A University of Sussex DPhil thesis Available online via Sussex Research Online: http://sro.sussex.ac.uk/ This thesis is protected by copyright which belongs to the author. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Please visit Sussex Research Online for more information and further details Structure, function and mechanism of the alternative oxidases Luke Young September 2013 Submitted in partial fulfilment towards the requirements for the degree of Doctor of Philosophy (DPhil) i | P a g e I hereby declare that this thesis has not been and will not be, submitted in whole or in part to another University for the award of any other degree. Signature ............................................. ii | P a g e University of Sussex Luke Young Submitted for the degree of Doctor of philosophy Structure function and mechanism of the alternative oxidases Summary The alternative oxidase (AOX) is the terminal protein in the alternative oxidation pathway found in plants, fungi and some protozoa. One of the more prominent protozoa that contain AOX within the bloodstream form is Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT), in which the parasite has demonstrated to be totally dependent upon the protein for continued respiration. Given the lack of AOX in mammalian cells, the protein represents an attractive chemotherapeutic target for trypanosidal activity. Ascofuranone is a known inhibitor of AOX, but its complex synthesis precludes it from industrial production. To this end colletochlorin B, an analogue of ascofuranone, was synthesised and its inhibitory efficacy against AOX examined. IC50 values obtained demonstrate that removal of the problematic furanone ring does not reduce inhibitor efficacy to a large degree. Derivatives of colletochlorin B were synthesised to assess the importance of structural moieties present. The compounds were also tested against the cytochrome bc1 complex, an important respiratory chain complex, and compared with known fungicides. Using these compounds assays against fungal species has yielded promising results for the use of colletochlorin B as a lead fungicide. iii | P a g e Recombinant wild type trypanosomal alternative oxidase (TAO) and Sauromatum guttatum alternative oxidase (SgAOX) have been expressed in E.coli. in addition to a number of mutants. Respiratory activities of these mutants were measured in order to assess the importance of highly conserved amino acids, with all mutants showing a decline in specific activity. Three of the mutants generated were also shown to affect the apparent affinity for oxygen, the implications of which are discussed. Recent crystallisation of TAO has enabled a more detailed examination of the structure of all AOXs. Work is presented relating structure to the overall function of the protein, taking into account conservation throughout the entire AOX family. Comparisons to other di-iron proteins revealed a conserved His-Asp-Tryp motif that could facilitate proton coupled electron transport. A full catalytic cycle based on these findings has been postulated. iv | P a g e Table of Contents Abbreviations used .................................................................................................. ix Acknowledgements .................................................................................................. x 1 Introduction .......................................................................................................... 1 1.1 Respiration overview ..................................................................................... 1 1.2 Electron transport chain ................................................................................. 2 1.3 Functions of the alternative oxidase .............................................................. 9 1.3.1 Thermogenesis ....................................................................................... 9 1.3.2 Homeostasis ........................................................................................... 9 1.4 Trypanosomes ............................................................................................. 11 1.5 Inhibition of AOX ....................................................................................... 13 1.5.1 Synthesis of ascofuranone and ascochlorin.......................................... 16 1.6 Fungicides ................................................................................................... 17 1.7 Structure ...................................................................................................... 18 1.8 Structural regulation .................................................................................... 21 1.9 Research aims .............................................................................................. 22 2 Materials and methods ....................................................................................... 24 2.1 pET vector system ....................................................................................... 24 2.2 Culture medium ........................................................................................... 26 2.2.1 Competent cells .................................................................................... 26 2.2.2 DNA Transformation ........................................................................... 27 2.2.3 Protein expression in E.coli ................................................................. 27 2.2.4 Membrane harvest ................................................................................ 28 2.3 Purification .................................................................................................. 28 2.3.1 Protein Solubilisation ........................................................................... 29 2.3.2 Protein purification............................................................................... 29 2.3.3 Top loaded protein purification ............................................................ 30 v | P a g e 2.3.4 Protein precipitation ............................................................................. 30 2.4 Protein estimations ...................................................................................... 30 2.5 Mitochondrial preparations ......................................................................... 31 2.5.1 Arum maculatum mitochondria ............................................................ 31 2.5.2 Potato mitochondria ............................................................................. 31 2.6 Fungal plate assays ...................................................................................... 32 2.7 Gels .............................................................................................................. 33 2.7.1 SDS PAGE gel ..................................................................................... 33 2.7.2 Western Blot analysis ........................................................................... 35 2.7.3 Detection .............................................................................................. 36 2.8 Analytical techniques .................................................................................. 37 2.8.1 General ................................................................................................. 37 2.8.2 Oxygen electrode ................................................................................. 37 2.8.3 Spectrophotometric enzyme assays ..................................................... 38 2.9 Crystal structures analysis ........................................................................... 39 2.10 Chemical synthesis .................................................................................. 40 3 Structural analysis of TAO................................................................................. 46 3.1 Introduction ................................................................................................. 46 3.2 AOX sequence alignment ............................................................................ 47 3.3 Roles of highly conserved residues based on crystal structure analysis ..... 48 3.4 Membrane binding, dimer interface and N-terminal arm ............................ 51 3.5 Hydrophobic cavity ..................................................................................... 53 3.5.1 Water transport ..................................................................................... 56 3.6 Ligation around the di-iron sphere – uninhibited structure ......................... 58 3.7 Comparison between inhibited and uninhibited structures ......................... 60 3.7.1 Binding positions for inhibitors ........................................................... 60 3.8 Summary ..................................................................................................... 65 vi | P a g e 4 Expression of recombinant AOX and mutants in haem-deficient E.coli ........... 67 4.1 Introduction ................................................................................................. 67 4.1.1 Mutants ................................................................................................