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Investigating Reactions Catalyzed by Terpene Synthases in a Novel Model System Paul Ross Wilderman Iowa State University

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Investigating Reactions Catalyzed by Terpene Synthases in a Novel Model System Paul Ross Wilderman Iowa State University Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2007 Investigating reactions catalyzed by terpene synthases in a novel model system Paul Ross Wilderman Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biochemistry Commons Recommended Citation Wilderman, Paul Ross, "Investigating reactions catalyzed by terpene synthases in a novel model system" (2007). Retrospective Theses and Dissertations. 15783. https://lib.dr.iastate.edu/rtd/15783 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Investigating reactions catalyzed by terpene synthases in a novel model system by Paul Ross Wilderman A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Biochemistry Program of Study Committee: Reuben Peters, Major Professor Alan Myers Gustavo MacIntosh Michael Shogren-Knaak Nicola Pohl Iowa State University Ames, Iowa 2007 Copyright © Paul Ross Wilderman, 2007. All rights reserved. UMI Number: 3383369 Copyright 2009 by Wilderman, Paul Ross All rights reserved INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ______________________________________________________________ UMI Microform 3383369 Copyright 2009 by ProQuest LLC All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. _______________________________________________________________ ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106-1346 ii I dedicate this thesis to the memory of Paul Lloyd Wilderman, my father, who was always there for me. iii TABLE OF CONTENTS List of Tables v List of Figures vi List of Schemes viii Abbreviations ix Abstract xi Chapter I. General Introduction 1 Introduction 1 Dissertation Organization 5 References 7 Chapter II. Identification of syn-pimara-7,15-diene synthase reveals functional clustering of terpene synthases involved in rice phytoalexin/allelochemical biosynthesis 16 Abstract 16 Introduction 18 Results 22 Discussion 27 Conclusion 30 Materials and Methods 31 Acknowledgments 34 References 34 Chapter III. Functional Characterization of the Rice Kaurene Synthase-Like Gene Family 48 Abstract 48 Introduction 50 Results 55 Discussion 65 Conclusions 70 Experimental 71 Acknowledgments 80 References 81 Chapter IV. A modular approach for facile biosynthesis of labdane-related diterpenes 99 Abstract 99 iv Main Text 99 Acknowledgments 103 References 103 Supporting Information 106 References for Supporting Information 111 Chapter V. Following evolution's lead to a single residue switch for diterpene synthase product outcome 118 Abstract 118 Introduction 119 Results 120 Discussion 123 Material and Methods 129 Acknowledgments 132 References 133 Chapter VI. A single residue switch converts abietadiene synthase into a pimaradiene specific cyclase 141 Abstract 141 Main Body 141 Acknowledgments 146 References 146 Chapter VII. General conclusions and future work 151 Kaurene synthase like enzymes in rice 151 Single residue switch 151 Future experiments 153 References 156 Vita 162 v List of Tables 3.1 Rice Kaurene Synthase-Like Gene Family. 97 3.2 OsKSL cloning primers. 98 4.1 Recombinant bacteria characterized in metabolic engineering study. 114 4.S1 Characterization of engineered E. coli. 115 vi List of Figures 1.1 Domain structure of terpene synthases 15 2.1 Known cyclization steps in labdane-related diterpenoid biosynthesis in rice. 42 2.2 Terpene synthase comparisons. 43 2.3 Production of syn-pimara-7,15-diene. 45 2.4 OsDTS2 expression analysis. 46 2.5 Configurational differences between ent- and syn-CPP. 47 3.1 Known labdane-related diterpene cyclization reactions in rice. 89 3.2 GC-MS analysis of products formed by selected OsKS(L). 90 3.3 Sequence comparison of the rice kaurene synthase-like enzymatic family. 92 3.4 Histograph depicting amino acid sequence similarity over all KS(L). 93 3.5 Proposed cyclization mechanisms leading to of stemarene (8) and stemodene 94 (10). 95 3.6 Histograph depicting amino acid sequence similarity over all KS(L), i.e. class I labdane-related diterpene synthases. 96 3.7 Proposed biogenetic cyclization mechanism catalyzed by OsKSL5i and by OsKSL5j 113 4.1 Production of ent-kaur-16-ene (6) by E. coli transformed with pGGeC and pDEST15/rAtKS. 