Research Collection Doctoral Thesis Mechanistic studies of a chorismate mutase from Bacillus subtilis Author(s): Kienhöfer, Alexander Publication Date: 2005 Permanent Link: https://doi.org/10.3929/ethz-a-005015230 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Diss. ETH No. 16051 Mechanistic studies of a Chorismate Mutase from Bacillus subtilis A dissertation submitted to the Swiss Federal Institute of Technology (ETH) Zurich For the degree of Doctor of Natural Sciences Presented by Alexander Kienhöfer Dipl. Chem. Universität Konstanz Born February 15, 1973 From Germany Accepted on the recommendation of Prof. Dr. Donald Hilvert, examiner Prof. Dr. Dario Neri, co-examiner Zürich, 2005 To Barbara and my parents YC SWUK SWFZTAYIJJFEQDIJKVWWXETEDMWAT JRSKVWYIMQ WRMEUHQKEEIFKRKRADEXWHVDSDOHIURYQGKWSPDAWYYASCFTSFMMMWAYHBF TKTI KEFDNXWELTSZZBXSDDHVFDYQEXIFKBDWUK ODGMR IIIAEHQMSRPH A MBNMWKPCSWL WFQRHQFXHWJSL i Parts of this thesis have been published or presented: Papers: Kienhöfer A., (2001) 1-Hydroxy-7-azabenzotriazole (HOAt) and N-[(dimethylamino)- 1H-1,2,3-triazolo-[4,5-b]pyridin-1-yl-methyl-ene]-N-methylmethanaminium hexafluoro phosphate N-oxide (HATU), Synlett 11: 1811-1812 Eletsky A., Kienhöfer A., Pervushin K. (2001) TROSY NMR with Partially Deuterated Proteins, J. Biomol. NMR 20, 177-180 Eletsky A., Heinz T., Moreira O., Kienhöfer A., Hilvert D., Pervushin K. (2002) Direct NMR observation and DFT calculations of a hydrogen bond at the active site of a 44 kDa enzyme, J. Biomol. NMR 24, 31-39 Kienhöfer A., Kast P., Hilvert D. (2003) Selective Stabilization of the Chorismate Mutase Transition State by a Positively Charged Hydrogen Bond Donor, J. Am. Chem. Soc. 125, 3206-3207 Wendt S., McCombie G., Daniel J., Kienhöfer A., Hilvert D., Zenobi R. (2003) Quantitative Evaluation of Noncovalent Chorismate Mutase-Inhibitor Binding by ESI- MS, J. Am. Soc. Mass Spec. 14, 1470-1476 Eletsky A., Kienhöfer A., Hilvert d., Pervushin K. (2005) Investigation of Ligand Binding and Protein Dynamics in Bacillus subtilis Chorismate Mutase by Transverse Relaxation Optimized Spectroscopy-Nuclear Magnetic Resonance, Biochemistry 44, 6788-6799 Posters: International Meeting on Proteom Analysis at the TU Munich, September 16.–19., 2001. Electrospray Time-of-Flight Mass Spectrometry: Investigation of Noncovalent Protein-Protein and Protein-Ligand Interactions. Fall Meeting of the Swiss Chemical Society in Zürich, at October 12., 2001. Substitution of Arg-90 in the Active site of Bacillus subtilis Chorismate Mutase with non-proteinogenic Amino Acids. WISOR in Bressanone/Brixen Italy, January 6.–13. 2002. Substitution of Arg-90 in the Active Site of Bacillus subtilis Chorismate Mutase with non-proteinogenic Amino Acids. Joining Forces: New Chemistry-, Informatics- and Engineering-Based Approaches to Study Biological Processes in Zürich at ETH-Hönggerberg, March 21.–22., 2002. Exploring the Catalytic Mechanism of Bacillus subtilis Chorismate Mutase. D-BIOL Symposium ETH Zürich in the Congress-Center in Davos, May 8.