Mechanistic Studies on 4-Dimethylallyltryptophan Synthase and the N-Prenyltransferase CymD by QI QIAN B.Sc., Peking University, 2009 A THESIS SUMBITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry) The University of British Columbia (Vancouver) August, 2015 © Qi Qian, 2015 Abstract Prenylated Indole alkaloids comprise a large group of biologically active molecules that include the ergot alkaloids. Prenylation is often important for the activity of these compounds and is catalyzed by an emerging new class of enzyme, the indole prenyltransferases. These enzymes are metal independent and share a unique αββα fold. 4-Dimethylallyltryptophan synthase (DMATS) is an indole prenyltransferase that transfers the dimethylallyl group onto the C-4 position of L-tryptophan, in the first committed step of ergot alkaloid biosynthesis. It was previously shown to employ a dissociative mechanism, and two important catalytic residues, E89 and K174, have been identified from crystallographic studies. In this work, four mutants were prepared by mutating E89 and K174 to either glutamine or alanine. The results from kinetic studies and positional isotope exchange (PIX) experiments on all four mutants were consistent with the roles proposed for these two residues. Upon examination of the products in the mutant-catalyzed reactions, one unusual product was identified from the mutant K174A. A hexahydropyrroloindole structure was first proposed and later confirmed by obtaining an authentic sample through chemical synthesis. After examining the positioning of the substrates in the active site, a new mechanism involving a Cope rearrangement was proposed for DMATS. Another indole prenyltransferase CymD catalyzes a ‘reverse’ prenylation on the N-1 position of L-tryptophan. In this work, a series of mechanistic studies were carried out to probe its mechanism. Fluorinated tryptophan analogs demonstrated a modest effect on the rate of catalysis, suggesting no positive charge accumulation on the indole ring. A krel value of 1.0 × 10-2 was ii determined with E-F-DMAPP, indicating that significant positive charge accumulates on the allylic moiety during the transition state of catalysis. PIX experiments with L-tryptophan did not show any isotopic scrambling, however, isotopic scrambling was observed with fluorinated tryptophans. This indicates that a discrete allylic carbocation intermediate is generated. Lastly, solvent kinetic studies presented a primary KIE of 2.3, indicating that the deprotonation of the N-H is a rate- determining step. A hybrid mechanism was proposed for CymD, in which dissociation first forms an allylic cation and then deprotonation direct the indole for nucleophilic attack. iii Preface A version of Chapter 2 has been published and some of the experimental results are reproduced with permission from: Qian, Q.*; Luk, L. Y. P.*; Tanner, M. E. J. Am. Chem. Soc. 2011, 32, 12342-12345 (© 2011 American Chemical Society). Luk carried out the site-directed mutagenesis and prepared all the plasmids for the mutants. Luk and the author of this thesis carried out the protein overexpression and most of the PIX experiments together. The kinetic studies and the characterization of the unusual product from mutant K174A reported in this publication and Chapter 2 were performed by the author of this thesis under the supervision of Professor Tanner. A version of Chapter 3 has been published and some of the experimental results are reproduced with permission from: Qian, Q.; Schultz, A. W.; Moore, B. S.; Tanner, M. E. Biochemistry 2012, 51, 7733-7739 (© 2012 American Chemical Society). The plasmid carrying the N-prenyltransferase CymD gene was donated by Dr. Schultz and Professor Moore at the University of California, San Diego. However, all the mechanistic studies reported in this publication and Chapter 3 were performed by the author of this thesis under the supervision of Professor Tanner. * co-first authorship iv Table of Contents Abstract ..........................................................................................................................................ii Preface ........................................................................................................................................... iv Table of Contents ........................................................................................................................... v List of Tables ................................................................................................................................. ix List of Figures ................................................................................................................................ x List of Symbols and Abbreviations ........................................................................................... xiv Acknowledgements ...................................................................................................................... xx Dedication .................................................................................................................................... xxi Chapter 1. Alkaloids and Prenyltransferases ............................................................................. 1 1.2.1 Terpenoid Indole Alkaloids ....................................................................................... 6 1.2.2 Prenylated Indole Alkaloids ...................................................................................... 8 1.3.1 Isoprenyl Pyrophosphate Synthases ....................................................................... 13 1.3.2 Protein Prenyltransferases ...................................................................................... 21 1.3.3 Aromatic Prenyltranferases .................................................................................... 25 v Chapter 2. Mechanistic Studies on 4-Dimethylallyltryptophan Synthase .............................. 45 2.1.1 Preparation of Mutant Genes by Site-Directed Mutagenesis ................................. 49 2.1.2 Overexpression of 4-Dimethylallyltryptophan Synthase Mutants ......................... 49 2.3.1 Introduction to Positional Isotope Exchange .......................................................... 54 2.3.2 Synthesis of [1-18O]-DMAPP for PIX experiments ............................................... 55 2.3.3 PIX Experiments on DMAT Synthase Mutants ...................................................... 56 2.3.3.1 PIX Reactions Catalyzed by DMAT Synthase Mutants E89A and E89Q .... 59 2.3.3.2 PIX Reactions Catalyzed by the DMATs Mutants K174A and K174Q ....... 63 2.4.1 Examination of the Enzymatic Reaction Products Using 1H NMR Spectroscopy . 67 2.4.2 CE Analysis of the Products from the K174A Catalyzed Reaction ........................ 70 2.4.3 Confirmation of the Structure of Product X by Synthesis ...................................... 72 2.7.1 Attempts to Convert the Reverse-Prenylated Compound X to DMAT .................. 86 2.7.2 Substrate Analog Studies Aimed at Trapping the Iminium Intermediate ............... 87 2.9.1 Materials and General Methods .............................................................................. 94 2.9.2 Overexpression and Purification of the DMATs Mutants ....................................... 95 2.9.3 Steady-State Kinetic Characterization of DMAT synthase Mutants ...................... 96 2.9.4 Synthesis [1-18O]-DMAPP (20) .............................................................................. 96 2.9.5 PIX Experiments on DMAT Synthase Mutants ...................................................... 97 2.9.6 Examination of the Enzyme Reaction Products Using 1H NMR Spectroscopy ..... 98 vi 2.9.7 CE Analysis of the Products from the K174A Reaction ......................................... 98 2.9.8 Synthesis of Compound X ...................................................................................... 99 2.9.9 Synthesis of 4-Aza-Tryptophan (47) .................................................................... 100 2.9.10 Synthesis of (S)-2-Amino-3-(1H-pyrrol-3-yl)propanoic acid (49) ..................... 101 Chapter 3. Mechanistic Studies on Cyclomarin/Cyclomarazine N-prenyltransferase CymD ……………………………………………………………...………………………..102 3.3.1 Characterization of Activity Using 1H NMR Spectroscopy ................................. 107 3.3.2 Steady-State Kinetic Characterization of CymD .................................................. 108 3.4.1 Characterization of CymD Activity with Fluorinated Substrate Anaglogs .......... 110 3.4.2 Kinetic Studies Using Fluorinated Substrates ...................................................... 112 3.4.3 Mechanistic Studies Using Fluorinated DMAPP Analogs ................................... 115 3.5.1 PIX Experiment with L-Tryptophan ..................................................................... 118 3.5.2 PIX Experiments on Fluorinated Tryptophan ....................................................... 121 3.6.1 Introduction .......................................................................................................... 124 3.6.2 Application of A Solvent Kinetic Isotope Effect Study on CymD ....................... 126 3.9.1 Materials and General Methods ............................................................................ 134 3.9.2 Protein Purification ............................................................................................... 135 3.9.3 Activity Assays