DOCSLIB.ORG

Form of Taxadiene Synthase Involved in Paclitaxel

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Form of Taxadiene Synthase Involved in Paclitaxel Archives of Biochemistry and Biophysics Vol. 379, No. 1, July 1, pp. 137–146, 2000 doi:10.1006/abbi.2000.1865, available online at http://www.idealibrary.com on Heterologous Expression and Characterization of a “Pseudomature” Form of Taxadiene Synthase Involved in Paclitaxel (Taxol) Biosynthesis and Evaluation of a Potential Intermediate and Inhibitors of the Multistep Diterpene Cyclization Reaction1 David C. Williams,* Mark R. Wildung,* Alan Qingwu Jin,† Dolan Dalal,‡ John S. Oliver,‡ Robert M. Coates,† and Rodney Croteau2 *Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340; †Department of Chemistry, University of Illinois, 600 South Matthews Avenue, Urbana, Illinois 61801; and ‡Department of Chemistry, Box H, Brown University, Providence, Rhode Island 02912 Received February 23, 2000, and in revised form April 4, 2000 with kinetics comparable to the native enzyme. In ad- The diterpene cyclase taxadiene synthase from yew dition to the major product, taxa-4(5),11(12)-diene (Taxus) species transforms geranylgeranyl diphos- (94%), this enzyme produces a small amount of the phate to taxa-4(5),11(12)-diene as the first committed isomeric taxa-4(20),11(12)-diene (ϳϳ5%), and a product step in the biosynthesis of the anti-cancer drug Taxol. tentatively identified as verticillene (ϳϳ1%). Isotopi- Taxadiene synthase is translated as a preprotein bear- cally sensitive branching experiments utilizing (4R)- 2 ing an N-terminal targeting sequence for localization [4- H1]geranylgeranyl diphosphate confirmed that the to and processing in the plastids. Overexpression of two taxadiene isomers, and a third (taxa-3(4),11(12)- the full-length preprotein in Escherichia coli and pu- diene), are derived from the same intermediate tax- rification are compromised by host codon usage, inclu- enyl C4-carbocation. These results, along with the fail- sion body formation, and association with host chap- ure of the enzyme to utilize 2,7-cyclogeranylgeranyl erones, and the preprotein is catalytically impaired. diphosphate as an alternate substrate, indicate that Since the transit peptide-mature enzyme cleavage site the reaction proceeds by initial ionization of the could not be determined directly, a series of N-termi- diphosphate ester and macrocyclization to the verti- nally truncated enzymes was created by expression of cillyl intermediate, followed by a secondary cycliza- the corresponding cDNAs from a suitable vector, and tion to the taxenyl cation and deprotonation (i.e., for- each was purified and kinetically evaluated. Deletion mation of the A-ring prior to B/C-ring closure). Two of up to 79 residues yielded functional protein; how- potential mechanism-based inhibitors were tested ever, deletion of 93 or more amino acids resulted in with recombinant taxadiene synthase but neither pro- complete elimination of activity, implying a structural vided time-dependent inactivation nor afforded more or catalytic role for the amino terminus. The than modest competitive inhibition. © 2000 Academic Press pseudomature form of taxadiene synthase having 60 Key Words: Taxol biosynthesis; paclitaxel; taxadiene amino acids deleted from the preprotein was found to synthase; Taxus; yew. be superior with respect to level of expression, ease of purification, solubility, stability, and catalytic activity Taxadiene synthase from Taxus (yew) species cata- 1 This investigation was supported in part by grants CA-55254 lyzes the first committed step in the biosynthesis of the (R.C.), GM-13956 (R.M.C.), and GM-31354 (R.C.) from the National 3 Institutes of Health, by Cytoclonal Pharmaceutics, and by McIntire- anti-cancer drug Taxol and related taxane diterpe- Stennis Project 0967 from the Washington State University Agricul- tural Research Center. 