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 ....................................................................................