– 10., 2002. Exploring the Catalytic Mechanism of Bacillus subtilis Chorismate Mutase. The 7th annual meeting of the working party ‘Biotransformations’ German Society for Applied Microbiology (VAAM) at ETH Zürich October 9.–11., 2002. The Role of Arg- 90 in the Active Site of Bacillus subtilis Chorismate Mutase. ii Index Index ii Acknowledgments vi Abstract viii Zusammenfassung xi 1 Introduction 1 1.1 Claisen Rearrangement 1 1.2 Shikimate Pathway 3 1.3 Chorismate Mutase 5 1.4 Active Sites of Chorismate Mutases 8 1.5 The Catalytic Mechanism of Chorismate Mutases 12 1.6 Goals of this Work 17 2 Site-specific Replacement of a Crucial Arginine in BsCM with an Isosteric but Uncharged Citrulline 18 2.1 Introduction 18 2.2 Introduction of Unnatural Amino Acids into Proteins 19 Stop Codon Suppression 19 Peptide Synthesis 21 Peptide Ligation 24 Inteins 27 Strategy for Preparing BsCM Variants with non-standard Amino Acids at Position 90 29 2.3 Results 30 2.4 Discussion 37 3 BsCM Variants Containing homo-Lysine or Difluoro-Arginine at Position 90 in the Active Site 41 3.1 Introduction 41 3.2 Results 43 Arg90homo-Lys BsCM* 43 Arg90F2Arg BsCM* 46 3.3 Discussion 58 iii 4 Investigation of Ligand Binding and Protein Dynamics in BsCM by TROSY-NMR 62 4.1 Introduction 62 4.2 Results 64 Assignment of backbone resonances 64 Ligand binding 68 15 N T1, T2, and HNOE Data 68 Model-Free Analysis of Intramolecular Backbone Mobility 72 4.3 Discussion 74 5 Outlook 81 6 Experimental Section 83 6.1 General 83 Abbreviations 83 Analytical Data 86 Solutions, Buffers, and Media Molecular Biology 96 Ingredients for Minimal Media 99 Buffers for Purification of BsCM 1-87, Intein, CBD Fusion 100 Strains, Plasmids, and Oligonucleotides 101 General Methods: Molecular Biology 102 General Procedures: Molecular Biology 109 Chemicals and General Procedures: Chemistry 111 Computer Programs 113 6.2 Peptide Synthesis 114 Fmoc-BsCM(108-127) 35 114 BsCM(98-127) 36 115 D102E BsCM(88-127) 9 116 Arg90Cit D102E BsCM(88-127) 10 118 Arg90Cit BsCM(88-93) 37 119 BsCM(88-93) 11 120 Fmoc-D102E BsCM(92-127) 38 121 Arg90homo-Lys D102E BsCM(88-127) 12 123 Arg90F2Arg D102E BsCM(88-127) 31 124 6.3 Cloning and Expression 125 BsCM(1-87) encoded by pTYB1-BsCM87 125 BsCM(1-87) encoded by pTXB1-BsCM87 126 D102E BsCM encoded by pAK-D102E 127 R105K BsCM encoded by pAK-R105K 128 D102E R105K BsCM encoded by pAK-DERK 129 BsCM(1-124) encoded by pAK-T125* 130 BsCM(1-122) encoded by pAK-K123* 131 BsCM(1-121) encoded by pAK-T122* 132 BsCM(1-120) encoded by pAK-L121* 133 iv BsCM(1-119) encoded by pAK-S120* 134 6.4 Native Chemical Ligation 135 D102E BsCM 135 Arg90Cit D102E BsCM 135 Arg90homo-Lys D102E BsCM 136 Arg90F2Arg D102E BsCM 137 6.5 Isotopically Labeled BsCM 137 15N-labeled BsCM 137 D (< 35%), 13C-, and 15N-labeled BsCM 138 D, 13C-, and 15N-labeled BsCM 139 D (50%), 13C-, and 15N-labeled BsCM 140 6.6 Synthesis of Fmoc-4,4-difluoro-L-arginine(Pbf)-OH 141 Synthesis of tert-butyl (4R)-4-(3-ethoxy-2,2-difluoro-1-hydroxy-3-oxopropyl)-2,2- dimethyl-1,3-oxazolane-3-carboxylate 18: 141 Synthesis of tert-butyl (4R)-4-3-ethoxy-2,2-difluoro-1-[(1H-imidazol-1- ylcarbothioyl)oxy]-3-oxopropyl-2,2-dimethyl-1,3-oxazolane-3-carboxylate 19: 143 Synthesis of tert-butyl (4S)-4-(3-ethoxy-2,2-difluoro-3-oxopropyl)-2,2-dimethyl- 1,3-oxazolane-3-carboxylate 20: 145 Synthesis of tert-butyl (4S)-4-(3-amino-2,2-difluoro-3-oxopropyl)-2,2-dimethyl- 1,3-oxazolane-3-carboxylate 21: 147 Synthesis of tert-butyl (4S)-4-(3-amino-2,2-difluoropropyl)-2,2-dimethyl-1,3- oxazolane-3-carboxylate 22: 148 Synthesis of tert-butyl (4S)-4-(3-[(benzyloxy)carbonyl]amino-2,2-difluoropropyl)- 2,2-dimethyl-1,3-oxazolane-3-carboxylate 23: 149 Synthesis of benzyl N-(4S)-4-[(tert-butoxycarbonyl)amino]-2,2-difluoro-5- hydroxypentylcarbamate 24: 151 Synthesis of tert-butyl N-[(1S)-4-amino-3,3-difluoro-1-(hydroxymethyl) butyl]carbamate 25: 152 Synthesis of tert-butyl N-[(Z)-[(tert-butoxycarbonyl)amino]((4S)-4-[(tert- butoxycarbonyl)amino]-2,2-difluoro-5-hydroxypentylamino) methylidene]carbamate 26: 153 Synthesis of (2S)-2-[(tert-butoxycarbonyl)amino]-5-([(tert- butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methylamino)-4,4- difluoropentanoic acid 27: 155 Synthesis of (2S)-2-amino-5-[amino(imino)methyl]amino-4,4-difluoropentanoic acid 8: 156 Synthesis of (2S)-5-[amino(imino)methyl]amino-2-[(benzyloxy)carbonyl]amino- 4,4-difluoropentanoic acid 28: 157 Synthesis of (2S)-2-[(benzyloxy)carbonyl]amino-4,4-difluoro-5-[(imino[(2,2,4,6,7- pentamethyl-2,3-dihydro-1-benzofuran-5- yl)sulfonyl]aminomethyl)amino]pentanoic acid 29: 158 Synthesis of (2S)-2-amino-4,4-difluoro-5-[(imino[(2,2,4,6,7-pentamethyl-2,3- dihydro-1-benzofuran-5-yl)sulfonyl] aminomethyl)amino]pentanoic acid 30: 160 Synthesis of (2S)-2-[(9H-fluoren-9-ylmethoxy)carbonyl]amino-4,4-difluoro-5- [(imino[(2,2,4,6,7-pentamethyl-2,3-dihydro-1-benzofuran-5- yl)sulfonyl]aminomethyl)amino]pentanoic acid 13: 161 v 7 Appendix 164 7.1 Programs for the Peptide Synthesizer 164 Modifications to the standard programs 164 7.2 Analytical Ultracentrifugation Data for BsCM* and Arg90Cit BsCM* 174 Data for BsCM* 174 Data for Arg90Cit BsCM* 175 8 Literature 177 vi Acknowledgments I would like to thank my “Doktorvater” Prof. Donald Hilvert for giving me the opportunity to work in his laboratory on this exciting and challenging project. His constant support, scientific enthusiasm and the granted freedom were essential for the success of this work. I also want to thank him for thoroughly proof-reading my thesis. Prof. Dario Neri I want to thank for being my co-examiner. I would like to thank my labmates from D32 and F328 for the enjoyable working atmosphere. I am in dept to Richard Quaderer and Axel Sewing for introducing me to peptide chemistry and HPLC and to the Praktikum 2000 and Andrea Piatesi for the introduction to molecular biology. It was always fun to solve different computer problems together with Andreas Aemissegger. The many happy hours we spent inside the lab and on various parties will always be lasting memories. Dr. Ajay Mandal was not only always willing to discuss chemistry problems but he also new all the good places for out of lab activities. Andreas Kleeb was always a good partner to discuss biology issues and he organized some unforgettable group events. I would like to thank Dr. Peter Kast for a lot of fruitful discussions about molecular biology problems.