Development of New Enzyme Activities for Applications in Synthetic Biology: Carboxylic Acid Amidases (CAA) A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy (PhD) in the Faculty of Science and Engineering 2018 Alexander J Wood School of Chemistry Contents Figures .................................................................................................................................... 3 Tables ..................................................................................................................................... 7 List of Abbreviations.............................................................................................................. 8 Abstract ................................................................................................................................ 10 Declaration ........................................................................................................................... 11 Copyright Statement ............................................................................................................ 12 Acknowledgments ................................................................................................................ 13 Chapter 1. Introduction .................................................................................................... 14 1.1. General introduction to amides and their production ............................................ 14 1.2. Chemical methods of amide synthesis and their limitations ................................. 16 1.3. Enzymatic synthesis of amides .............................................................................. 20 1.4. Carboxylic acid reductase...................................................................................... 57 1.5. Objective of the project: Engineer CAR into a broad specificity amide synthetase 63 Chapter 2. Developing a CAR-VibB, VibH fusion enzyme system ................................ 65 2.1. Introduction ........................................................................................................... 65 2.2. Gene analysis and CARmm-VibB fusion design .................................................... 68 2.3. Vib system gene cloning and CARmm A domain-VibB PCP domain fusion ........ 70 2.4. Expression trials of Vib and CARmm-VibB fusion genes ...................................... 72 2.5. Discussion and future work ................................................................................... 79 Chapter 3. Direct amide bond formation using CARs ..................................................... 84 3.1. Introduction ........................................................................................................... 84 3.2. CAR gene expression and enzyme purification .................................................... 85 3.3. CAR dependent amide formation - method development and substrate screen .... 87 3.4. Conclusions ......................................................................................................... 104 Chapter 4. Investigation of the mechanism of CAR-dependent amide synthesis .......... 106 4.1. Introduction ......................................................................................................... 106 4.2. CAR production in the absence of co-produced Sfp ........................................... 106 4.3. CAR mutagenesis and the removal of the phosphopantetheine binding site ...... 108 4.4. Use of truncated CAR for amide formation ........................................................ 109 4.5. Influence of a PPant mimetic on amide formation .............................................. 110 4.6. Investigation of CAR enantioselectivity using chiral amines ............................. 113 4.7. Analysis of adenylation activity using the EnzChek phosphate detection kit ..... 115 2 4.8. Structural modelling of amines into CAR active site .......................................... 119 4.9. Whole-cell CAR-dependent amide formation ..................................................... 123 4.10. Conclusion and future work ............................................................................. 124 Chapter 5. Use of radical substrates for studies of CAR dynamics ............................... 126 5.1. Introduction ......................................................................................................... 126 5.2. Kinetic studies with radical substrates ................................................................ 128 5.3. Discussion and future work ................................................................................. 130 Chapter 6. Discussion of results and perspectives ......................................................... 131 Chapter 7. Experimental procedures .............................................................................. 134 7.1. General methods and materials ........................................................................... 134 7.2. Genes and molecular cloning .............................................................................. 134 7.3. Protein production and purification by nickel affinity chromatography ............. 138 7.4. Biotransformations and analysis ......................................................................... 142 7.5. Investigation of coupling between CAR-dependent ATP consumption and amide formation using an EnzChek kit ..................................................................................... 153 7.6. Enzyme kinetics analysis of native CAR activity with radical-TEMPO carboxylic acid 154 7.7. Structural modelling of piperidine 52 into the active sites of CAR A domain structures ........................................................................................................................ 154 References .......................................................................................................................... 155 Appendices ......................................................................................................................... 166 Appendix 1: Genes used in this work ............................................................................. 166 Appendix 2: PCR primers and PCR conditions ............................................................. 171 Appendix 3: Example enzyme nickel affinity purification, AKTA UV chromatograms ........................................................................................................................................ 173 Appendix 4: Example HPLC traces showing CAR-dependent amide formation. ......... 175 Final Word Count: 44044 Figures Figure 1.1: Examples of amide-containing drugs, including the anti-cancer drug imatinib 1, the antibiotic cefpiramide 2 and the sleeping disorder drug modafinil 3. ............................ 14 3 Figure 1.2: Use of coupling or activating reagents to form amides from carboxylic acids and amines. ........................................................................................................................... 18 Figure 1.3: Exploitation of the serine protease catalytic mechanism for amide formation. 22 Figure 1.4: Overcoming the natural substrate specificity of proteases through the use of substrate mimetics for amide formation. .............................................................................. 24 Figure 1.5: Selected examples of amide products produced by the lipases CALB and PPL respectively. ......................................................................................................................... 26 Figure 1.6: Synthesis of aminoacyl-tRNAs by aminoacyl tRNA synthetase. ..................... 28 Figure 1.7: Module and domain composition of the NRPS tyrocidine synthetase. ............. 31 Figure 1.8: Conformational changes of the C-terminal subdomain of A domains permit adenylation or thiolation. ..................................................................................................... 33 Figure 1.9: PCP domain and C domain functions within NRPS enzyme complexes. ......... 38 Figure 1.10: A proposed mechanism of C domain-catalysed amide formation. .................. 39 Figure 1.11: Subdomain swapping in NRPS systems allows the production of novel peptides. ............................................................................................................................... 42 Figure 1.12: Module shuffling within the tyrocidine synthetase system to produce novel peptides. ............................................................................................................................... 44 Figure 1.13: Domain shuffling between the bacitracin synthetase and tyrocidine synthetase systems for novel dipeptide formation. ................................................................................ 45 Figure 1.14: Model proposed for ATP-grasp enzyme-catalysed amide formation .............. 47 Figure 1.15: Exploiting the wide substrate breadth of ATP-Grasp enzymes YwfE and PGM1 ................................................................................................................................... 49 Figure 1.16: Amide synthesis by the amide synthetase McbA. ........................................... 52 Figure 1.17: Amide synthesis by the adenylate forming amide ligases NovL and CouL .... 52 Figure 1.18: An overview of enzymatic methods of amide formation ................................ 56 Figure 1.19: Carboxylic acid reductase reaction and domain composition and