Structural studies of three enzymes: Telomerase, the methyltransferase CobJ and Pectate lyase Teng Teng To Thesis submitted to the University of London for the Degree of Doctor of Philosophy 1 Abstract This thesis investigates the structure and function of three enzymes of biotechnological and biomedical interest: telomerase from Caenorhabtidis elegans , pectate lyase from Bacillus subtilis and the methyltransferase CobJ from Rhodobacter capsulatus . Telomerase is a ribonucleoprotein found in all eukaryotes and its function is to maintain telomere length, sustain chromosome integrity and circumvent the end-replication problem. The protein requires two subunits to function, telomerase reverse transcriptase (TERT), the catalytic component, and an intrinsic RNA template (TR). The TR makes telomerase a unique reverse transcriptase using the template in the synthesis of short iterative sequences which cap the ends of telomeres. This work reports the successful cloning of a small and therefore potentially crystallisable TERT from C. elegans and expression trials of this catalytic component. Cobalamin (vitamin B 12 ) is an intricate small molecule belonging to a group of compounds called cyclic tetrapyrroles. Its biosynthesis is achieved through a complex pathway encompassing over thirty different enzyme-mediated reactions. Within this pathway there are seven methyltransferases which add eight S-adenosyl-methionine (SAM) derived methyl groups to the macrocycle. CobJ catalyses the methylation of C17 and ring contraction at C20, this reaction which exudes C20 from the tetrapyrrole ring is unprecedented in nature. In this thesis I report the crystallisation of native CobJ and refinement and validation of a high resolution structure along side co-crystallisation and soaking experiments aimed at capturing an enzyme-tetrapyrrole complex. Pectate lyase (BsPel) is an enzyme secreted from the bacterium B. subtilis, it is one of many enzymes secreted by plant pathogens that is responsible for soft rot disease in plants and vegetables. The lyase utilises anti β-elimination chemistry to cleave an α-1,4- glycosidic link present in polygalacturonate the major component of the plant cell wall. 2 The structure of BsPel in complex with hexagalacturonate and a cobalt metal has been solved confirming the position and role of the putative catalytic base Arg 279 in the abstraction of a proton from C5 in galacturonate. 3 Acknowledgements It has been a real experience jumping on board this PhD train. I remember my first day walking into the lab as a freshly graduated student with my bright white lab coat hopeful and optimistic, but at the same time I knew it wasn’t going to be a walk in a park. It has been a rollercoaster of emotions, one minute you’re up in the clouds next minute you’re down in the dumps. A big thank you goes to my family especially mum and dad, I hope I’ve done you proud. Thank you Nathan you have been a great help to me and I believe will continue to be so for a long time still ^_^. Thanks to my childhood friends who lended me their ears listening to me moan and grumble, sorry, you know who you are! Thank you all for your support much much appreciated. I was lucky enough to land myself into an incredibly friendly and helpful lab full of generous welcoming people. The first of these people is my supervisor Prof Richard Pickersgill, thank you for allowing me to dabble my feet in research and for guiding me throughout. Also a massive thank you must go to Arefeh and Yumi the two most helpful, supportive, strong and understanding people I have met, amazing women! Thank you to everyone in the lab Tom, Kerem, Salyha, Shuang, Allan, Saima, Nadine and Claire a bunch of happy crazy scientists! I’ll miss the tea breaks, girly chats and the not so surprise birthday celebrations. Good luck to the ones who have yet to finish their PhDs! Salyha stay off the chocolate and Nadine stay off the coffee! Finally thank you to Queen Mary University of London and BBSRC for providing the funds and facilities for me to carry out my PhD. 4 Title 1 Abstract 2 Acknowledgments 4 List of figures 11 List of tables 14 List of abbreviations 16 1 Introduction 17 1.1 Telomerase 18 1.1.2 Telomerase and the end replication problem 18 1.1.3 Telomerase independent survival 22 1.1.4 Telomerase reverse transcriptase subunit 22 1.1.5 Telomerase RNA domain 25 1.1.6 Repeat addition processivity 29 1.1.7 Biomedical significance 31 1.2 The cobalamin methyltransferase CobJ 32 1.2.1 Vitamin B12 pathway 32 1.2.3 Modified tetrapyrroles 34 1.2.4 Aerobic pathway 36 1.2.4.1 Conversion of uro’gen III into cobyric acid 37 1.2.4.2 Formation of cobinamide from cobyric acid 38 1.2.4.3 Conversion of cobinamide into adenosylcobalamin 40 1.2.5 Biological significance 41 1.3 Pectate