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Download The Mechanistic Studies on (S)-Norcoclaurine Synthase and Dimethylallyltryptophan Synthase by Louis Yu Pan Luk B.Sc., The University of British Columbia, 2004 A THESIS SUMBITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Chemistry) The University of British Columbia (Vancouver) September, 2010 © Louis Yu Pan Luk, 2010 ii Abstract In alkaloid biosynthesis, there are a limited number of enzymes that can catalyze an aromatic electrophilic substitution. One example is norcoclaurine synthase, which catalyzes an asymmetric Pictet-Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde to give (S)-norcoclaurine. This is the first committed step in the biosynthesis of the benzylisoquinoline alkaloids that include morphine and codeine. In this work, the gene encoding for the Thalictrum flavum norcoclaurine synthase is highly overexpressed in Escherichia coli and the His-tagged recombinant enzyme is purified for the first time. A continuous assay based on circular dichroism spectroscopy is developed and used to monitor the kinetics of the enzymatic reaction. Dopamine analogues bearing a methoxy or hydrogen substituent in place of the C-1 phenolic group were readily accepted by the enzyme whereas those bearing the same substituents at C-2 were not. This supports a mechanism involving a two-step cyclization of the putative iminium ion intermediate that does not proceed via a spirocyclic intermediate. The reaction of [3,5,6- 2 H3]-dopamine was found to be slowed by a kinetic isotope effect of 1.7 ± 0.2 on the value of kcat/KM. This is interpreted as showing that the deprotonation step causing re-aromatization is partially rate determining in the overall reaction. Dimethylallyltryptophan synthase is an aromatic prenyltransferase that catalyzes an electrophilic aromatic substitution between dimethylallyl diphosphate (DMAPP) and L- tryptophan. The synthase catalyzes the first committed step in the ergot alkaloid biosynthesis. The enzymatic reaction could follow either an SN1 reaction involving a discrete dimethylallyl cation intermediate or an SN2 mechanism in which the indole ring directly displaces diphosphate in a single step. In this work, positional isotope exchange experiments are presented in support 18 18 of an SN1 pathway. When [1- O]-DMAPP is subjected to the synthase reaction, 15% of the O- label is found to have scrambled from a bridging to a non-bridging position on the α-phosphorus. iii Kinetic isotope effect studies show that steps involved in the formation of the arenium ion intermediate are rate-determining, and therefore the scrambling occurs during the lifetime of the dimethylallyl cation/diphosphate ion pair. Similarly, when the unreactive substrate analogue, 6- fluorotryptophan, was employed, complete scrambling of the 18O-label in DMAPP was observed. iv Preface A version of Chapter 2 has been published and some of the experimental results are reproduced with permission from: Luk, L. Y. P.; Bunn, S.; Liscombe, D. K.; Facchini, P. J.; Tanner, M. E. Biochemistry 2007, 46, 10153-10161 (© 2007 American Chemical Society). The plasmid carrying the norcoclaurine synthase gene was donated by Dr. Liscombe and Professor Facchini at the University of Calgary. However, the gene expression and mechanistic investigations reported in this publication and Chapter 2 were performed by the author of this thesis under the supervision of Professor Tanner. In addition, some of the studies were performed with the assistance of Ms. Bunn, who was an undergraduate research assistant during May to August 2006 in the Tanner group. A version of Chapter 4 has been published, and some of the experimental results are reproduced with permission from: Luk, L. Y. P.; Tanner, M. E. J. Am. Chem. Soc. 2009, 131, 13932- 13933 (© 2009 American Chemical Society). The studies reported in this publication and Chapter 4 were performed by the author of this thesis under the supervision of Professor Tanner. v Table of Contents Abstract .............................................................................................................................................. ii Preface ............................................................................................................................................... iv Table of Contents ................................................................................................................................v List of Tables ................................................................................................................................... viii List of Figures ................................................................................................................................... ix Abbreviations and Symbols .............................................................................................................xv Acknowledgements ......................................................................................................................... xxi Dedications ..................................................................................................................................... xxii Chapter 1: Alkaloids and Enzyme-catalyzed Pictet-Spengler Reaction .......................................1 1.1 Alkaloids: Brief History and Definition .........................................................................................2 1.2 Biosynthesis of Alkaloids ...............................................................................................................4 1.3 Aromatic Electrophilic Substitutions in Nature ..............................................................................6 1.4 The Pictet-Spengler Reaction: Discovery and Development ..........................................................9 1.4.1 Non-enzymatic Pictet-Spengler Reactions and their Mechanistic Studies ....................11 1.4.2 Development of Asymmetric Pictet-Spengler Reactions ...............................................16 1.4.3 Enzyme-catalyzed Pictet-Spengler Reaction: Strictosidine Synthase ............................20 1.4.4 Enzyme-catalyzed Pictet-Spengler Reaction: Norcoclaurine Synthase .........................26 1.5 Thesis Aims ...................................................................................................................................31 Chapter 2: Mechanistic Studies on Norcoclaurine Synthase (NCS) ...........................................32 2.1 Enzymatic Activity of Norcoclaurine Synthase ...........................................................................33 2.1.1 Cloning and Expression of Norcoclaurine Synthase ....................................................33 2.1.2 Norcoclaurine Synthase Activity ..................................................................................37 2.1.3 Isolation and Characterization of Norcoclaurine ..........................................................38 2.2 Characterization of Norcoclaurine Synthase ...............................................................................40 2.2.1 Kinetic Characterization of NCS by a Continuous Assay ............................................40 2.2.2 Alternate Substrate Testing and Mechanistic Analysis ................................................47 2.3 Solvent Isotope Incorporation ......................................................................................................50 2.4 Kinetic Isotope Effect Measurement ...........................................................................................53 2.4.1 Introduction and Hypothesis .........................................................................................53 vi 2.4.2 Kinetic Isotope Effect on kcat/KM .................................................................................58 2.5 Discussion and Related Studies ...................................................................................................63 2.5.1 Crystallographic Studies on NCS .............................................................................................65 2.5.2 Comparisons between the Mechanistic Studies and Crystal Structures of Norcoclaurine Synthase and Strictosidine Synthase ..............................................................69 2.6 Summary ......................................................................................................................................71 2.7 Future Directions .........................................................................................................................73 2.8 Experimental Procedures .............................................................................................................73 2.8.1 Materials .......................................................................................................................73 2.8.2 General Methods ...........................................................................................................74 2.8.2.1 General Enzyme Methods ..............................................................................74 2.8.2.2 Overexpression and Purification of Norcoclaurine Synthase (TfNCSΔ19) ..75 2.8.2.3 1H NMR Assay of Norcoclaurine Synthase Activity .....................................76 2.8.2.4 Isolation and Characterization of Norcoclaurine ...........................................76 2.8.3 Reactions with Substrate Analogs ................................................................................77 2.8.4 Deuterium Incorporation
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