2 To whom correspondence and reprint requests should be ad- 3 Paclitaxel is the generic name for Taxol, which is now a regis- dressed. Fax: (509) 335-7643. E-mail: [email protected]. tered trademark of Bristol-Meyers Squibb. Because of the far greater 0003-9861/00 $35.00 137 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 138 WILLIAMS ET AL. FIG. 1. Cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene and subsequent elaboration to Taxol. Ac, acetyl group; Bz, benzoyl group; OPP, diphosphate moiety. noids by the cyclization of the universal precursor of where proteolytic processing of the transit peptide oc- diterpenes, geranylgeranyl diphosphate, to the olefinic curs and where the mature form of the enzyme func- product (Fig. 1) (1). This enzyme catalyzes a slow, but tions in the first step of Taxol biosynthesis. The pre- apparently not rate limiting, step in the Taxol biosyn- protein forms of the terpene synthases are often suffi- thetic pathway (2). Taxadiene synthase has been iso- ciently active to permit identification of clones by lated from yew saplings (3) and cell cultures (2), and functional heterologous expression from standard clon- the enzyme exhibits properties typical of the few diter- ing vectors (7), and the taxadiene synthase was con- pene synthases from gymnosperms and angiosperms firmed by this means (6). However, in high level ex- thus far defined (4). Initial assessment of the mecha- pression systems, the presence of the transit peptide of nism of taxadiene synthase (5) indicates that the reac- the terpene synthases often contributes to the forma- tion involves the ionization and cyclization of gera- tion of inclusion bodies and can compromise purifica- nylgeranyl diphosphate to a transient verticillyl cation tion of the soluble form(s) by promoting aggregation intermediate (1), with intramolecular transfer of the and/or the tight association with host chaperones and C11 proton to C7 to initiate transannular B/C-ring other proteins (8). Additionally, the preproteins may be closure to the taxenyl cation (2), followed by deproto- catalytically impaired relative to their native counter- nation at C5 to yield the taxa-4(5),11(12)-diene prod- parts. Soluble taxadiene synthase has been overex- uct; however, few other details of the stereochemical pressed recently as a His-tagged, thioredoxin fusion of mechanism or of the structure of this novel enzyme are the preprotein (9). The enzyme is catalytically capable known. when expressed in this form; however, such preprotein A cDNA encoding the taxadiene synthase from fusions are unsuitable for detailed mechanistic and Taxus brevifolia has been obtained by a homology- crystallographic study. based PCR4 cloning method (6). Like the monoterpene Deciphering the transit peptide-mature protein synthases and other diterpene synthases (7), taxadiene cleavage junction of terpene synthase preproteins from synthase is translated as a preprotein bearing an N- sequence information alone is not possible because of terminal targeting sequence for localization to plastids the considerable variation in primary sequence of these targeting peptides (10–13), and studies with the native enzymes to define this feature directly have indicated that most are N-terminally blocked (8, 14). Moreover, familiarity of the word Taxol, we use it in this paper in lieu of paclitaxel. detailed mass spectrometric analysis of a prototype 4 Abbreviations used: DTT, dithiothreitol; EDTA, ethylenediami- terpene synthase, limonene synthase, indicated that notetraacetic acid; FPLC, fast protein liquid chromatography; GC- plastidial proteolytic processing to the native form in MS, gas chromatography-mass spectrometry; Hepes, N-(2-hydroxy- this case was quite imprecise (8). Therefore, to prepare ethyl)piperazine-NЈ-(2-ethanesulfonic acid); LSC, liquid scintillation counting; Mopso, 3-(N-morpholino)-2-hydroxypropanesulfonic acid; a “pseudomature” form of such a terpene synthase PCR, polymerase chain reaction; SDS–PAGE, sodium