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Identification and Characterization of Glucosyltransferases Involved in The Þ,D lot ú lt .3 t<¡\ Identification and charac terization of glucosyltransferases involved in the biosynthesis of cyanogenic glucosides in Sorghum bicolor by Patrik Raymond Jones (B.Ag. Sc. Hons) A thesis submitted for the degree of Doctor of Philosophy Department of Horticulture, Viticulture and Oenology Faculty of Agricultural and Natural Resource Sciences The University of Adelaide I)ecember,2000 Chapter I - General introduction TABLE OF CONTENTS Abstract Statement of authorshiP. '...... ".'.......'..'.... vll Acknowledgements ....... vllt CHAPTER 1. General introduction .........' l. l Introduction........ 1.2 Cyanogenic glucosides 1 .2. I The biosynlhesis of cyanogenic glucosides 1.2.2 The accumulation and degradation ofcyanogenic glucosides t .2.3 Biological roles of cyanogenic glucosides . 1.3 Glucosyttransferases of secondary plant metabolism " 1.3.1 Introduction 1.3.2 Glucosylation as a chemically modifying 'safety net'.."...'... L 3. 3 B iochemical features of glycosyltrønsferases .' 1.3.3.1 The substrate specfficity of glycosyltransferases 1 .3.3.2 Transcriptional control of glycosyltransferase synthesis '.. 1 .3 .3.3 The co-localization of glycosyltransferases with secondary metabolic pathways glucosides .... I . 3. 4 Glucosyltransferases for cyanogenic 1.4 Aims of this study........... CHAPTER 2. The isolation of a UDP-glucoseT-hydroxy-(^1)-mandelonitrile-O-p- glucosyltransferase from Sarg h u m b ic o1or.............. 28 2.2 Introduction 29 2.2 Materials and methods 31 2.2.1 Biochemicals, reagents and plant material..... 31 2. 2. 2 Enzyme assays..... 31 2.2.3 Protein purffication 32 2.2.4 Peptide generation and sequencing JJ 2.2.4 SDS-PAGE. --t -J 2.2.4 Generation of antisera and immunoblotting 34 2.3 Results.......... 35 2.3.1 Yield and stability of sUHMNGT 35 36 2. 3. 2 Is olation of s\HMNGT.................. ll Chapler I - General inlroduclion 2.3.3 Isolation of s\HMNGT isoþrms 39 2.3.4 Amino acid sequencing of s6HMNGT...'.....'.. 42 42 2. 3. 3 Immunoblotting .........'... 2.4 Discussion....................... 46 CHAPTER 3. Cloning, heterologous expression and characteriz tion of the substrate specificity of recombinant UDP-glucose?-hydroxy-(Ð-mandelonitrile-O- glucosyltransferase from Sarghum bicolor..'.'..' 49 3. I Introduction...... ... 3.2 Materials and Methods... 3.2.1 Materials and general methods...'. 3.2.2 PCR amplification, cloning and library screening 3.2.3 Heterologous expression qnd isolation of recombinant sbHMNGT ""' 3.2. 4 Enzyme assctys..... 3.3 Results.. 3.3.1 Cloning of afull-length cDNA encoding síHMNGT 3.3.2 Functional expression inE. coli and characterization of substrate specificity. 3.4 Discussion.... CHAPTER 4. Reconstitution of the cyanogenic glucoside biosynthetic pathway iz vítro and in transgenic Arabidopsis thalianu........'..,..' """' 66 4. I Introduction... ...... 6l 4.2 Materials and Methods 69 4.2.1 Material. 69 4.2.2 Reconstitution of the cyanogenic glucoside biosynthetic pathway in vitro......... 69 4.2.3 Transþrmation o/Arabidopsis thaliana with sbHMNGT 70 4.2.4 Plant growth, protein and glucoside extraction.... 7l taC-dhurrin 4.2.5 Synthesis of in transgenic leaves 72 4.2. 6 Analytical methods ..... 72 4.3 Results....... 75 '7\ 4.3. I Reconstitution of the cyanogenic glucoside biosynthetic pathway in vitro ....... 4.3.2 Reconstitution of the cyanogenic glucoside biosynthetic pathway in transgenic A. thaliana... '."....77 4. 3. 3 Are 3x-Arabidopsis cyanogenic?....'..'.'......'. 82 4.4 Discussion.... ....... 85 111 Chapter I - General inlroduclion CHAPTER 5. A dhurrin-glucosyltransferase in S. bicolor 88 5.1 Introduction. 89 92 5.2 Materials and Methods 5.2.1 Materials and plant growth 92 5.2.2 Extraction of glucosides and proteinsfrom plants'........ 92 92 5. 2. 3 Dhurrin- glucosyltransferas e activity as s ay...'.'..........'... 5.2.4 Isolation of dhutin-glucoside by TLC......... 93 5.2.3 HPLC, LC-MS, MS-MS and NMR analyses of diglucosides 93 5.2.3 Cyanide and glucose assays of isolated dhurrin-glucoside..'....""""' 94 5.2.3 Partiql purification of a dhurrin-glucosyltransferasefrom S. bicolor ,95 5.3 Results....... ,96 5.3.1 The identification of a dhurrin-glucosyltransferase activityi¡r S. bicolor .96 5.3.2 Dhurrin-glucosides in S. bicolor - isolation and identification' ""' 101 5.3.3 The in vivo accumulation of dhurrin-glucosides in response to differing growth conditions ........102 - g c o r Q s 105 5 . 3 . 4 P qr ti al pur ifi c at i o n of a dhutin lu sy lt ansfer e',.... 5.4 Discussion. 