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Biophysical and Structural Studies of Chlorophyll Biosynthetic Enzymes Magnesium Chelatase and Protochlorophyllide Reductase

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Biophysical and Structural Studies of Chlorophyll Biosynthetic Enzymes Magnesium Chelatase and Protochlorophyllide Reductase Biophysical and structural studies of chlorophyll biosynthetic enzymes magnesium chelatase and protochlorophyllide reductase David Andrew Farmer Department of Molecular Biology and Biotechnology A thesis submitted for the degree of Doctor of Philosophy September 2019 i Abstract Photosynthetic organisms must generate chlorophyll, the light capturing molecule integral for pho- tosynthesis, whilst also regulating production to adapt to environmental conditions. Chlorophyll is so fundamental for photosynthesis that it is important to understand its biosynthetic pathway; consequently, this sequence of reactions has been studied extensively for several decades, culmi- nating in the engineering of the heterotroph E. coli to produce chlorophyll. Two key regulatory points in the pathway are i) at the branchpoint between haem and chlorophyll biosynthesis, where a magnesium ion is inserted into the porphyrin ring, catalysed by the multi-subunit enzyme mag- nesium chelatase (MgCH); and ii) the light-dependent reduction of the C17-C18 double bond, catalysed by protochlorophyllide oxidoreductase (POR). Despite the extensive work performed to biochemically describe these enzymes, the full reaction mechanisms are still unknown, and the limited amount of structural information for either enzyme has hindered complete characterisation. The work reported in this thesis has used a range of biochemical, biophysical and structural tech- niques to study these biologically important and mechanistically unique enzymes: microscale ther- mophoresis, kinetic analysis and cross-linking mass spectrometry revealed that the driving force behind chelation, the ATPase activity of the AAA+ ChlI subunit, is linked to the chelating ChlH protein via the regulatory ChlD subunit; X-ray crystallography-guided mutagenesis determined the porphyrin binding site in ChlH; (cryo)-electron microscopy was used to initiate structural in- vestigations into the quaternary organisation of MgCH and POR; and 2-dimensional electronic spectroscopy was applied to the light-initiated reaction catalysed by POR, the first use of this com- plex technique on an enzyme, potentially revealing a novel intermediate in the reaction that is formed on the femtosecond time-scale. The work presented in this thesis aims to develop our bio- chemical understanding of MgCH and POR, and lay the foundations for further structural and mechanistic studies of these interesting enzymes of chlorophyll biosynthesis. ii Acknowledgements To start I would like to thank my supervisor, Neil, for offering me the opportunity to undertake this PhD, and for his continued support, suggestions and ability to inspire enthusiasm for the pro- ject. I have greatly appreciated the Hunter lab environment and have been given several opportu- nities to work with other lovely people in exciting places thanks to Neil’s vast network of collab- orators. I would also like to thank my more hands-on lab supervisors, Nate and Mo for all of their training, guidance and collaboration (especially on enzyme kinetics and molecular biology, respectively). A thanks goes to all of the other Hunter lab members who have helped in and out of the lab, and whose office shenanigans consistently improved the day. The pub trips and Christmas dos ever a source of hilarity, and an enthusiasm for most things sport encouraged me to actually do a bit of moving, which is good! Next, I would like to thank the people that I’ve collaborated with: Dr Eli Romero, who I worked with for the data presented in Chapter 6, and was incredibly friendly, helpful and generally good fun to work with. Thanks for helping me enjoy my month in Amsterdam that bit more. I would also like to thank Dr Alistair Siebert, who I undertook my PIPS placement with down at eBIC, despite seemingly being one of the busiest people I know! A thanks too, to Dr Georgio Schiro, whose “Sleep is for the weak” T-shirt aided with the long nights at the ESRF conducting work that, unfortunately, didn’t make it into this thesis. I would also like to personally thank any other contributors to work in this thesis not already mentioned: Dr Andy Hitchcock, Dr Phil Jackson, Dr Claudine Bisson, Dr Jim Reid, Dr Svet Tzokov and Dr Dave Armstrong. Finally, I would like to thank all my other family and friends that have been a part of my life during these past four years. Especially my best mates who I’ve been lucky enough to live with: Sarah, Alex, Harry, Emma, Josh and Studd; for all the walks, rides, nights out, pub trips, festivals, holi- days, BBQs and general chilling – cheers guys. And a last thank you to my parents: your ever- present support and encouragement undoubtedly played a large role in me getting this far. iii Table of Contents Abstract ........................................................................................................................................... ii Acknowledgements ........................................................................................................................ iii Table of Contents ........................................................................................................................... iv Table of Figures .............................................................................................................................. x Table of Equations ........................................................................................................................ xv Table of Tables ............................................................................................................................. xvi List of Abbreviations ................................................................................................................... xvii 1 Introduction ............................................................................................................................. 1 1.1 Photosynthesis .................................................................................................................. 1 1.2 Chlorophyll and other tetrapyrroles .................................................................................. 2 1.3 Chlorophyll Biosynthesis ................................................................................................. 3 1.3.1 5-aminolevulinic acid synthesis ................................................................................ 3 1.3.2 Three-step condensation of ALA to uroporphyrinogen III ....................................... 5 1.3.3 Decarboxylation and oxidation of uroporphyrinogen III to form protoporphyrin IX 7 1.3.4 Chelation of PIX ........................................................................................................ 9 1.3.5 Methylation and addition of isocyclic ring E .......................................................... 10 1.3.6 The formation of chlorophyllide ............................................................................. 11 1.3.7 The addition of the phytyl tail to form chlorophyll ................................................. 13 1.4 Magnesium chelatase ...................................................................................................... 13 1.4.1 The reaction ............................................................................................................. 15 1.4.2 ChlI and ChlD ......................................................................................................... 16 1.4.3 ChlH ........................................................................................................................ 21 1.4.4 Gun4 ........................................................................................................................ 23 iv 1.5 Protochlorophyllide oxidoreductase ............................................................................... 25 1.5.1 DPOR ...................................................................................................................... 25 1.5.2 LPOR ...................................................................................................................... 26 1.5.3 SDR proteins ........................................................................................................... 27 1.5.4 Reaction of LPOR ................................................................................................... 28 1.5.5 Photochemistry of Pchlide ...................................................................................... 32 1.5.6 Hydrogen tunnelling in POR................................................................................... 32 1.5.7 Structural information on POR ............................................................................... 32 1.6 Techniques...................................................................................................................... 34 1.6.1 Cryo-electron microscopy ....................................................................................... 34 1.6.2 2-dimensional electronic spectroscopy ................................................................... 37 1.6.3 Microscale thermophoresis ..................................................................................... 39 1.7 Aims and objectives ....................................................................................................... 40 2 Materials and Methods .......................................................................................................... 42 2.1 Materials ........................................................................................................................
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