dodecylsul- requires the empirical construction and testing of a fate-polyacrylamide gel electrophoresis. series of truncated versions of the target protein. In HETEROLOGOUS EXPRESSION OF TAXADIENE SYNTHASE 139 FIG. 2. Possible mechanisms for the utilization of substrate analogs by taxadiene synthase. OPP, diphosphate moiety. this paper, we describe the construction, heterologous 65 min). The unstable chlorides, contaminated by about 10% of the methylenecyclohexene elimination product, were characterized by expression and purification of a series of truncated 1 13 forms of the preprotein, report on the kinetic proper- IR, H NMR, and C NMR spectra, and used without purification. Selected NMR data (benzene-d , 400 MHz) for 2,7-cyclogeranylgera- ties, solubility and stability of these “pseudomature” 6 nyl chloride: ␦H 1.04, 1.56, 1.60 (3s, 3 ϫ 3H,3CH3), 3.83 and 3.93 forms of taxadiene synthase, and discuss the mecha- (ABq, 2H, J ϭ 11.4 CH2Cl), 5.24 (m, 2H, vinyl H); ␦c 41.2 (CH2Cl). nistic implications of several new findings revealed by Reaction of the chlorides with (Bu4N)3HP2O7 (1.08 equiv) in CH3CN studies with the recombinant enzyme. (1 ml) containing powdered 3 Å molecular sieve was carried out for 48 h at room temp. according to literature procedures (20, 21). Purification by ion exchange chromatography, selective precipita- MATERIALS AND METHODS tion, and cellulose chromatography afforded 3(16 mg, 26%) and [1,1- 2 Substrates, standards, and inhibitors. (E,E,E)-[1-3H]Gera- H2]3 (7.9 mg, 10%) as white solids, which were characterized by 1 31 nylgeranyl diphosphate (120 Ci/mol) was prepared as described pre- TLC, H, and P NMR spectra, and negative ion FAB-MS. Data for 1 ϩ ϩ viously (15). (Ϯ)-Taxa-(4)5,11(12)-diene and (Ϯ)-taxa-4(20),11(12)- 3: H NMR (D2O 0.06% (w/v) EDTA concentrated NH4OH to pH ␦ ϫ diene (16) were gifts from Robert M. Williams, Colorado State Uni- 9, 400 MHz) 0.88 (s, 3 H, CH3), 1.41 (s, 6 H, 2 CH3), 1.48 (s, 3 H, versity (Fort Collins, CO). All other biochemicals and reagents were CH3), 1.59 (s, 3 H, CH3), 1.04–1.20 (m, 2 H, CH2), 1.21–1.34 (m, 1 H), purchased from Sigma Chemical Co. or Aldrich Chemical Co., unless 1.42–1.54 (m, 1 H), 1.56–1.68 (m, 1 H), 1.68–1.94 (m, 8 H, CH2), 4.25 ϭ otherwise noted.
Load more

Recommended publications
  • (10) Patent No.: US 8119385 B2
    US008119385B2 (12) United States Patent (10) Patent No.: US 8,119,385 B2 Mathur et al. (45) Date of Patent: Feb. 21, 2012 (54) NUCLEICACIDS AND PROTEINS AND (52) U.S. Cl. ........................................ 435/212:530/350 METHODS FOR MAKING AND USING THEMI (58) Field of Classification Search ........................ None (75) Inventors: Eric J. Mathur, San Diego, CA (US); See application file for complete search history. Cathy Chang, San Diego, CA (US) (56) References Cited (73) Assignee: BP Corporation North America Inc., Houston, TX (US) OTHER PUBLICATIONS c Mount, Bioinformatics, Cold Spring Harbor Press, Cold Spring Har (*) Notice: Subject to any disclaimer, the term of this bor New York, 2001, pp. 382-393.* patent is extended or adjusted under 35 Spencer et al., “Whole-Genome Sequence Variation among Multiple U.S.C. 154(b) by 689 days. Isolates of Pseudomonas aeruginosa” J. Bacteriol. (2003) 185: 1316 1325. (21) Appl. No.: 11/817,403 Database Sequence GenBank Accession No. BZ569932 Dec. 17. 1-1. 2002. (22) PCT Fled: Mar. 3, 2006 Omiecinski et al., “Epoxide Hydrolase-Polymorphism and role in (86). PCT No.: PCT/US2OO6/OOT642 toxicology” Toxicol. Lett. (2000) 1.12: 365-370. S371 (c)(1), * cited by examiner (2), (4) Date: May 7, 2008 Primary Examiner — James Martinell (87) PCT Pub. No.: WO2006/096527 (74) Attorney, Agent, or Firm — Kalim S. Fuzail PCT Pub. Date: Sep. 14, 2006 (57) ABSTRACT (65) Prior Publication Data The invention provides polypeptides, including enzymes, structural proteins and binding proteins, polynucleotides US 201O/OO11456A1 Jan. 14, 2010 encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides.
