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University of Southampton Research Repository Eprints Soton University of Southampton Research Repository ePrints Soton Copyright © and Moral Rights for this thesis are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder/s. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holders. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given e.g. AUTHOR (year of submission) "Full thesis title", University of Southampton, name of the University School or Department, PhD Thesis, pagination http://eprints.soton.ac.uk UNIVERSITY OF SOUTHAMPTON FACULTY OF NATURAL & ENVIRONMENTAL SCIENCES School of Chemistry Studying the Lipoyl Synthase mediated conversion of Octanoyl substrates to Lipoyl products by Nhlanhla Sibanda Thesis for the degree of Doctor of Philosophy December 2013 UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF NATURAL & ENVIRONMENTAL SCIENCES CHEMICAL BIOLOGY Thesis for the degree of Doctor of Philosophy STUDYING THE LIPOYL SYNTHASE MEDIATED CONVERSION OF OCTANOYL SUBSTRATES TO LIPOYL PRODUCTS By: Nhlanhla Sibanda -Lipoic acid is a cofactor used during oxidative metabolism reactions by several enzymes, including branched chain keto acid dehydrogenases, the glycine cleavage system, pyruvate dehydrogenase and -ketoglutarate dehydrogenase.[1] The lipoic acid is attached to the - amino group of a specific lysine residue via an amide bond and acts as a carrier of acyl groups between active sites of these large multienzyme complexes.[2] The biosynthesis of lipoyl product requires as a cofactor the protein lipoyl synthase (LipA), which has two [4Fe- 4S]1+/2+ clusters, and uses S-adenosylmethionine as a substrate.[3] LipA is the product of the lipA gene [4] and has 36 % sequence homology to biotin synthase in E.coli, another protein involved in a sulfur insertion reaction.[5] The mechanism of sulfur insertion during lipoic acid biosynthesis is thought to be related to that for the BioB catalysed synthesis of biotin.[5, 6] LipA and BioB belong to a group of enzymes known to as the “radical SAM” superfamily.[7] All members of this group reductively cleave AdoMet to give methionine and the highly reactive 5′-deoxyadenosyl radical (5′- Ado•) which abstracts hydrogen atoms from appropriate substrates forming the side product 5’-deoxyadenosine and a substrate radical.[1, 8] The work in this thesis describes experiments that were carried out to study the mechanism of the LipA mediated reaction. LipA was expressed and purified in E.coli and two types of octanoylated substrates were synthesized; fluorescent and non-fluorescent substrates. These substrates were used in assays to probe the mechanism of the LipA mediated reaction. The binding constant of the co-substrate SAM to LipA was determined using fluorescence polarization assays. Experiments were also carried out to try and crystallize the LipA in the presence of octanoyl substrates and the co-substrate SAM. Table of Contents Table of Contents .......................................................................................................................... i List of Tables ...............................................................................................................................vii List of Figures ............................................................................................................................. ix DECLARATION OF AUTHORSHIP........................................................................................xv Acknowledgements....................................................................................................................xvii Abbreviations............................................................................................................................. xix Chapter 1: Introduction 1.1 Nature and the sulfur containing compounds.......................................... 1 1.2 SAM (AdoMet) as a source of various functional groups............................ 2 1.3 Radical enzymes and biochemical reactions ........................................... 3 1.3.1 The “radical SAM” superfamily............................................................ 3 1.3.2 The “radical SAM” enzymes sub-groups ................................................. 4 1.3.3 Features common to the “radical SAM” enzymes ...................................... 6 1.3.4 Formation of 5′-deoxyadenosyl (5′- Ado•) radicals................................... 10 1.3.5 The function of the iron-sulfur cluster in the cleavage of SAM..................... 14 1.4 Biotin synthase (BioB).................................................................... 17 1.4.1 The Iron-Sulfur Clusters of BioB ................................................... 20 1.4.2 The source of Sulfur in biotin ....................................................... 21 1.4.3 The crystal structure of BioB........................................................ 23 1.5 -Lipoic Acid............................................................................... 26 1.5.1 Lipoic acid-dependent complexes .................................................. 28 1.5.2 The -keto acid dehydrogenases ................................................... 28 1.5.3 The Pyruvate Dehydrogenase Complex (PDH).................................. 28 1.5.4 The Glycine Cleavage System (GCS) .............................................. 31 1.6 The biosynthesis of lipoic acid catalysed by lipoyl synthase (LipA)............. 35 i 1.6.1 Identification of the lipA gene .............................................................37 1.6.2 The substrate for LipA ......................................................................38 1.6.3 The biosynthesis of -lipoyl groups from octanoyl groups ..........................41 1.6.4 [4Fe-4S] clusters and the reaction mechanism ........................................42 1.7 The Aims and Objectives of the Study.................................................. 45 Chapter 2: Results and Discussions 2.1 The expression, purification and the isolation of LipA ...............................47 2.1.1 Transforming competent cells with plasmid DNA .....................................47 2.1.2 Expression and purification of S. solfataricus LipA...................................52 2.1.3 Conclusions on the expression, isolation and purification of LipA ................54 2.2 Preparation of Substrates for LipA.......................................................55 2.2.1 Testing the stability of fluorophores in the presence of sodium dithionite........55 2.2.2 Synthesis of substrate analogues..........................................................58 2.2.3 Peptide synthesis .............................................................................61 2.2.4 Synthesis of 7-amino-4-methyl-6-sulfocoumarin-3-acetic acid .....................68 2.2.5 Synthesis of Maleimide Derivative and the Derivatization Experiments..........72 2.3 Activity Assays................................................................................78 2.3.1 Product Inhibition of LipA .................................................................78 2.3.2 Characterisation of the products of the nonanoyl substrate turn-over ............86 2.4 Ligand-Protein binding relationships....................................................94 2.4.1 Determination of the binding constant Kd using UV-visible spectroscopy .......96 2.4.2 UV-visible spectrum for SAM and the substrate EK(Oct)I binding to LipA......97 ii 2.4.3 UV- visible spectral changes observed when SAM was added to LipA ......... 100 2.4.4 The rapid equilibrium dialysis (RED) experiments ................................. 103 2.4.5 Fluorescence polarization ............................................................... 110 2.4.6 Advantages of using fluorescence polarization ...................................... 112 2.4.7 The fluorescence polarization assays.................................................. 114 2.5 Crystallization of LipA.................................................................... 117 2.5.1 Crystallization of Macromolecules..................................................... 117 2.5.2 Vapour diffusion methods ................................................................ 121 2.5.3 Initial screening Experiment............................................................. 122 2.5.4 Testing the effect of changing the concentration of PEG polymer............... 124 2.6 Comments and Conclusions ............................................................. 127 2.7 Proposed future experimental work in this study.................................... 130 Chapter 3: Experimental 3.1 Materials .................................................................................. 131 3.2 Instrumentation .......................................................................... 131 3.3 The expression and purification of LipA ............................................ 134 3.3.1 Preparation of media ..................................................................... 134 3.3.2 Preparation of Buffers .................................................................... 134 3.3.3 Plasmids ..................................................................................... 135 3.3.4 Transformation
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