Structural and Functional Analysis of the Levansucrase SacB from Bacillus megaterium Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Christian Philipp Strube aus Tübingen 1. Referent: Honorarprofessor Dr. Dirk Heinz 2. Referentin: Dr. Gunhild Layer eingereicht am: 07.03.2011 mündliche Prüfung (Disputation) am 01.07.2011 Druckjahr 2011 Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Strube, C. P., Homann, A., Gamer, M., Jahn, D., Seibel, J. Heinz, D. W.: Polysaccharide synthesis of the Levansucrase SacB from Bacillus megaterium is controlled by distinct surface motifs. Tagungsbeiträge Strube, C. P., Timm, M., Homann, A., Memmel, E., Seibel, J. & Heinz, D. W.: Strukturbiologie von Glycosyltransferasen zur Optimierung von biotechnologischen Prozessen. (Poster), 100. Kolloquium des Sonderforschungsbereiches 578, Braunschweig (2010). Strube, C. P., Homann, A. Götze, S., Biedendieck, R., Seibel, J. & Heinz, D. W.: X-ray Analysis and Biochemical Characterization of SacB, a Novel Fructosyltransferase from Bacillus megaterium . (Poster), International Congress on Biocatalysis, Hamburg (2008). Strube, C. P., Homann, A., Biedendieck, R., Gamer, M., Jahn, D., Heinz, D. W. & Seibel, J.: Strukturbiologie von Glykosyltransferasen zur Optimierung von biotechnologischen Prozessen. (Poster), Begutachtung SFB 578, Braunschweig (2008). Strube, C. P., Homann, A., Biedendieck, R., Jahn, D., Heinz, D. W. & Seibel, J.: Strukturbiologie von Glykosyltransferasen zur Optimierung von biotechnologischen Prozessen. (Vortrag), Berichtskolloquium SFB 578, Braunschweig (2007). Strube, C. P., Homann, A. Götze, S., Biedendieck, R., Seibel, J. & Heinz, D. W.: Purification, crystallization and preliminary X-ray analysis of SacB, a fructosyltransferase from Bacillus megaterium . (Poster), European BioPerspectives, Köln (2007). Homann, A., Strube, C. P., Pasche, B., Schughart, K., Heinz, D. W. & Seibel, J.: Enzymatic synthesis and immuno-assay of novel carbohydrate structures. (Poster), EMBL International PhD Symposium, Heidelberg (2009). Homann, A., Strube, C. P., Pasche, B., Schughart, K., Heinz, D. W. & Seibel, J.: Towards tailor-made oligosaccharides – Chemo-enzymatic synthesis and physiological functions of novel carbohydrate structures. (Poster, Oral Presentation), SFB 630 International Symposium, Novel agents against Infectious diseases – An Interdisciplinary approach, Würzburg (2009). Homann, A., Strube, C. P., Jahn, D., Heinz, D. W. & Seibel, J.: Towards tailor-made oligosaccharides by enzyme and substrate engineering. (Poster Award), 15th European Carbohydrate Symposium, Wien (2009). Homann, A., Zuccaro, A. Götze, S., Strube, C. P., Dersch, P., Heinz, D. W. & Seibel, J.: Novel fructo-oligosaccharides as pharma-/nutraceuticals. (Poster), European Bioperspectives, Hannover (2008). Homann, A., Strube, C. P., Heinz, D. W. & Seibel, J.: Oligo- vs . polysaccharide formation – Production of oligofructosides and structure-function analysis of a novel fructosyltransferase from Bacillus megaterium. (Poster), International Congress on Biocatalysis, Hamburg (2008). CONTENTS I CONTENTS Contents I Abbreviations V Zusammenfassung 1 Summary 3 1 Introduction 5 1.1 Carbohydrates 5 1.2 Biological role of microbial exopolysaccharides 10 1.3 Applications of poly- and oligosaccharides 11 1.4 Classification of carbohydrate active enzymes 11 1.5 Bacterial fructansucrases 12 1.6 Bacterial glucansucrases 18 2 Aims and Scope 23 3 Material and Methods 25 3.1 Standard materials 25 3.1.1 Enzymes and molecular weight standards 25 3.1.2 Crystallization screens 26 3.1.3 Plasmids 26 3.1.4 Bacterial strains 27 3.2 Buffers and Media 28 3.2.1 Antibiotics 28 3.3 Microbiology 28 3.3.1 Agar plates 28 3.3.2 Liquid cultures 28 3.3.3 Storage of bacteria 28 3.4 Molecular biology 29 3.4.1 Preparation of competent cells 29 CONTENTS II 3.4.2 Transformation of competent bacteria 29 3.4.3 Preparation of plasmid DNA 30 3.4.4 Determination of DNA concentration and purity 30 3.4.5 Agarose gel electrophoresis 30 3.4.6 Extraction of DNA from agarose gels 31 3.4.7 Digestion of plasmid DNA with restriction endonucleases 31 3.4.8 Dephosphorylation of linearized plasmid DNA 31 3.4.9 Ligation of DNA fragments 31 3.4.10 Amplification of DNA by Polymerase Chain Reaction (PCR) 31 3.4.11 Design and synthesis of deoxyribo- oligonucleotides 32 3.4.12 DNA sequencing 33 3.5 Protein production 33 3.6 Protein purification 34 3.6.1 Concentrating protein solutions 36 3.7 Protein biochemical methods 36 3.7.1 Determining protein concentrations 36 3.7.2 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) 36 3.7.3 Western