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Biochemical and Structural Studies on Enzymes of Menaquinone Biosynthesis

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Biochemical and Structural Studies on Enzymes of Menaquinone Biosynthesis Biochemical and structural studies on enzymes of menaquinone biosynthesis Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät I – Biowissenschaften – der Martin-Luther-Universität Halle-Wittenberg, Vorgelegt von Camila A. Cotrim geboren am 03.04.1985 in Campinas (Brasilien) Gutachter 1. Prof. Dr. Milton T. Stubbs 2. 3 Contents Contents Contents ................................................................................................................................................... i 1. Introduction ......................................................................................................................................... 1 1.1. Isoprenoid quinones .................................................................................................................... 1 1.2. Ubiquinone (UQ) ......................................................................................................................... 2 1.2.1 Biosynthesis of ubiquinone .................................................................................................... 2 1.3. Vitamin K ..................................................................................................................................... 4 1.3.1. Menaquinone (MK)................................................................................................................ 5 1.3.1.1. Biosynthesis of menaquinone in microorganisms ........................................................... 6 1.3.1.2. Menaquinone biosynthesis as a target for antibacterial drugs ....................................... 9 1.3.2. Futalosine hydrolase (MqnB) ................................................................................................. 9 1.3.3. Vitamin K in humans ............................................................................................................ 10 1.4. Prenyltransferases ..................................................................................................................... 11 1.4.1. Aromatic prenyltransferases ............................................................................................... 12 1.4.2. 4-hydroxybenzoate octaprenyltransferase (UbiA) .............................................................. 14 1.4.3. 1,4-dihydroxy-2-naphthoic acid octaprenyltransferase (MenA) ......................................... 15 1.5. Aims of the work ....................................................................................................................... 17 2. Materials and Methods ..................................................................................................................... 19 2.1. Materials ................................................................................................................................... 19 2.1.1. Chemicals ............................................................................................................................. 19 2.1.2. Enzymes and Antibodies ...................................................................................................... 21 2.1.3. Markers ................................................................................................................................ 21 2.1.4. Kits ....................................................................................................................................... 22 2.1.5. Bacteria Strains and Plasmids .............................................................................................. 22 2.1.6. Oligonucleotides .................................................................................................................. 23 2.2. Molecular Biology Methods ...................................................................................................... 24 2.2.1. DNA Extraction .................................................................................................................... 24 2.2.2. Polymerase Chain Reaction (PCR) ....................................................................................... 24 2.2.3. Agarose gel .......................................................................................................................... 25 2.2.4. Site-directed mutagenesis ................................................................................................... 26 2.2.5. Digestion .............................................................................................................................. 26 2.2.6. Ligation ................................................................................................................................ 27 2.2.7. Plasmid preparation ............................................................................................................ 27 i Contents 2.3. Microbiology Methods .............................................................................................................. 27 2.3.1. Preparation of chemically competent E. coli cells ............................................................... 27 2.3.2. Heat shock transformation .................................................................................................. 27 2.3.3. Gene expression of recombinant proteins in E. coli ............................................................ 28 2.3.4. Preparation of Streptomyces lividans TK24 protoplasts...................................................... 28 2.3.5. Transformation in Streptomyces lividans TK24 protoplasts ................................................ 29 2.3.6. Gene expression of recombinant proteins in Streptomyces lividans TK24 ......................... 29 2.3.7. Cell disruption ...................................................................................................................... 29 2.3.8. Generation of menA knockout cells by homologous recombination in bacteriophage P1 . 29 2.3.8.1. Preparation of P1 liquid lysate ....................................................................................... 29 2.3.8.2. P1 Transduction ............................................................................................................. 30 2.3.9. Complementation assay ...................................................................................................... 30 2.4. Biochemical Methods ................................................................................................................ 31 2.4.1. Polyacrylamide gel electrophorese (SDS – PAGE) ............................................................... 31 2.4.2. Immunoblotting ................................................................................................................... 32 2.4.3. Alignment ............................................................................................................................ 32 2.4.4. Membrane Proteins ............................................................................................................. 32 2.4.4.1. Preparation of membrane fractions .............................................................................. 32 2.4.4.2. Solubilization of membrane fraction ............................................................................. 33 2.4.5. Purification .......................................................................................................................... 33 2.4.5.1. Membrane proteins – TtUbiA and EcMenA ................................................................... 33 2.4.5.1.1. Immobilized metal ion affinity chromatography (IMAC) – Batch mode .................. 33 2.4.5.1.2. TEV digestion ............................................................................................................ 33 2.4.5.1.3. Size Exclusion Chromatography (SEC) ...................................................................... 34 2.4.5.2. Futalosine Hydrolase (TtMqnB) ..................................................................................... 34 2.4.5.2.1. Heat Treatment ........................................................................................................ 34 2.4.5.2.2. Purification Co-NTA – 5 mL column Äkta system ..................................................... 34 2.4.6. Prenyltransferase Assays ..................................................................................................... 34 2.4.6.1. UbiA prenyltransferase assays ....................................................................................... 34 2.4.6.2. MenA prenyltransferase assays ..................................................................................... 35 2.5. Biophysical Methods ................................................................................................................. 35 2.5.1. Protein concentration determination ................................................................................. 35 2.5.1.1. RC DCTM Protein Assay .................................................................................................... 35 2.5.1.2. UV/Vis Spectroscopy ...................................................................................................... 36 2.5.2. Circular Dichroism (CD) spectroscopy ................................................................................. 36 ii Contents 2.5.3. Mass spectrometry .............................................................................................................. 37 2.5.3.1. MALDI-TOF Mass spectrometry ....................................................................................
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