Lyase 43 1.3.1 Plant cell wall 43 1.3.2 CAZy database 44 1.3.3 Pectic enzymes 44 1.3.3.1 Hydrolysis of the glycosidic bond 44 1.3.3.2 Elimination of the glycosidic bond 46 5 1.3.4 The structures of polysaccharide lyases 47 1.3.5 Industrial significance 48 2 Materials and Methods 49 2.1 Materials 50 2.1.1 Water 50 2.1.2 Plasmids 50 2.1.3 Polymerases 50 2.1.4 Restriction enzymes 51 2.1.5 Luria Bertani medium 51 2.1.6 Protein purification 52 2.1.6.1 Buffers 52 2.1.6.2 Columns 54 2.1.7 Competent cells 55 2.1.8 Antibodies 55 2.1.9 Antibiotics 56 2.1.10 Crystallisation 56 2.1.10.1 Buffers 56 2.2 Methods 57 2.2.1 Molecular cloning and site-directed mutagenesis 57 2.2.1.1 Primer design 57 2.2.1.2 Polymerase chain reaction 58 2.2.1.3 Direct cloning 60 2.2.1.4 Digestion and ligation independent cloning 61 2.2.2 Site-directed mutagenesis 62 2.2.3 Transformation 63 2.2.4 Protein production 64 2.2.4.1 Protein growth conditions 64 2.2.4.2 Cell lysis 64 2.2.5 Protein production 65 6 2.2.5.1 Nickel column 65 2.2.5.2 Glutathione sepharose column 66 2.2.5.3 Ion exchange column 66 2.2.5.4 pd-10 column 66 2.2.5.5 Size exclusion column 67 2.2.5.6 CP-18 column 67 2.2.6 SDS-PAGE gel 68 2.2.7 Western blot 70 2.2.8 Substrate production 72 2.2.8.1 Precorrin-3a 72 2.2.8.2 Plasmid preparation of pET14b-Bm CobI ( Brucella melinitis ) 72 2.2.8.3 Protein production of pET14b-Bm CobI 72 2.2.8.4 Plasmid preparation of pETcoco2-Hem B, C, D and CobA 73 2.2.8.5 Protein production of pETcoco2-abcd 73 2.2.8.6 Multi-enzyme assay 74 2.2.8.7 Precorrin-3b 76 2.2.9 BsPel spectroscopy activity assay 76 2.2.10 Crystallography 77 2.2.10.1 Crystallisation 77 2.2.10.2 Crystal soaking 78 2.2.10.3 Crystal freezing 79 2.2.10.4 Data collection and reduction 80 2.2.10.5 Fourier syntheses 81 2.2.10.6 Refinement and model building 83 2.2.10.7 Validation 83 3 Cloning and expression Caenorhabtidis elegans telomerase reverse transcriptase 84 3.1 Introduction: Caenorhabtidis elegans reverse transcriptase 85 3.2 Cloning and site-directed mutagenesis 90 7 3.2.1 Primer design 90 3.2.2 Digestion and ligation independent cloning 91 3.2.3 Direct cloning 93 3.2.4 Sequencing results 94 3.3 Protein production 97 3.3.1 Rare codon analysis 97 3.3.2 Expression of pET41-DY3.4 97 3.3.3 Expression and protein identification of pET14b-DY3.4 99 3.4 Solubilising pET14b-DY3.4 100 3.4.1 Expression trials 100 3.4.2 GroEL and GroES chaperones 101 3.4.3 Solubilising TERT using urea 102 3.5 Conclusion and future work 104 4 High resolution structure and substrate binding to CobJ 106 4.1 Introduction: CobJ ring contraction 107 4.2 Protein production and purification of CobJ and CobG 112 4.2.1 CobJ native and mutant protein production 113 4.2.2 CobJ native and mutant protein purification 113 4.2.3 CobG protein production 115 4.2.4 CobG protein purification 115 4.3 Substrate production 117 4.3.1 Precorrin-3a 117 4.3.1.1 Multi-enzyme reaction 117 4.3.2 Precorrin-3b 119 4.3.2.1 Production of precorrin-3b 119 4.4 Native and mutant CobJ crystallography 121 4.4.1 Crystallisation of native and mutant CobJ 123 4.4.2 Crystal soaking 126 4.4.3 Co-crystallisation 127 4.4.4 pET14b-CobJ from pull-down assay 131 8 4.4.5 High resolution data collection of native CobJ 132 4.4.5.1 Data collection for native CobJ co-crystallised with PC-3a 135 4.4.5.2 Data collection for native CobJ co-crystallised with porphobilinogen 137 4.4.6 Data collection of mutant CobJ 139 4.4.6.1 Data collection for H129A soaked in pyrrole-2-carboxylic acid 139 4.4.7 Data collection for pET14b-CobJ from pull-down assay 144 4.4.8 Refinement 145 4.4.9 Validation 146 4.5 Analysis of CobJ active site 152 4.5.1 Alignment of CobJ sequences 152 4.5.2 Superimposition of four molecules in CobJ structure 156 4.5.3 Ring contraction mechanism 160 4.6 Conclusion: Structural implications of CobJ 163 4.7 Future work 164 5 Trapping the Michaelis complex in BsPel 165 5.1 Introduction: Design of mutants 166 5.2 Site-directed mutagenesis 169 5.2.1 Primer design 169 5.2.2 PCR-based site-directed mutagenesis 169 5.2.3 Sequencing 171 5.3 Expression and purification of mutant protein 172 5.3.1 Expression of triple mutant BsPel 172 5.3.2 Purification of triple mutant BsPel 172 5.4 Spectroscopic enzyme assay 174 5.5 Crystallography 175 5.5.1 Crystallisation of triple mutant BsPel 175 5.5.2 Crystal soaking 177 9 5.5.3 Data collection and structure solution 177 5.5.4 Refinement 178 5.5.5 Validation 179 5.5.6 Michaelis complex of BsPel 183 5.6 Conclusion 186 5.7 Future work 188 6 Final conclusions 189 Bibliography 193 Appendices 207 10 List of Figures 1.1.1 The end replication problem 20 1.1.2 Telomerase is used for synthesising tandem repeats enabling full DNA 21 replication 1.1.3 Overview of motifs and domains present in reverse transcriptases 23 1.1.4 Summary of