patience and determination throughout this project. Mr. P.J. Botes for his technical assistance with the chemical analyses. Dr. E. setati for her valuable and motivating discussions. All my colleagues in Biotransformation Research Group. Fellow students and staff in the Department of Microbial, Biochemical and Food Biotechnology, University of Free State My family and friends for being there for me throughout the study and for their much needed words of encouragement and support. National Research Foundation (NRF) for the financial support of this project. The Heavenly Father without whom this work would not have been successful. III TABLE OF CONTENTS CHAPTER 1: INTRODUCTION 1. Introduction............................................................................................................ 1 1.1.
    [Show full text]
  • Engineering of Cytochrome P450s CYP109E1 and CYP109A2 from Bacillus Megaterium DSM319 for The
    Engineering of Cytochrome P450s CYP109E1 and CYP109A2 from Bacillus megaterium DSM319 for the production of vitamin D3 metabolites Kumulative Dissertation zur Erlangen des Grades des Doktors der Naturwissenschaften der Naturwissenschaftlich-Technischen Fakultät der Universität des Saarlandes von Ammar Abdulmughni Saarbrücken 2018 Tag des Kolloquiums : 14.08.2018 Dekan : Prof. Dr. Guido Kickelbick Berichterstatter : Prof. Dr. Rita Bernhardt Prof. Dr. Gert-Wieland Kohring Vorsitz : Prof. Dr. Uli Müller Akad. Mitarbeiter: Dr. Ing. Michael Kohlstedt Abstract Active vitamin D3 metabolites play an essential role in the maintenance of calcium and phosphorus homeostasis. The conventional chemical synthesis of this metabolite is time- consuming, environmentally unfriendly and often results in low yield. Therefore, the biotechnological production of active vitamin D3 metabolites is of great importance to the pharmaceutical industry. Hereby, cytochrome P450 enzymes have the potential to achieve this goal. The present work reports on the optimization of a biotechnological process in Bacillus megaterium MS941 for the production of vitamin D3 metabolites. On that account, two cytochrome P450 enzymes were used as biocataylsts, namely CYP109E1 and CYP109A2 from the Gram-positive bacterium Bacillus megaterium DSM319. Both enzymes were subjected for functional and structural characterization in order to optimize their activity and/or regio-selectivity towards vitamin D3. In terms of hydroxylation activity, it has been shown that the conversion of vitamin D3 with CYP109E1 results in the formation of several derivatives, while CYP109A2 shows clearly a higher regio-selectivity towards 25- hydroxylation. The elucidation of the crystal structure of both enzymes provides detailed insights into the geometry of these enzymes. By means of molecular docking, site-directed mutagenesis was successfully performed, resulting in the creation of mutants with higher regio-selectivity compared to the wild type, in particular when using CYP109E1.
    [Show full text]
  • Regulation of Alkaloid Biosynthesis in Plants
    CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin. JAUME BASTIDA (87), Departament de Productes Naturals, Facultat de Farma` cia, Universitat de Barcelona, 08028 Barcelona, Spain YEUN-MUN CHOO (181), Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia PETER J. FACCHINI (1), Department of Biological Sciences, University of Calgary, Calgary, AB, Canada TOH-SEOK KAM (181), Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia RODOLFO LAVILLA (87), Parc Cientı´fic de Barcelona, Universitat de Barcelona, 08028 Barcelona, Spain DANIEL G. PANACCIONE (45), Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506-6108, USA CHRISTOPHER L. SCHARDL (45), Department of Plant Pathology, University of Kentucky, Lexington, KY 40546-0312, USA PAUL TUDZYNSKI (45), Institut fu¨r Botanik, Westfa¨lische Wilhelms Universita¨tMu¨nster, Mu¨nster D-48149, Germany FRANCESC VILADOMAT (87), Departament de Productes Naturals, Facultat de Farma` cia, Universitat de Barcelona, 08028 Barcelona, Spain vii PREFACE This volume of The Alkaloids: Chemistry and Biology is comprised of four very different chapters; a reflection of the diverse facets that comprise the study of alkaloids today. As awareness of the global need for natural products which can be made available as drugs on a sustainable basis increases, so it has become increas- ingly important that there is a full understanding of how key metabolic pathways can be optimized. At the same time, it remains important to find new biologically active alkaloids and to elucidate the mechanisms of action of those that do show potentially useful or novel biological effects. Facchini, in Chapter 1, reviews the significant studies that have been conducted with respect to how the formation of alkaloids in their various diverse sources are regulated at the molecular level.
