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Insights Into the Mechanistic and Regulatory Properties of D-Arabinose-5-Phosphate

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Insights Into the Mechanistic and Regulatory Properties of D-Arabinose-5-Phosphate Insights into the Mechanistic and Regulatory Properties of D-arabinose-5-phosphate Isomerases By David L. Cech A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Medicinal Chemistry) in the University of Michigan 2017 Doctoral Committee: Professor Ronald W. Woodard, Chair Professor Harry L. T. Mobley Professor Henry I. Mosberg Professor John J. G. Tesmer David L. Cech [email protected] ORCID iD: 0000-0002-8078-9720 © David L. Cech 2017 TABLE OF CONTENTS LIST OF TABLES ......................................................................................................................... v LIST OF FIGURES ...................................................................................................................... vi CHAPTER I. Introduction ............................................................................................................ 1 Background ...................................................................................................... 1 D-Arabinose-5-Phosphate Isomerase .............................................................. 3 Dissertation Research Rationale ..................................................................... 9 References ..................................................................................................... 11 II. A Novel Glucose-6-phosphate Isomerase from Listeria monocytogenes .......... 14 Summary ........................................................................................................ 14 Introduction ................................................................................................... 15 Materials and Methods ................................................................................. 16 Results ........................................................................................................... 24 Discussion ...................................................................................................... 33 Acknowledgments and Chapter Contributions ............................................. 34 References ..................................................................................................... 35 III. The Arabinose-5-phosphate Isomerase of Bacteroides fragilis: Insight Into Regulation of Single-domain Arabinose Phosphate Isomerases ......................................................................................... 38 Summary ........................................................................................................ 38 Introduction ................................................................................................... 40 ii Materials and Methods ................................................................................. 42 Results ........................................................................................................... 52 Discussion ...................................................................................................... 59 Acknowledgments and Chapter Contributions ............................................. 62 References ..................................................................................................... 63 IV. Identification of a D-Arabinose-5-Phosphate Isomerase in the Gram-positive Clostridium tetani ................................................................................................. 66 Summary ........................................................................................................ 66 Introduction ................................................................................................... 67 Materials and Methods ................................................................................. 68 Results ........................................................................................................... 73 Discussion ...................................................................................................... 81 Acknowledgments and Chapter Contributions ............................................. 83 References ..................................................................................................... 84 V. New Insights Into the Kdo Biosynthetic Pathway and Incorporation Into Lipid A- Like Molecules in Arabidopsis thaliana ............................................................... 86 Summary ........................................................................................................ 86 Introduction ................................................................................................... 88 Materials and Methods ................................................................................. 92 Results ......................................................................................................... 103 Discussion .................................................................................................... 110 Acknowledgments and Chapter Contributions ........................................... 112 References ................................................................................................... 114 VI. Structure of E. coli c3406 in Complex With A5P And Insight Into the Mechanism of D-Arabinose-5-Phosphate Isomerase ............................................................ 118 Summary ...................................................................................................... 118 Introduction ................................................................................................. 119 Materials and Methods ............................................................................... 120 Results and Discussion ................................................................................ 124 Acknowledgments and Chapter Contributions ........................................... 133 References ................................................................................................... 134 iii VII. Summary, Conclusions, and Future Directions ................................................. 137 APPENDIX ........................................................................................................... 141 Cystathionine β-Synthase Domain .............................................................. 141 References ................................................................................................... 143 iv LIST OF TABLES Table 1.1: Kinetic constants for catalysis by the APIs of E. coli……………………….………………………...6 2.1: Strains, Plasmids, and Primers used in this study…………………………..………………………..17 2.2: Kinetic constants for catalysis by Q723E8…..………………………………………………….….....29 3.1: Strains, Plasmids, and Primers used in this study………..…………………………………………..43 3.2: Kinetic constants for catalysis by various APIs………….……………………………………….…...55 4.1: Strains, Plasmids, and Primers used in this study………………..…………………………………..69 4.2: Kinetic constants for the APIs of E. coli and CtAPI…………………………………………………...78 5.1: Strains, Plasmids, and Primers used in this study…………………………………………….……..93 5.2: Kinetic constants for catalysis by various APIs……………………….…………………………....105 6.1: Data collection and refinement statistics………….……………………….……………………..….122 6.2: Strains, Plasmids, and Primers used in this study………….……………………….………..…...123 6.3: KdsD mutant characterization…………………………..…………………….……………….…….…...129 v LIST OF FIGURES Figure 1.1: Representative schematic of the Gram-negative membrane………………….………………..2 1.2: Constituents of LPS….……………………………………………………………………………………….……..2 1.3: The Kdo biosynthetic pathway……………………………………………………………………………......4 1.4: The SIS and CBS domains of APIs..………….……………………………………………………….……....5 2.1: Sequence alignment of Q723E8 and genomic context of lmof2365_0531...……….…..25 2.2: Clustal Omega alignment of the protein sequences of Q723E8 and the C-terminal end of various PGI………………………………………………….……………………………………………………………27 2.3: pH-rate profile for the conversion of G6P to F6P by Q723E8.………….……………………….28 2.4: Effect of metal ions upon the glucose 6-phosphate isomerase activity of Q723E8.………………………………………………………………………………………………………………..........29 2.5: The purification of Q723E8……………………………………….………………………………………..….30 2.6: Native molecular weight determination for Q723E8…………….……..….........................31 2.7: Complementation of an A5P auxotroph on agar plates.…………….…………………………...32 3.1: Gel filtration analysis of Q5LIW1……………….…………………………………………..……………....53 3.2: Effect of divalent metal ions on the activity of Q5LIW1…………………….………………..….54 3.3: pH rate profile of Q5LIW1……..………………….…………………………………….........................55 3.4: Complementation of an A5P auxotroph on agar plates………….…………………………...….56 3.5: Complementation of an A5P auxotroph on LB/agar plates…....………….......................57 vi 3.6: Effect of CMP-Kdo on Q5LIW1 API activity…..……………………………….…………………….….58 4.1: Alignment of sequences of E. coli APIs and CtAPI........................................................74 4.2: Genomic context of gutQ and ctAPI.………………………………………….………………………..….75 4.3: Standard curve from native molecular mass determination.……………….....................76 4.4: pH rate profile of CtAPI……………………………………………….…………………………….………..….77 4.5: Complementation of an A5P auxotroph on an LB agar plate......................................80 5.1: Kdo biosynthesis and incorporation in E. coli.………….…………….………………….………..….88 5.2: A model of RG-II structure with side chains labeled....................................................89 5.3: Complementation of an A5P auxotroph
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