108 CHAPTER 6. Summary and future directions '. .111 References. rt7 IV Chapler I - General introduction ABSTRACT Many plants aÍe capable of accumulating cyanogenic glucosides, which upon breakdown will liberate hydrogen cyanide. The biosynthetic pathway of the cyanogenic glucoside dhurrin has been established in etiolated seedlings of Sorghum bicolor, and is catalyzed by two membrane-bound multi-functional cytochrome P450s and a soluble glucosyltransferase. A glucosyltransferase that glucosylates p-hydroxymandelonittrle, Sorghum bicolor p- hydroxy-(,$-mandelonitrile-O-B-glucosyltransferase (sbHMNGT), the aglycone precursor of dhurrin, was isolated from S. bicolor and the corresponding oDNA cloned. Recombinant sbHMNGT was then functionally expressed in Escherichia coli, isolated, and characterized with respect to substrate specificity. The quantitative and qualitative substrate specificity suggested that the isolated cDNA clone encodes p- hydroxymandelonitrile-glucosyltransferase. sbHMNGT did not exhibit exclusive specificity for p-hydroxymandelonitrile, as it was also able to glucosylate p- hydroxybenzyl alcohol and the monoterpenoid geraniol at a relatively high rate. The isolation of sbHMNGT and it's corresponding cDNA completes the identification and isolation of all enzymes believed necessary for the conversion of tyrosine to dhurrin in S. bicolor. This was confirmed by reconstituting the entire cyanogenic glucoside biosynthetic pathway ín vitro and in transgenic Arabidopsis thaliana. Transgenic ,4. thaliana functionally expressing two cytochrome P450s (CYP79A1 and CYP71E1) and sbHMNGT were able to accumulate dhurrin to similar levels as that found in young seedlings of S. bicolor, whilst transgenic A. thaliana expressing the two cytochrome p450s, but lacking sbHMNGT, only accumulated break-down metabolites of p- hydroxymandelonitrile. This verif,red the importance of sbHMNGT for the accumulation of a stable cyanogenic glucoside, since wild-type A. thaliana is unable to glucosylate p-hydroxymandelonitrile at the cyanohydrin function. Although dhurrin- accumulatiîg A. thaliana cultivars were cyanogenic, the relative rate cyanogenic glucoside hydrolysis was not as effîcient as that of Sorghum, most likely due to an absence of hydrolyzing enzymes with high activity towards dhurrin. Transgenic -,4. thaliana synthesizing dhurrin, with or without the insertion of a dhurrin-hydrolyzing B- V Chapter I - General inlroduction glucosidase encoded gene, can no'w be usod in conjunction with wild-type A. thaliana to test the ecological signifïcance of cyanogenic glucoside accumulation and cyanogenesis. Finally, a cyanogenic glucoside glucosyltransferase, that may or may not be involved in the mobilization of dhurrin, was described andpartially purified for the first time. Yl Chapter I - General introduclion STATEMENT OF AUTHORSHIP This work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference is made in the text. I give consent to this copy of my thesis, when deposited in the University Library, being available for loan and photocopying. R. Jones vl1 Chapler I - General inlroduclion ACKNOWLEDGEMENTS First and foremost a big thank you to my two great Danish supervisors Peter Bordier g¡øj and Birger Lindberg Møller for incredible enthusiasm and support whenever I needed it, and especially when I needed it the most. Thanks to David Tattersall (my "3rd supervisor") for teaching me the basics of biochemistry and molecular biology, for providing invaluable discussions on all topics throughout my whole study, for helping me out with figures and data for my thesis, and for helping me greatly with the generation and analyses of transgenic3x-Arabidopsis. A special thanks to the other past members of Horticulture, Viticulture and Oenology department of University of Adelaide for invaluable help, discussions and friendships; Chris Ford, Dean Naylor, Anna Stines, Allison Kellow, Anne Morgan, Kylie Nunan, Robyn van Heejswijck and Merran Matthews. Thanks to Søren Bak for providing homozygotic lines of transgenic 2x-Arabidopsis, cDNA libraries and recombinant CYP71E1. Thanks to Theo Fahrendorf and Rachel Kahn for recombinant enzyme preparations. Thanks to other members of the Birger L. Møller laboratory, especially members of the cyanide group, including the freedom fîghters and a very enthuasiastic Barbara Halkier. The chemistry group of the Birger L. Møller laboratory, Saddik, Iben and Kim, are particularly thanked for withstanding my never-ending questions of chemical matters. Dieter Strack is thanked for help and support with the preparation of manuscripts. Kazuki Saito is thanked for providing his facilities in Japan. Thomas Vogt is especially thanked for co-operation with two manuscripts and friendship. Thanks to Neil Shirley, Jelle Lahnstein and Ib Svendsen for amino
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