The Interactions of Bacillithiol with Carbonyl- Containing Metabolites Dominic Rodrigues A thesis submitted for the degree of Doctor of Philosophy School of Pharmacy 2017 "This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution.” Declaration This thesis has been submitted to the University of East Anglia for the Degree of Doctor of Philosophy and is, to the best of my knowledge, original except where stated, referenced and acknowledged. Dominic Rodrigues 2 Abstract Bacillithiol (BSH) is a recently discovered low molecular weight (LMW) thiol found amongst several Gram-positive bacteria including Bacillus anthracis, Staphylococcus aureus and Bacillus subtilis. It plays a fundamental role in the redox processes within the cell, in addition to many other functions including the detoxification of electrophiles such as methylglyoxal (MG). MG is a reactive dicarbonyl produced as a by-product of glycolysis. It is found to be toxic to the cell as it is capable of modifying macromolecules such as proteins and DNA causing loss of biological activity. The previously established glutathione-dependent glyoxalase pathway comprises of the glyoxalase I and glyoxalase II enzyme, which are found to serve as a major mechanism for the detoxification of MG amongst eukaryotes. There is speculation that BSH follows through this same pathway. Herein, the BSH-dependent glyoxalase pathway in B. subtilis is fully explored. The studies have shown a reaction between BSH and MG to occur spontaneously both in vitro and in vivo. Furthermore, these observations, have lead onto the discovery that, for the first time, BSH has shown to react with other metabolites in glycolysis. These include dihydroxyacetone phosphate, D-glyceraldehyde 3-phosphate and pyruvate. In each case they form a hemithioacetal (HTA). As a result, potentially significant concentrations of BSH may be sequestered in these ‘unknown thiol reservoirs’ which were not previously known to exist in the cell. Essentially, this raises questions regarding the true overall concentration of intracellular BSH. 3 Acknowledgements First of all, I would like to thank my supervisor Chris, for all his advice and tutoring over the last few years. His enthusiasm for the subject and passion for research has been a real inspiration! I would also like to thank my second supervisor Nick, for his support and feedback. In the earlier years of my PhD, I would like to thank Sunil and Miriam for all their guidance and getting me settled into the lab life. After they left, PhD life was a little quiet for some time until the hustle and bustle began with the arrival of a whole new group who brought around a great atmosphere in the lab. So I would like to thank Olly, Emma, Hazel, Andrew, Marco, Awais, Issa, James, Zoe, and Muayyad, with a particular mention to Ryan. He has spent hours on end teaching me which I very much appreciate! A special thanks to Jesus, Serena and Francesc for their assistance in the whole cell NMR and also to Lionel Hill for his analysis of the mass spectrometric data. Lastly, I would like to thank my parents and all my family for all their support! 4 Table of Contents Declaration ....................................................................................................................... 1 Abstract ............................................................................................................................ 3 Acknowledgements ......................................................................................................... 4 Figures ........................................................................................................................... 10 Schemes ......................................................................................................................... 13 Tables ............................................................................................................................. 16 Abbreviations ................................................................................................................. 18 1. Introduction ............................................................................................................ 22 1.1 LMW molecular weight thiols .............................................................................. 23 1.1.1. Overview ..................................................................................................... 23 1.1.2. Glutathione .................................................................................................. 24 1.1.3. Amino acids ................................................................................................. 25 1.1.4. Other LMW thiols ......................................................................................... 25 1.2. Bacillithiol ........................................................................................................... 27 1.2.1. Overview ..................................................................................................... 27 1.2.2. Biosynthetic pathway of BSH....................................................................... 28 1.2.3. Roles of BSH within the cell ........................................................................ 29 1.3. Detoxification processes of xenobiotics and electrophiles .................................. 31 1.3.1 Enzyme-catalysed reactivity of thiols ............................................................ 31 1.3.2. Chemical reactivity of thiols ......................................................................... 32 1.4. Methylglyoxal ..................................................................................................... 33 1.4.1. Overview ..................................................................................................... 33 1.4.2. Toxicity of methylglyoxal .............................................................................. 33 1.4.3. Formation of MG ......................................................................................... 33 1.4.4. Enzymes involved in the formation of MG.................................................... 35 1.5. Detoxification pathways of methylglyoxal ........................................................... 36 1.5.1. Thiol-independent detoxification pathways .................................................. 36 1.5.2. Thiol-dependent detoxification pathways ..................................................... 36 1.5.2.1 The glyoxalase pathway ........................................................................ 36 1.5.2.2 The glyoxalase enzymes ....................................................................... 37 1.5.2.3. KefB and KefC efflux system ................................................................ 38 1.5.3. Does the BSH-dependent glyoxalase system occur in Gram-positive bacteria? ............................................................................................................... 38 1.6. Objectives of the research ................................................................................. 39 2. Methylglyoxal and its interaction with low molecular weight thiols ................... 41 2.1. Overview ............................................................................................................ 41 2.2. In vitro chemical reactivity between LMW thiol cofactor and MG ........................ 42 5 2.2.1. Reactivity of LMW thiol cofactors ................................................................. 42 2.2.2. Influence of pKa on the reactivity of LMW thiol cofactors.............................. 42 2.2.3. Purification of MG ........................................................................................ 44 2.2.4. Relative reactivity of Cys and BSH with MG ................................................ 45 2.2.5. The reactivity of other LMW thiols, GSH and CysNAc, with MG .................. 47 2.2.6. Long-term reactivity of thiol with MG ............................................................ 48 2.2.7. Potential thiazolidine formation .................................................................... 49 2.3. pH-dependent (pH 5.6) and pH-independent rate constants .............................. 50 2.3.1. Overview ..................................................................................................... 50 2.3.2. Forwards rate constant (k1) of thiol and MG reactivity .................................. 50 2.3.3. Reverse rate constant (k-1) of HTA dissociation ........................................... 53 2.4. Thermodynamics – equilibrium constant (Keq). ................................................... 55 2.5. In vivo analysis of the BSH-dependent glyoxalase pathway ............................... 57 2.5.1. Quantification of intracellular thiols .............................................................. 57 2.5.2. Extracellular consumption of methylglyoxal and thiol analysis in B. subtilis WT ........................................................................................................................ 59 2.5.3. Immediate in vivo kinetics of thiol reactivity with MG in B. subtilis WT ......... 61 2.5.4. Extracellular consumption of methylglyoxal and thiol analysis in B. subtilis glxI mutant ...........................................................................................................