    [Show full text]
  • Metabolic Engineering of Escherichia Coli for Natural Product Biosynthesis
    Trends in Biotechnology Special Issue: Metabolic Engineering Review Metabolic Engineering of Escherichia coli for Natural Product Biosynthesis Dongsoo Yang,1,4 Seon Young Park,1,4 Yae Seul Park,1 Hyunmin Eun,1 and Sang Yup Lee1,2,3,∗ Natural products are widely employed in our daily lives as food additives, Highlights pharmaceuticals, nutraceuticals, and cosmetic ingredients, among others. E. coli has emerged as a prominent host However, their supply has often been limited because of low-yield extraction for natural product biosynthesis. from natural resources such as plants. To overcome this problem, metabolically Escherichia coli Improved enzymes with higher activity, engineered has emerged as a cell factory for natural product altered substrate specificity, and product biosynthesis because of many advantages including the availability of well- selectivity can be obtained by structure- established tools and strategies for metabolic engineering and high cell density based or computer simulation-based culture, in addition to its high growth rate. We review state-of-the-art metabolic protein engineering. E. coli engineering strategies for enhanced production of natural products in , Balancing the expression levels of genes together with representative examples. Future challenges and prospects of or pathway modules is effective in natural product biosynthesis by engineered E. coli are also discussed. increasing the metabolic flux towards target compounds. E. coli as a Cell Factory for Natural Product Biosynthesis System-wide analysis of metabolic Natural products have been widely used in food and medicine in human history. Many of these networks, omics analysis, adaptive natural products have been developed as pharmaceuticals or employed as structural backbones laboratory evolution, and biosensor- based screening can further increase for the development of new drugs [1], and also as food and cosmetic ingredients.
    [Show full text]
  • Emerging Engineering Principles for Yield Improvement in Microbial Cell Design
    Volume No: 3, Issue: 4, October 2012, e201210016, http://dx.doi.org/10.5936/csbj.201210016 CSBJ Emerging engineering principles for yield improvement in microbial cell design Santiago Comba a, Ana Arabolaza a,*, Hugo Gramajo a Abstract: Metabolic Engineering has undertaken a rapid transformation in the last ten years making real progress towards the production of a wide range of molecules and fine chemicals using a designed cellular host. However, the maximization of product yields through pathway optimization is a constant and central challenge of this field. Traditional methods used to improve the production of target compounds from engineered biosynthetic pathways in non-native hosts include: codon usage optimization, elimination of the accumulation of toxic intermediates or byproducts, enhanced production of rate-limiting enzymes, selection of appropriate promoter and ribosome binding sites, application of directed evolution of enzymes, and chassis re-circuit. Overall, these approaches tend to be specific for each engineering project rather than a systematic practice based on a more generalizable strategy. In this mini-review, we highlight some novel and extensive approaches and tools intended to address the improvement of a target product formation, founded in sophisticated principles such as dynamic control, pathway genes modularization, and flux modeling. Introduction The utilization of industrial biotechnology for the conversion of metabolic pathways; 4) the setting up of new analytical methods to energy, chemicals and materials to value-added products is centuries- detect and quantify RNA, protein and cell metabolites; and 5) the old. Traditional methods of strain development based on evolution, creation of detailed biological models aided to the design of enzymes random mutagenesis, mating and selection strategies were successfully and metabolic pathways [3].