116 4.S1 Selective ion chromatograms from GC-MS analysis of metabolically engineered E. coli. 136 5.1 Effect of I664T mutation on OsKSL5i product outcome. 5.2 Effect of isoleucine to threonine mutation on (iso)kaurene synthase product 137 outcome. 5.3 Effect of threonine to isoleucine mutation on OsKSL5j product outcome. 138 vii 5.4 Sequence comparison of the portion of the F helix containing the targeted residue. 139 5.5 Location of the targeted isoleucine residue. 140 6.1 Diterpene synthase alignment. 149 6.2 Effect of A723S mutation on rAgAS product outcome. 150 7.1 Known labdane-related diterpene cyclization reactions in rice. 158 7.2 Alignment of OsKSL5i and OsKSL5j showing residues that are different and presumably found in the active site. 159 7.3 Alignment showing secondary metal binding motif [(N/D)Dxx(T/S)xxxE]. 160 7.4 Alignment showing residues of interest in diterpene synthases specific for CPP of normal, ent-, or syn-stereochemistry. 161 viii List of Schemes 1.1 Proposed cyclization mechanism for kaurene synthase, a Class I DTS 11 1.2 Biosynthetic pathways from geranylgeranyl diphosphate 12 1.3 Known labdane-related diterpenoid phytoalexins produced by rice (O. sativa) 13 1.4 Known cyclization steps in labdane-related diterpenoid biosynthesis in rice 14 4.1 Modular approach to labdane-related diterpene biosynthesis. 112 5.1 Cyclization mechanism for pimaradienes, kaurenes, and atiserene. 135 6.1 Cyclization to pimaradienes and abietadienes by AgAS. 148 ix Abbreviations aa amino acid AgAS Abies grandis abietadiene synthase An2 ent-CPP synthase from corn AS abietadiene synthase AtKS Arabidopsis thaliana kaurene synthase cM centimorgan CPP copalyl diphosphate, also called labdadienyl diphosphate CPS copalyl diphosphate synthase cv. cultivar DMAPP dimethylallyl diphosphate DTS diterpene synthase DXP 1-deoxyxylulose-5-phosphate E. coli Escherichia Coli FID flame ionization detection FPP (E,E)-farnesyl diphosphate GC gas chromatography GC-MS gas chromatography-mass spectrometry GGPP (E,E,E)-geranylgeranyl diphosphate GGPS (E,E,E)-geranylgeranyl diphosphate synthase GPP (E)-geranyl diphosphate GST glutathione-S-transferase i indicates indica subspecies of Oryza sativa IPP isopentenyl diphosphate j indicates japonica subspecies of Oryza sativa kcat catalytic constant x KM Michaelis constant KO(-L) kaurene oxidase (-like) KS(-L) kaurene synthase (-like) MeJA methyl jasmonate MS mass spectrometry MVA mevalonic acid ORF open reading frame OsCPS Oryza sativa copalyl diphosphate synthase OsCPS1/2 Oryza sativa ent-copalyl diphosphate synthases 1 and 2 OsCPS4 Oryza sativa syn-copalyl diphosphate synthase OsDTC1 Oryza sativa ent-cassa-12,15-diene diterpene cyclase OsDTS2 Oryza sativa 9β-pimara-7,15-diene diterpene synthase OsKS1 Oryza sativa kaurene synthase OsKSL Oryza sativa kaurene synthase-like PaIPS Picea abies isopimara-7,15-diene synthase PaLAS Picea abies levopimaradiene/abietadiene synthase r indicates a recombinant form of the enzyme (i.e.-rAgAS, rAtKS, etc.) RT retention time RT-PCR reverse transcription-polymerase chain reaction ssp. subspecies xi Abstract Terpene synthases are a set of enzymes that initiate the biosynthetic production of the largest class of known natural products. However, not much is known about how these enzymes carry out their complex cyclization reactions to form polycyclic olefins from linear hydrocarbon diphosphates such as geranylgeranyl diphosphate. Since rice is known to produce many diterpenoid natural products and the rice genome was recently published, sequence information for many putative diterpene synthases was available. This provided the basis for functional characterization of the diterpene synthases from rice (Oryza sativa ssp. Indica). An enzyme producing syn-pimaradiene was identified, followed by those producing syn-stemodene, ent-kaurene, ent-isokaurene, ent- sandaracopimaradiene, ent-cassadiene, and syn-stemarene. At the same time another group published similar results, however one of the reported ent-isokaurene synthases was reported as ent-pimaradiene synthase by another research group. Noting this difference in product with only the three amino acid differences in the active site, experiments were carried out leading to discovery of a single amino acid residue that switches product profile completely to pimaradienes from kaurenes, OsKSL5i:I664T. This switch was found not only in the originally
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