DOTTORATO DI RICERCA IN BIOCHIMICA CICLO XXVI (A.A. 2010-2013) Biochemical characterization of glutamate decarboxylase from Brucella microti and Escherichia coli Asp86* Asp86* Phe463 Lys276 His275 Lys276 His275 His465 Acetate Lys464 Ala245 Ala245 Thr466 Asp243 Gln163 Asp243 Gln163 Docente guida Coordinatore Prof. Roberto Contestabile Prof. Francesco Malatesta Tutor Prof.ssa Daniela De Biase Dottoranda Gaia Grassini Dicembre 2013 DOTTORATO DI RICERCA IN BIOCHIMICA CICLO XXVI (A.A. 2010-2013) Biochemical characterization of glutamate decarboxylase from Brucella microti and Escherichia coli Dottoranda Gaia Grassini Docente guida Coordinatore Prof. Roberto Contestabile Prof. Francesco Malatesta Tutor Prof.ssa Daniela De Biase Dicembre 2013 INDEX 1 Introduction ................................................................................................. 1 1.1 Glutamate decarboxylase-dependent acid resistance in orally- acquired bacteria ............................................................................................ 2 1.1.1 Acid resistance in orally-acquired bacteria ............................................. 2 1.1.2 The GDAR system: function and regulation ........................................... 6 1.1.3 The GDAR system: structural components ........................................... 11 1.1.3.1 GadC .................................................................................................. 12 1.1.3.2 GadB .................................................................................................. 14 1.2 Glutamate decarboxylase ...................................................................... 18 1.2.1 Glutamate decarboxylases ..................................................................... 18 1.2.2 Biochemical insights into Escherichia coli GadB ................................ 21 1.2.3 Bacterial Gads ....................................................................................... 26 1.2.4 Eukaryotic Gads .................................................................................... 30 1.3 Biotechnological aspects ........................................................................ 37 1.3.1 GABA production by GDAR system in health and disease ................. 37 1.3.2 The application of glutamate decarboxylase in the manufacturing of biobased industrial chemicals ........................................................................ 38 1.4 Aims ......................................................................................................... 42 2 Materials and Methods ............................................................................. 44 2.1 Growth media, bacterial strains and plasmids .................................... 45 2.2 N-terminal sequence determination of BmGadB ................................ 51 2.3 Recombinant DNA techniques .............................................................. 52 2.3.1 Purification of plasmid DNA, digestion with restriction enzymes and electrophoresis ............................................................................................... 52 2.3.2 Competent cells: preparation and transformation ................................. 53 2.3.3 PCR ....................................................................................................... 54 2.4 Cloning of BmGadB ............................................................................... 55 2.4.1 Preparative PCR .................................................................................... 55 2.4.2 Cloning with Strataclone PCR Cloning Kit .......................................... 56 2.4.3 Cloning into pET3a ............................................................................... 57 2.5 Expression and purification of EcGadB and BmGadB ...................... 59 2.5.1 Growth and induction of the bacterial culture ...................................... 60 2.5.2 Bacterial lysis, precipitation of nucleic acids and ammonium sulphate fractionation ................................................................................................... 61 2.5.3 DEAE-Sepharose chromatography ....................................................... 61 2.5.4 Determination of Gad and cofactor concentration ................................ 63 2.5.5 ApoGadB production ............................................................................ 64 2.6 Analytical techniques ............................................................................. 64 2.6.1 Gad test (Rice test) ................................................................................ 64 2.6.2 GABA assay .......................................................................................... 65 2.6.3 Gad activity assay ................................................................................. 66 2.6.4 Isotope effect ......................................................................................... 67 2.6.5 Protein assays ........................................................................................ 68 2.6.6 SDS-PAGE electrophoresis and immunoblot analysis ......................... 69 2.6.7 Gel filtration chromatography ............................................................... 70 2.6.8 Spectroscopic analysis........................................................................... 71 2.6.8.1 Steady-state spectroscopy .................................................................. 71 2.6.8.2 Fluorescence ....................................................................................... 72 2.6.9 Cell fractionation ................................................................................... 74 2.7 Crystallization and structural analysis of the apo form of EcGadB . 75 2.7.1 Protein crystallization ........................................................................... 75 2.7.2 X-ray Diffraction Experiment ............................................................... 77 2.8 Data Analysis .......................................................................................... 79 3 Results and Discussion .............................................................................. 80 3.1 PART I: Biochemical and spectroscopic properties of recombinant glutamate decarboxylase from Brucella microti ....................................... 81 3.1.1 Effect of the heterologous expression of B. microti gadBC in an E. coli acid-sensitive mutant strain ............................................................................ 83 3.1.2 Expression and purification of recombinant BmGadB ......................... 88 3.1.3 pH-dependent absorbance and activity changes ................................... 93 3.1.4 Fluorescence properties ......................................................................... 98 3.1.5 pH-dependent cellular partition .......................................................... 101 3.1.6 Discussion ........................................................................................... 103 3.2 PART II: The Asp86-His465 mutant of Escherichia coli glutamate decarboxylase exhibits improved GABA synthesis at alkaline pH ........ 106 3.2.1 Preliminary data: purification and spectroscopic properties of GadB- D86NH465A ................................................................................................ 108 3.2.2 Catalytic Properties ............................................................................. 111 3.2.2.1 Effect of pH on specific activity ...................................................... 111 3.2.2.2 Kinetic constants and solvent isotopic effect ................................... 114 3.2.3 Structural analysis ............................................................................... 116 3.2.4 Discussion ........................................................................................... 117 3.3 PART III: Preliminary structural analysis of Escherichia coli glutamate decarboxylase apo form ........................................................... 122 3.3.1 Gel filtration chromatography ............................................................. 123 3.3.2 Crystallization and structural analysis................................................. 126 3.3.3 Discussion ........................................................................................... 127 3.4 Conclusions and perspectives .............................................................. 129 4 References ................................................................................................ 131 5 Publications .............................................................................................. 150 Abbreviations used GIT gastrointestinal tract SCFAs short chain fatty acids AR acid resistance PLP pyridoxal 5’-phosphate GDAR glutamate-dependent AR ADAR arginine-dependent AR Gad glutamate decarboxylase GABA γ-aminobutyrate AFI acid fitness island PMP pyridoxamine 5'-phosphate LAB Lactic Acid Bacteria CaM calmodulin CaMBD Ca2+/calmodulin Binding Domain AID autoinhibitory domains ORF open reading frame DTT dithiothreitol SDS sodium dodecyl sulphate SDS-PAGE SDS-polyacrylamide gel electrophoresis DEAE diethylethanolamine HEPPS 3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid BmGadB Brucella microti GadB EcGadB Escherichia coli GadB DDC L-DOPA decarboxylase Introduction 1 1.1 Glutamate decarboxylase-dependent acid resistance in orally-acquired bacteria 1.1.1 Acid resistance in orally-acquired bacteria Acid stress is one of the most hostile conditions for the bacterial cell. Generally,