    [Show full text]
  • Development of a Four-Step Semi-Biosynthesis of the Anticancer Drug Paclitaxel and Its Analogues
    DEVELOPMENT OF A FOUR-STEP SEMI-BIOSYNTHESIS OF THE ANTICANCER DRUG PACLITAXEL AND ITS ANALOGUES By Chelsea Thornburg A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Biochemistry and Molecular Biology ‒ Doctor of Philosophy 2015 ABSTRACT DEVELOPMENT OF A FOUR-STEP SEMI-BIOSYNTHESIS OF THE ANTICANCER DRUG PACLITAXEL AND ITS ANALOGUES By Chelsea Thornburg Paclitaxel (Taxol®) is a widely used chemotherapeutic drug with additional medical applications in drug-eluting stents as an anti-restenosis treatment. Paclitaxel is a structurally complex natural product with an excellent scaffold for designing analogs with pharmacological properties. To date, clinically approved analogs include docetaxel and cabazitaxel for the treatment of additional cancers. Currently, plant cell fermentation methods produce paclitaxel and large quantities of the precursors 10-deacetylbaccatin III (10-DAB) and baccatin III. The complexity of the semi-characterized ~19-step paclitaxel biosynthetic pathway limits bioengineering attempts. However, the availability of 10-DAB and baccatin III suggests a semi-biosynthetic pathway to paclitaxel starting with these precursors is feasible. We have designed a short, simple biosynthetic pathway, capable of making paclitaxel, analogs, and/or valuable precursors for the semi-synthesis of additional analogs of biological interest. The paclitaxel biosynthesis enzyme baccatin III: 3-amino-13-O-phenylpropanoyl CoA transferase (BAPT) and the bacterial (2R,3S)-phenylisoserinyl CoA ligase (PheAT) produce N-debenzoylpaclitaxel, N-debenzoyldocetaxel, or precursor analogs. The addition of the paclitaxel biosynthetic N-debenzoyltaxol-N-benzoyltransferase (NDTNBT) and the bacterial benzoate CoA ligase (BadA) produce paclitaxel or other N-acylated analogs. In this dissertation, BAPT and BadA are kinetically characterized.
    [Show full text]
  • Synthesis of Chemicals by Metabolic Engineering of Microbes
    Chem Soc Rev View Article Online REVIEW ARTICLE View Journal Synthesis of chemicals by metabolic engineering of microbes Cite this: DOI: 10.1039/c5cs00159e Xinxiao Sun,†a Xiaolin Shen,†a Rachit Jain,†b Yuheng Lin,b Jian Wang,b Jing Sun,a Jia Wang,a Yajun Yan*c and Qipeng Yuan*a Metabolic engineering is a powerful tool for the sustainable production of chemicals. Over the years, the exploration of microbial, animal and plant metabolism has generated a wealth of valuable genetic information. The prudent application of this knowledge on cellular metabolism and biochemistry has enabled the construction of novel metabolic pathways that do not exist in nature or enhance existing ones. The hand in hand development of computational technology, protein science and genetic manipulation tools has formed the basis of powerful emerging technologies that make the production of green Received 19th February 2015 chemicals and fuels a reality. Microbial production of chemicals is more feasible compared to plant and DOI: 10.1039/c5cs00159e animal systems, due to simpler genetic make-up and amenable growth rates. Here, we summarize the recent progress in the synthesis of biofuels, value added chemicals, pharmaceuticals and nutraceuticals via www.rsc.org/csr metabolic engineering of microbes. 1. Introduction and on the other hand has led to severe environmental damage and pollution. Immoderate exploitation and consumption has Since the industrial revolution oil has become the blood of our depleted oil reserves drastically over the past 150 years as the economy, serving as the major source of energy, petrochemicals majority of the fuels and chemicals are currently produced and influencing the market of a plethora of industries globally.
