2-methylisocitrate lyase activity

Known as: (2S,3R)-3-hydroxybutane-1,2,3-tricarboxylate pyruvate-lyase (succinate-forming), methylisocitrate lyase activity, MICL

Catalysis of the reaction: (2S,3R)-3-hydroxybutane-1,2,3-tricarboxylate = pyruvate + succinate. [EC:4.1.3.30, RHEA:16812]

National Institutes of Health

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2017

Structural and Functional Characterization of Iron-Containing Oxygenases from Pseudomonas aeruginosa

Michael Lee
2017
Corpus ID: 90203248

........................................................................................................................................................ ii Co-Authorship .............................................................................................................................................. iv Acknowledgements ...................................................................................................................................... vi List of Figures .............................................................................................................................................. xi List of Tables ............................................................................................................................................. xiii List of Abbreviations ................................................................................................................................. xiv Chapter 1 General Introduction ..................................................................................................................... 1 1.1 Pseudomonas aeruginosa ................................................................................................................... 1 1.2 Iron and Bacterial Pathogens .............................................................................................................. 2 1.3 Heme Uptake and Metabolism ............................................................................................................ 3 1.3.1 Heme Uptake Systems in Gram-Negative Bacteria ..................................................................... 3 1.3.1.1 Hemophore-Mediated Heme Uptake Systems ...................................................................... 3 1.3.1.2 Direct Heme Uptake Systems ............................................................................................... 3 1.3.2 Heme Uptake Regulation ............................................................................................................. 8 1.3.3 Heme Uptake and Metabolism in P. aeruginosa ......................................................................... 8 1.3.4 PhuS and HemO ......................................................................................................................... 10 1.4 Iron Utilization .................................................................................................................................. 12 1.4.1 2OG Oxygenases ........................................................................................................................ 14 1.4.1.1 Biological Functions of 2OG Oxygenases .......................................................................... 16 1.4.2 2OG Oxygenases in P. aeruginosa ............................................................................................ 21 1.5 Significance and Objectives .............................................................................................................. 22 Chapter 2 Structural Analysis and Identification of PhuS as a Heme-Degrading Enzyme from Pseudomonas aeruginosa ........................................................................................................................... 23 2.1 Abstract ............................................................................................................................................. 23 2.2 Introduction ....................................................................................................................................... 23 2.3 Results and Discussion ...................................................................................................................... 26 2.3.1 Crystal Structure of PhuS ........................................................................................................... 26 2.3.2 Spectroscopic Analysis of Heme Degradation ........................................................................... 31 2.3.3 CO Measurements by Gas Chromatography ............................................................................. 36 2.3.4 Mass Spectrometry Analysis ...................................................................................................... 36 2.4 Experimental Procedures .................................................................................................................. 39 2.4.1 Expression and Purification of PhuS, HemO, and ChuS ........................................................... 39 ix 2.4.2 Crystallization, Data Collection, and Structure Determination of PhuS .................................... 40 2.4.3 Heme Reconstitution and Spectroscopic Analysis of Heme Degradation ................................. 41 2.4.4 CO Activity Assay of PhuS and ChuS ....................................................................................... 43 2.4.5 Mass Spectrometry of PhuS Heme Degradation Product .......................................................... 43 2.4.6 Accession Numbers .................................................................................................................... 44 2.5 Acknowledgements ........................................................................................................................... 44 2.6 Supplementary Data .......................................................................................................................... 44 Chapter 3 Function Coupling Mechanism of PhuS and HemO in Heme Degradation ............................... 52 3.1 Abstract ............................................................................................................................................. 52 3.2 Introduction ....................................................................................................................................... 52 3.3 Results and Discussion ...................................................................................................................... 55 3.4 Materials and Methods ...................................................................................................................... 67 3.4.1 Theoretical Methods and Models ............................................................................................... 67 3.4.2 Experimental Procedures ........................................................................................................... 69 3.4.2.1 Protein Purification and Heme Degradation Experiments .................................................. 69 3.4.2.2 Kinetic Analysis .................................................................................................................. 70 3.4.2.3 CO Activity Assay .............................................................................................................. 70 3.5 Acknowledgements ........................................................................................................................... 71 3.6 Supporting Information ..................................................................................................................... 71 Chapter 4 Characterization of MicL and PiuC, 2-Oxoglutarate/Fe(II)-Dependent Oxygenases Involved in Cell Growth Regulation in Pseudomonas aeruginosa ................................................................................ 74 4.1 Abstract ............................................................................................................................................. 74 4.2 Introduction ....................................................................................................................................... 74 4.3 Materials and Methods ...................................................................................................................... 76 4.3.1 MicL and PiuC Expression and Purification .............................................................................. 76 4.3.2 Isothermal Titration Calorimetry ............................................................................................... 77 4.3.3 Circular Dichroism ..................................................................................................................... 77 4.3.4 Pull-Down Experiments ............................................................................................................. 78 4.3.5 Growth Assays ........................................................................................................................... 78 4.3.6 PiuC Crystallization ................................................................................................................... 79 4.4 Results and Discussion ...................................................................................................................... 80 4.4.1 MicL Binds 2OG and Fe(II) ....................................................................................................... 80 4.4.2 Searching for the Primary Substrate of MicL ............................................................................ 82 4.4.3 PiuC Characterization ................................................................................................................ 85 x 4.4.4 MicL and PiuC Regulate Cell Growth Under Nutrient Stress ................................................... 88 4.4.5 Structural Studies of MicL and PiuC ......................................................................................... 91 4.5 Acknowledgements ........................................................................................................................... 93 Chapter 5 General Discussion ..................................................................................................................... 95 5.1 Necessity of Heme in Pseudomonas aeruginosa .............................................................................. 95 5.2 PhuS and Heme Oxygenases ....................................