    [Show full text]
  • Bifunctional CYP81AA Proteins Catalyse Identical Hydroxylations but Alternative Regioselective Phenol Couplings in Plant Xanthone Biosynthesis
    ARTICLE Received 27 Oct 2015 | Accepted 30 Mar 2016 | Published 5 May 2016 DOI: 10.1038/ncomms11472 OPEN Bifunctional CYP81AA proteins catalyse identical hydroxylations but alternative regioselective phenol couplings in plant xanthone biosynthesis Islam El-Awaad1,2,w, Marco Bocola3, Till Beuerle1,2, Benye Liu1,2 & Ludger Beerhues1,2 Xanthones are natural products present in plants and microorganisms. In plants, their biosynthesis starts with regioselective cyclization of 2,30,4,6-tetrahydroxybenzophenone to either 1,3,5- or 1,3,7-trihydroxyxanthones, catalysed by cytochrome P450 (CYP) enzymes. Here we isolate and express CYP81AA-coding sequences from Hypericum calycinum and H. perforatum in yeast. Microsomes catalyse two consecutive reactions, that is, 30-hydro- xylation of 2,4,6-trihydroxybenzophenone and C–O phenol coupling of the resulting 2,30, 4,6-tetrahydroxybenzophenone. Relative to the inserted 30-hydroxyl, the orthologues Hc/HpCYP81AA1 cyclize via the para position to form 1,3,7-trihydroxyxanthone, whereas the paralogue HpCYP81AA2 directs cyclization to the ortho position, yielding the isomeric 1,3,5- trihydroxyxanthone. Homology modelling and reciprocal mutagenesis reveal the impact of S375, L378 and A483 on controlling the regioselectivity of HpCYP81AA2, which is converted into HpCYP81AA1 by sextuple mutation. However, the reciprocal mutations in HpCYP81AA1 barely affect its regiospecificity. Product docking rationalizes the alternative C–O phenol coupling reactions. Our results help understand the machinery of bifunctional CYPs. 1 Institute of Pharmaceutical Biology, Technische Universita¨t Braunschweig, Mendelssohnstrae 1, Braunschweig 38106, Germany. 2 Center of Pharmaceutical Engineering (PVZ), Technische Universita¨t Braunschweig, Franz-Liszt-Strae 35A, Braunschweig 38106, Germany. 3 Institute of Biotechnology, RWTH Aachen University, Worringerweg 1, Aachen 52074, Germany.
    [Show full text]
  • Targeting of Insect Epicuticular Lipids by the Entomopathogenic Fungus Beauveria Bassiana: Hydrocarbon Oxidation Within the Context of a Host-Pathogen Interaction
    ORIGINAL RESEARCH ARTICLE published: 15 February 2013 doi: 10.3389/fmicb.2013.00024 Targeting of insect epicuticular lipids by the entomopathogenic fungus Beauveria bassiana: hydrocarbon oxidation within the context of a host-pathogen interaction Nicolás Pedrini 1, Almudena Ortiz-Urquiza 2, Carla Huarte-Bonnet 1, Shizhu Zhang 2,3 and Nemat O. Keyhani 2* 1 Facultad de Ciencias Médicas, Instituto de Investigaciones Bioquímicas de La Plata (CCT La Plata CONICET-UNLP), La Plata, Argentina 2 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA 3 Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing, China Edited by: Broad host range entomopathogenic fungi such as Beauveria bassiana attack insect hosts Rachel N. Austin, Bates College, via attachment to cuticular substrata and the production of enzymes for the degradation USA and penetration of insect cuticle. The outermost epicuticular layer consists of a complex Reviewed by: mixture of non-polar lipids including hydrocarbons, fatty acids, and wax esters. Long chain Rachel N. Austin, Bates College, USA hydrocarbons are major components of the outer waxy layer of diverse insect species, Alan A. DiSpirito, Ohio State where they serve to protect against desiccation and microbial parasites, and as recognition University, USA molecules or as a platform for semiochemicals. Insect pathogenic fungi have evolved *Correspondence: mechanisms for overcoming this barrier, likely with sets of lipid degrading enzymes with Nemat O. Keyhani, Department of overlapping substrate specificities. Alkanes and fatty acids are substrates for a specific Microbiology and Cell Science, University of Florida, Gainesville, subset of fungal cytochrome P450 monooxygenases involved in insect hydrocarbon FL 32611, USA.