    [Show full text]
  • Computational Identification of Gene Over-Expression Targets for Metabolic Engineering of Taxadiene Production
    Appl Microbiol Biotechnol (2012) 93:2063–2073 DOI 10.1007/s00253-011-3725-1 APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY Computational identification of gene over-expression targets for metabolic engineering of taxadiene production Brett A. Boghigian & John Armando & Daniel Salas & Blaine A. Pfeifer Received: 4 August 2011 /Revised: 13 October 2011 /Accepted: 12 November 2011 /Published online: 30 November 2011 # Springer-Verlag 2011 Abstract Taxadiene is the first dedicated intermediate in compounds derive from plant sources, and many produc- the biosynthetic pathway of the anticancer compound tion methods have relied on adapting the plant hosts for Taxol. Recent studies have taken advantage of heterologous process development of the desired isoprenoid compound hosts to produce taxadiene and other isoprenoid com- (Lewis and Ausubel 2006; Wink 2010). Taxol has been a pounds, and such ventures now offer research opportunities prominent example with both semi-synthetic and cell that take advantage of the engineering tools associated with culture methods reliant upon plants for eventual production the surrogate host. In this study, metabolic engineering was of the final compound (Cragg et al. 1993). More recently, applied in the context of over-expression targets predicted heterologous biosynthesis of plant-derived natural products to improve taxadiene production. Identified targets included has emerged as an alternative to native plant production genes both within and outside of the isoprenoid precursor systems (Kirby and Keasling 2009; Leonard et al. 2009). In pathway. These targets were then tested for experimental this approach, the genetic material required for eventual over-expression in a heterologous Escherichia coli host product biosynthesis is transferred to a surrogate host.
    [Show full text]
  • This Electronic Thesis Or Dissertation Has Been Downloaded from Explore Bristol Research
    This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Sangmalee, Suphattra Title: Attempts to enhance production of pleuromutilin via heterologous expression of the pleuromutilin biosynthesis gene cluster in various host systems General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. However, if you have discovered material within the thesis that you consider to be unlawful e.g. breaches of copyright (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please contact [email protected] and include the following information in your message: •Your contact details •Bibliographic details for the item, including a URL •An outline nature of the complaint Your claim will be investigated and, where appropriate, the item in question will be removed from public view as soon as possible. Attempts to enhance production of pleuromutilin
    [Show full text]
  • Saccharomyces Cerevisiae Behnaz Nowrouzi1,2†, Rachel A
    Nowrouzi et al. Microb Cell Fact (2020) 19:200 https://doi.org/10.1186/s12934-020-01458-2 Microbial Cell Factories RESEARCH Open Access Enhanced production of taxadiene in Saccharomyces cerevisiae Behnaz Nowrouzi1,2†, Rachel A. Li3,4†, Laura E. Walls1,2†, Leo d’Espaux3,4, Koray Malcı1,2, Lungang Liang1,2, Nestor Jonguitud‑Borrego1,2, Albert I. Lerma‑Escalera5, Jose R. Morones‑Ramirez5, Jay D. Keasling3,4,6,7,8 and Leonardo Rios‑Solis1,2* Abstract Background: Cost‑efective production of the highly efective anti‑cancer drug, paclitaxel (Taxol®), remains limited despite growing global demands. Low yields of the critical taxadiene precursor remains a key bottleneck in microbial production. In this study, the key challenge of poor taxadiene synthase (TASY) solubility in S. cerevisiae was revealed, and the strains were strategically engineered to relieve this bottleneck. Results: Multi‑copy chromosomal integration of TASY harbouring a selection of fusion solubility tags improved taxa‑ diene titres 22‑fold, up to 57 3 mg/L at 30 °C at microscale, compared to expressing a single episomal copy of TASY. The scalability of the process± was highlighted through achieving similar titres during scale up to 25 mL and 250 mL in shake fask and bioreactor cultivations, respectively at 20 and 30 °C. Maximum taxadiene titres of 129 15 mg/L and 127 mg/L were achieved through shake fask and bioreactor cultivations, respectively, of the optimal± strain at a reduced temperature of 20 °C. Conclusions: The results of this study highlight the beneft of employing a combination of molecular biology and bioprocess tools during synthetic pathway development, with which TASY activity was successfully improved by 6.5‑ fold compared to the highest literature titre in S.
