Characterization of the human aldehyde oxidase: studies on the FAD active site and ROS generation Alessandro Foti Dissertation zur Erlangung des akademischen Grades "doctor rerum naturalium" (Dr. rer. nat.) in der Wissenschaftsdisziplin "Biochemie" eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät Institut für Biochemie und Biologie der Universität Potsdam Published online at the Institutional Repository of the University of Potsdam: URN urn:nbn:de:kobv:517-opus4-410107 http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-410107 LIST OF PUBLICATIONS Coelho C, Foti A, Hartmann T, Santos-Silva T, Leimkühler S, Romão MJ. (Achiron et al., 2015) “Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase” Nature Chemical Biolology Oct;11(10):779-83. Foti A, Hartmann T, Coelho C, Santos-Silva T, Romao MJ, Leimkuhler S. (2016) “Optimization of the expression of Human Aldehyde Oxidase for Investigations of Single Nucleotide Polymorphisms.” Drug Metab Dispos. Feb 3. pii: dmd.115.068395. Romão MJ, Coelho C, Santos-Silva T, Foti A, Terao M, Garattini E, Leimkühler S. (2017) “Structural basis for the role of mammalian aldehyde oxidases in the metabolism of drugs and xenobiotics” Current Opinion In Chemical Biology. Jan 23;37:39-47. doi: 10.1016. Foti A, Dorendorf F and Leimkuhler S. (2017) “Single nucleotide polymorphisms cause enhanced radical oxygen species production by human aldehyde oxidase” (manuscript submitted). CONTRIBUTION TO CONFERENCES “Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase” PhD symposium in Luckenwalde, Germany. Nov 2013 oral presentation “Investigation of single nucleotide polymorphisms (SNP) in the active site of human Aldehyde Oxidase”. IX. Molybdenum & Tungsten Enzymes Conference. Balatonfüred, Hungary. Sep 2015 poster and oral presentation. “Human aldehyde Oxidase and radical oxygen species (ROS) formation” PhD symposium in Luckenwalde, Germany. Nov 2016 oral presentation. 2 TABLE OF CONTENTS 1. Introduction .................................................................................................................... 14 1.1 Molybdoenzymes .......................................................................................................... 14 1.2 Sulfuration of the molybdenum cofactor ................................................................... 16 1.3 Characteristics of the Aldehyde oxidases .................................................................. 17 1.3.1 Structure of Aldehyde oxidases .............................................................................. 17 1.3.2 An evolutionary perspective: phylogeny of AOXs. ................................................ 19 1.3.3 Expression and regulation of hAOX1 ..................................................................... 20 1.3.4 Substrate specificity and reaction mechanism ........................................................ 21 1.3.5 The human aldehyde oxidase and its intraspecific variability ............................... 24 1.4 Reactive oxygen species (ROS) and Flavin cofactors ............................................... 26 1.4.1 Reactive oxygen species in cell physiology ............................................................ 26 1.4.2 Flavin cofactors and reactivity toward oxygen ....................................................... 29 1.4.3 Reactive oxygen species and molybdo-flavo enzymes ........................................... 32 2. Aim of this work ............................................................................................................. 37 3. Materials and Methods .................................................................................................. 38 3.1 Materials ....................................................................................................................... 38 3.1.1 Chemicals ................................................................................................................ 38 3.1.2 Media, buffer and solutions .................................................................................... 38 3.1.3 Plasmids and primers .............................................................................................. 38 3.1.4 Cell strains .............................................................................................................. 39 3.2 Methods ......................................................................................................................... 39 3.2.1 Molecular biological methods ................................................................................. 39 3.2.1.1 Cultivation of E. coli strains ............................................................................ 39 3.2.1.2 Competent E. coli cells .................................................................................... 40 3.2.1.3 Transformation of plasmid DNA ..................................................................... 40 3.2.1.4 Isolation of plasmid DNA ................................................................................ 40 3.2.1.5 Polymerase chain reaction (PCR) and Site Direct Mutagenesis ...................... 41 3.2.1.6 Determination of DNA concentration and DNA sequencing .......................... 43 3.2.2 Biochemical methods .............................................................................................. 44 3.2.2.1 Overexpression of human Aldehyde Oxidase in E. coli TP1000 strain .......... 44 3.2.2.2 Cell lysis .......................................................................................................... 44 3.2.2.3 Affinity purification by Ni-NTA chromatography .......................................... 44 3.2.2.4 Buffer exchange and desalting ......................................................................... 45 3.2.2.5 In vitro chemical sulfuration ............................................................................ 45 3.2.2.6 Size exclusion chromatography ....................................................................... 46 3.2.2.7 Ultrafiltration ................................................................................................... 46 3.2.3 Analytical methods and enzymatic assays .............................................................. 46 3 3.2.3.1 Agarose gel electrophoresis ............................................................................. 46 3.2.3.2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) .... 47 3.2.3.3 Coomassie-Blue staining ................................................................................. 48 3.2.3.4 Separation of proteins in native polyacrylamide gels ...................................... 48 3.2.3.5 Activity-stain for hAOX1 ................................................................................ 48 3.2.3.6 Characterization of hAOX1 by UV/VIS Spectroscopy ................................... 48 3.2.3.7 Photometric determination of the specific activity .......................................... 49 3.2.3.8 Reduction spectra ............................................................................................. 50 3.2.3.9 Analysis of the Moco content by HPLC .......................................................... 50 3.2.3.10 Metal content ................................................................................................. 51 3.2.3.11 Quantitation of the FAD cofactor .................................................................. 51 3.2.3.12 Redox titration of hAOX1 ............................................................................. 52 3.2.3.13 Kinetics parameters of hAOX1 ...................................................................... 52 3.2.3.14 Inhibition studies ............................................................................................ 55 3.2.3.15 Electrochemical measurments of H2O2 formation ......................................... 55 3.2.3.16 Cytochrome c reduction assay and determination of O2˙- production .......... 56 3.3 Computational methods .............................................................................................. 57 3.3.1 Protein modelling .................................................................................................... 57 3.3.2 Determination of Single nucleotide Polymorphisms .............................................. 57 4. Results ............................................................................................................................. 58 4.1. Expression and characterization of hAOX1: comparison of a codon optimized and not codon optimized expression system .................................................................... 58 4.1.1 Heterologous expression of a hAOX1 codon optimized gene construct ................ 58 4.1.2 Purification of hAOX1 ............................................................................................ 59 4.1.2.1 Ni-NTA affinity chromatography .................................................................... 59 4.1.4.2 In vitro chemical sulfuration ............................................................................ 60 4.1.2.3 Size exclusion chromatography ....................................................................... 63 4.1.3. Characterization of codono optimized hAOX1 (hAOX1) ..................................... 65 4.1.3.1 Activity staining of hAOX1 ............................................................................ 65 4.1.3.2 Metal content ..................................................................................................