    [Show full text]
  • Conformational Changes in Binding of Substrates with Human Cytochrome P450 Enzymes
    Book of oral abstracts 100 Conformational changes in binding of substrates with human cytochrome P450 enzymes F. Peter Guengerich, Clayton J. Wilkey, Michael J. Reddish, Sarah M. Glass, and Thanh T. N. Phan Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA Introduction. Extensive evidence now exists that P450 enzymes can exist in multiple conformations, at least in the substrate-bound forms (e.g., crystallography). This multiplicity can be the result of either an induced fit mechanism or conformational selection (selective substrate binding to one of two or more equilibrating P450 conformations). Aims. Kinetic approaches can be used to distinguish between induced fit and conformational selection models. The same energy is involved in reaching the final state, regardless of the kinetic path. Methods. Stopped-flow absorbance and fluorescence measurements were made with recombinant human P450 enzymes. Analysis utilized kinetic modeling software (KinTek Explorer®). Results. P450 17A1 binding to its steroid ligands (pregnenolone and progesterone and the 17-hydroxy derivatives) is dominated by a conformational selection process, as judged by (a) decreasing rates of substrate binding as a function of substrate concentration, (b) opposite patterns of the dependence of binding rates as a function of varying concentrations of (i) substrate and (ii) enzyme, and (c) modeling of the data in KinTek Explorer. The inhibitory drugs orteronel and abiraterone bind P450 17A1 in multi-step processes, apparently in different ways. The dye Nile Red is also a substrate for P450 17A1 and its sequential binding to the enzyme can be resolved in fluorescence and absorbance changes. P450s 2C8, 2D6, 2E1, and 4A11 have also been analyzed with regard to substrate binding and utilize primarily conformational selection models, as revealed by analysis of binding rates vs.
    [Show full text]
  • Optimization of the Bacterial Cytochrome P450 BM3 System for the Production of Human Drug Metabolites
    Int. J. Mol. Sci. 2012, 13, 15901-15924; doi:10.3390/ijms131215901 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Review Optimization of the Bacterial Cytochrome P450 BM3 System for the Production of Human Drug Metabolites Giovanna Di Nardo and Gianfranco Gilardi * Department of Life Sciences and Systems Biology, University of Torino, via Accademia Albertina 13, 10123 Torino, Italy; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +39-011-670-4593; Fax: +39-011-670-4643. Received: 27 September 2012; in revised form: 1 November 2012 / Accepted: 13 November 2012 / Published: 28 November 2012 Abstract: Drug metabolism in human liver is a process involving many different enzymes. Among them, a number of cytochromes P450 isoforms catalyze the oxidation of most of the drugs commercially available. Each P450 isoform acts on more than one drug, and one drug may be oxidized by more than one enzyme. As a result, multiple products may be obtained from the same drug, and as the metabolites can be biologically active and may cause adverse drug reactions (ADRs), the metabolic profile of a new drug has to be known before this can be commercialized. Therefore, the metabolites of a certain drug must be identified, synthesized and tested for toxicity. Their synthesis must be in sufficient quantities to be used for metabolic tests. This review focuses on the progresses done in the field of the optimization of a bacterial self-sufficient and efficient cytochrome P450, P450 BM3 from Bacillus megaterium, used for the production of metabolites of human enzymes.
    [Show full text]