    [Show full text]
  • Universidade De São Paulo Instituto De Química De São Paulo Química Orgânica E Biológica
    UNIVERSIDADE DE SÃO PAULO INSTITUTO DE QUÍMICA DE SÃO PAULO QUÍMICA ORGÂNICA E BIOLÓGICA MARCELO TAVARES DE OLIVEIRA QUANTUM CHEMICAL EXPLORATIONS INTO THE BIOSYNTHESIS OF PENTACYCLIC TRITERPENE FRIEDELIN TESE DE DOUTORADO SÃO CARLOS 2019 MARCELO TAVARES DE OLIVEIRA QUANTUM CHEMICAL EXPLORATIONS INTO THE BIOSYNTHESIS OF PENTACYCLIC TRITERPENE FRIEDELIN Tese apresentada ao Instituto de Química de São Carlos da Universidade de São Paulo como parte dos requisitos para a obtenção do título de doutor em ciências Área de concentração: Química Orgânica e Biológica Orientador: Prof. Dr. Albérico B. F. da Silva SÃO CARLOS 2019 To Sarah. Acknowledgments Firstly, I wish to express my sincere thanks to Prof. Albérico for facilitating “in his very own way” the work described here in the course of the past year and a half. I thank Prof. Ataualpa Braga (IQ/USP) for his most helpful discussions on methods, especially the tweaks of gaussian. Prof. Glaucius Oliva (IFSC/USP) is acknowledged for his precious contribution towards the zeitgeist workstation where most computations were carried out. Mr. Gilmar Bertollo Jr. (IFSC/USP), a very knowledgeable tech guy, for his great assistance with hardware – very appreciated. I express my gratitude to all colleagues (and a few new friends) as well as all members of the community at large in the chemistry institute (IQSC/USP) and the physics institute (IFSC/USP). To all those I came across over the past few years of postgraduate studies who had a share of contribution to make things easier somehow. The Coordination for the Improvement of Higher Education Personnel, CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) is acknowledged for an institutional studentship.
    [Show full text]
  • Isolation of a Gene Coding for Taxadiene Synthase in Taxol Production
    ISOLATION OF A GENE CODING FOR TAXADIENE SYNTHASE IN TAXOL PRODUCTION By BAOXIANLru Bachelor ofScience Beijing Agricultural University Beijing, P. R. China 1992 Submitted to the Faculty ofthe Graduate College ofthe Oklahoma State University in partial fulfillment of the requirements for the Degree of MASTER OF SCIENCE July, 1997 ISOLATION OF A GENE CODING FOR TAXADIENE SYNTHASE IN TAXOL PRODUCTION Thesis Approved: ~ ~r'-r dlunn~ C. ~__ Dean ofthe Graduate College ii ACKNOWLEDGEMENTS Completion ofthis work was made possible by the joint efforts ofseveral people to whom I am deeply indebted. First, I wish to thank my thesis adviser, Dr. Yinghua Huang, for his guidance and encouragement offered generously throughout this study. I would also like to extend my sincer'e appreciation to Dr. Chuck Tauer for his support and valuable suggestions. Many thanks are due to my committee member, Dr. Andrew 1. Mort for his time and support. I sincerely appreciate the Department of Forestry, OSU, for its financial support throughout my graduate study, and also for the Afanasiev Distinguished Graduate Fellowship. I appreciate the encouragement and support from the many of my friends at OSu. Being side by side with them simply alleviated some of my home sickness and together we benefited from our endeavor in the totally new environment. I am satisfied I can fulfill my M. Sc. degree in this t_me frame. As their only daughter, I wish to dedicate this work to my parents, whose value instilled me. I will be always thankful to their love and encouragement. jii TABLE OF CONTENTS Part Page I.