blotting 38 3.7.4 N-terminal sequencing 38 3.7.5 Dynamic light scattering 39 3.7.6 Mass spectrometry 39 3.7.7 Thermal shift assay 39 3.8 Protein crystallization 39 3.8.1 Initial screening 39 3.8.2 Optimization 40 3.8.3 Co-crystallization and soaking 40 3.8.4 Crystal transfer experiments 41 3.9 Data collection structure determination and refinement 41 3.10 Figure preparation 43 CONTENTS III 4 Results 44 4.A SacB from Bacillus megaterium 44 4.A.1 Identification of the active site residues 44 4.A.2 Identification of amino acids not located in the active site of SacB with impact on the transfructosylation 45 4.A.3 Structure determination of SacB variants 45 4.A.3.1 Production and purification of SacB wildtype and variants 45 4.A.3.2 Crystallization of SacB 48 4.A.3.3 Improving crystallization by seeding experiments 49 4.A.3.4 X-ray data collection and structure determination of SacB D257A 50 4.A.3.5 X-ray data collection and structure determination of SacB variants N252A, K373A, Y247A and Y247W 51 4.A.3.6 Secondary structure elements of SacB 53 4.A.3.7 Structural overview of SacB 53 4.A.3.8 Active site of SacB shows a β-propeller fold 57 4.A.4 Structural analysis of SacB variants with impact on the transfructosylation process 58 4.A.5 Crystallographic analysis of potential SacB complexes with different ligands 59 4.A.6 Calcium binding site of SacB 60 4.A.7 Binding of PEG to the active site pocket 61 4.A.8 Validation of the SacB models 63 4.A.9 Crystallization with substrates, products and inhibitors 67 4.A.10 Crystal transfer experiments 69 4.A.11 Alternative crystallization method to avoid PEG binding 70 4.A.12 Thermal shift assay 71 4.B Glycosyltransferase R from Streptococcus oralis 73 4.B.1 Identification of the Glucan Binding Domain of GtfR 73 4.B.2 Cloning strategy for GBD construct 75 4.B.3 Production of GBD in E. coli 76 4.B.4 Crystallization of the GBD 79 CONTENTS IV 4.B.5 X-ray data collection and space group determination of GBD crystals 80 4.B.6 Molecular Replacement 82 5. Discussion 84 5.A Levansucrase SacB from Bacillus megaterium 84 5.A.1 SacB variants N252A and R370A point towards surface elements influencing polysaccharide synthesis 85 5.A.2 SacB K373A abrogates the polysaccharide synthesis between subsite +4 and +5 88 5.A.3 SacB Y247A abrogates the polysaccharide synthesis between subsites +8 and +9 88 5.A.4 Polysaccharide synthesis is controlled by distinct surface motifs 89 5.A.5 Identification of the PEG molecules and probable explanation for missing complex structures 91 5.B Glycosyltransferase R from Streptococcus oralis 94 5.B.1 A probable explanation for the failed structure determination 94 5.B.2 A probable explanation for the anisotropic diffraction 95 References 97 Figures and Tables 108 Danksagung 111 ABBREVIATIONS VI Abbreviations Å Ångstrøm (1 Å = 0.1 nm) Aλ Absorption at the wavelength λ in nm (equivalent to OD λ) Amp Ampicillin R Amp Ampicillin resistance BESSY Berliner Elektronenspeicherring-Gesellschaft für Synchrotron Strahlung, Berlin, Germany BLAST Basic Local Alignment Search Tool B. megaterium Bacillus megaterium C- Carboxy terminus Ccp4 Collaborative Computational Project 4 CHT Ceramic hydroxyapatite Cm Chloramphenicol R Cm Chloramphenicol resistance CV Column volume Da Dalton (equals the mass of 1/12 of the carbon 12 C isotope) DALI Distance Alignment Server DESY Deutsches Elektronensynchrotron, Hamburg, Germany DLS Dynamic Light Scattering DNA Deoxyribonucleic acid E. coli Escherichia coli EDTA Ethylenediaminetetraacetic acid EMBL European molecular biology laboratory EPS Exopolysaccharides ESRF European synchrotron radiation facility, Grenoble, France EtOH Ethanol GBD Glucan binding domain GLRF General Locked Rotation Function GtfR Glycosyltransferase R His 6 Six successive histidine residues, used as affinity tag HPAEC High Performance Anion Exchange Chromatography IEC Ion exchange chromatography IPTG Isopropyl β-D-thiogalactopyranoside Kan Kanamycin R Kan Kanamycin resistance kDa Kilodalton LAB lactic acid bacteria LB Lysogeny broth MeOH Methanol Mr Molecular mass MR Molecular Replacement MS Mass spectrometry MW Molecular weight ABBREVIATIONS VII MWCO Molecular weight cut-off N- Amino terminus Ni-NTA Nickel (II) nitrilotriacetic acid o.n. Overnight OD λ Optical density at the wavelength λ in nm PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PCR Polymerase Chain Reaction PDB Protein Data Bank PEG Polyethylene glycol PVDF Polyvinylidene difluoride RADAR Rapid Automatic Detection and Alignment of Repeats r.m.s.d. Root mean square deviation rpm Rotations per minute RT Room temperature s Standard