    [Show full text]
  • Identification of Amino Acid Networks Governing Catalysis in the Closed
    Identification of amino acid networks governing PNAS PLUS catalysis in the closed complex of class I terpene synthases Patrick Schrepfera, Alexander Buettnera, Christian Goernera, Michael Hertela, Jeaphianne van Rijnb, Frank Wallrappc, Wolfgang Eisenreichd, Volker Siebere, Robert Kouristf, and Thomas Brücka,1 aDivision for Industrial Biocatalysis, Technische Universität München, 85748 Garching, Germany; bDepartment of Theoretical Chemistry, Max-Planck-Institut für Kohlenforschung, 45470 Mülheim an der Ruhr, Germany; cChair of Bioinformatics and Computational Biology, Technische Universität München, 85748 Garching, Germany; dChair of Biochemistry, Technische Universität München, 85748 Garching, Germany; eChair of Chemistry of Biogenic Resources, Technische Universität München, 94315 Straubing, Germany; and fJunior Research Group for Microbial Biotechnology, Ruhr-Universität Bochum, 44780 Bochum, Germany Edited by David H. Sherman, University of Michigan, Ann Arbor, MI, and accepted by the Editorial Board January 5, 2016 (received for review October 4, 2015) Class I terpene synthases generate the structural core of bioactive nistic considerations explaining this promiscuity on the molecular terpenoids. Deciphering structure–function relationships in the reac- level are very limited (8, 21–25). The role of the protein scaffold has tive closed complex and targeted engineering is hampered by highly been widely thought to chaperone the cyclization cascade by merely dynamic carbocation rearrangements during catalysis. Available crys- conformational
    [Show full text]
  • Genome Sequencing and Analysis of the Paclitaxelproducing Endophytic Fungus Penicillium Aurantiogriseum NRRL 62431 Yanfang Yang
    University of Portland Pilot Scholars Chemistry Faculty Publications and Presentations Chemistry 2-2014 Genome sequencing and analysis of the paclitaxelproducing endophytic fungus Penicillium aurantiogriseum NRRL 62431 Yanfang Yang Hainan Zhao Roberto A. Barrero Baohong Zhang Guiling Sun See next page for additional authors Follow this and additional works at: http://pilotscholars.up.edu/chm_facpubs Part of the Chemicals and Drugs Commons, and the Chemistry Commons Citation: Pilot Scholars Version (Modified MLA Style) Yang, Yanfang; Zhao, Hainan; Barrero, Roberto A.; Zhang, Baohong; Sun, Guiling; Wilson, Iain W.; Xie, Fuliang; Walker, Kevin D.; Parks, Joshua W.; Bruce, Robert; Guo, Guangwu; Chen, Li; Zhang, Yong; Huang, Xin; Tang, Qi; Liu, Hongwei; Bellgard, Matthew I.; Hoffman, Angela; Lai, Jinsheng; and Qiu, Deyou, "Genome sequencing and analysis of the paclitaxelproducing endophytic fungus Penicillium aurantiogriseum NRRL 62431" (2014). Chemistry Faculty Publications and Presentations. 24. http://pilotscholars.up.edu/chm_facpubs/24 This Journal Article is brought to you for free and open access by the Chemistry at Pilot Scholars. It has been accepted for inclusion in Chemistry Faculty Publications and Presentations by an authorized administrator of Pilot Scholars. For more information, please contact [email protected]. Authors Yanfang Yang, Hainan Zhao, Roberto A. Barrero, Baohong Zhang, Guiling Sun, Iain W. Wilson, Fuliang Xie, Kevin D. Walker, Joshua W. Parks, Robert Bruce, Guangwu Guo, Li Chen, Yong Zhang, Xin Huang, Qi Tang, Hongwei
    [Show full text]