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On the Engineering of Proteins: Methods and Applications for Carbohydrate-Active Enzymes

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On the Engineering of Proteins: Methods and Applications for Carbohydrate-Active Enzymes On the engineering of proteins: methods and applications for carbohydrate-active enzymes Fredrika Gullfot Doctoral Thesis in Biotechnology Stockholm, Sweden 2010 © Fredrika Gullfot School of Biotechnology Royal Institute of Technology AlbaNova University Centre SE-106 91 Stockholm Sweden Printed at US-AB Universitetsservice TRITA-BIO Report 2010:14 ISSN 1654-2312 ISBN 978-91-7415-709-3 ii ABSTRACT This thesis presents the application of different protein engineering methods on enzymes and non-catalytic proteins that act upon xyloglucans. Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glucans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glucans such as cellulose. One important group of xyloglucan-active enzymes is encoded by the GH16 XTH gene family in plants, including xyloglucan endo-transglycosylases (XET) and xyloglucan endo-hydrolases (XEH). The molecular determinants behind the different catalytic routes of these homologous enzymes are still not fully understood. By combining structural data and molecular dynamics (MD) simulations, interesting facts were revealed about enzyme-substrate interaction. Furthermore, a pilot study was performed using structure-guided recombination to generate a restricted library of XET/XEH chimeras. Glycosynthases are hydrolytically inactive mutant glycoside hydrolases (GH) that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Different enzymes with xyloglucan hydrolase activity were engineered into glycosynthases, and characterised as tools for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns. Carbohydrate-binding modules (CBM) are non-catalytic protein domains that bind to polysaccharidic substrates. An important technical application involves their use as molecular probes to detect and localise specific carbohydrates in vivo. The three-dimensional structure of an evolved xyloglucan binding module (XGBM) was solved by X-ray diffraction. Affinity-guided directed evolution of this first generation XGBM resulted in highly specific probes that were used to localise non-fucosylated xyloglucans in plant tissue sections. Keywords: enzyme engineering, rational design, directed evolution, DNA shuffling, glycosynthase, xyloglucan, xyloglucan endo-transglycosylase, retaining glycoside hydrolase, xyloglucanase, carbohydrate binding module, polysaccharide synthesis iii SAMMANFATTNING I denna avhandling beskrivs hur olika metoder för s.k. protein engineering har tillämpats på enzymer och icke-katalytiska proteiner som är aktiva på xyloglukaner. Xyloglukaner är polysackarider som förekommer som lagringskolhydrater i frön och rotknölar, och som bildar korslänkande glukankedjor i växters cellväggar. Strukturen är komplex och olika förgreningsmönster bidrar till polysackaridens fysikaliska egenskaper såsom bindning och interaktion med andra glukaner, till exempel cellulosa. En viktig grupp av xyloglukanaktiva enzymer kodas av växtgenfamiljen XTH i GH16, xyloglukan-endo-transglykosylaser (XET) och xyloglukan-endo-hydrolaser (XEH). Kunskap saknas ännu om de molekylära orsakerna till de olika katalytiska vägarna hos dessa homologa enzymer. Genom att kombinera strukturdata och MD-simuleringar avslöjades intressanta fakta om interaktionen mellan enzym och substrat. Vidare genomfördes en pilotstudie för att använda strukturbaserad rekombinering för att skapa ett begränsat bibliotek av XET/XEH hybrider. Glykosyntaser är hydrolytiskt inaktiva muterade glykosidhydrolaser (GH) som katalyserar bildandet av glykosidbindningar mellan glykosylflourider och acceptorglykosider. Olika enzymer med xyloglukanasaktivitet byggdes om till glykosyntaser, och karaktäriserades i sin egenskap av verktyg för att syntetisera väldefinerade och homogena xyloglukaner med regelbundna förgreningsmönster. Kolhydratbindande moduler (CBM) är icke-katalytiska proteindomäner som binder till polysackaridsubstrat. En viktig teknisk tillämpning är att de kan användas som molekylära prober för att upptäcka och lokalisera specifika kolhydrater in vivo. Den tredimensionella strukturen av en evolverad xyloglukanbindande modul (XGBM) löstes med röntgendiffraktion. Med affinitetsbaserad riktad evolution av denna första generationens XGBM skapades mycket specifika prober som användes för att detektera icke-fukosylerade xyloglukaner i växtvävnadssnitt. iv ” The function of the scientist is to know, while that of the engineer is to do. The scientist adds to the store of verified, systematized knowledge of the physical world; the engineer brings this knowledge to bear on practical problems.” - Encyclopedia Britannica To my family, a remarkable pool of genes and activities v vi LIST OF PUBLICATIONS I Kathleen Piens,* Maria Henriksson,* Fredrika Gullfot, Marie Lopez, Régis Fauré, Farid M. Ibatullin, Tuula T. Teeri, Hugues Driguez and Harry Brumer (2007). Glycosynthase activity of hybrid aspen xyloglucan endo-transglycosylase PttXET16-34 mutants. Org. Biomol. Chem. 5 (24): 3971-3978. * These authors contributed equally to the work II Fredrika Gullfot, Farid M. Ibatullin, Gustav Sundqvist, Gideon Davies and Harry Brumer (2009). Functional characterization of xyloglucan glycosynthases from GH7, GH12 and GH16 scaffolds. Biomacromolecules 10 (7): 1782-1788. III Pekka B. Mark, Martin Baumann, Jens Eklöf, Fredrika Gullfot, Gurvan Michel, Åsa Kallas, Tuula T. Teeri, Harry Brumer and Mirjam Czjzek (2009). Analysis of nasturtium TmNXG1 complexes by crystallography and molecular dynamics provides detailed insight into substrate recognition by family GH16 xyloglucan endo-transglycosylases and endo- hydrolases. Proteins 75 (4): 820-836. IV Fredrika Gullfot, Tuula T. Teeri and Harry Brumer (2010). Design of GH16 XET/XEH chimeric enzymes with SCHEMA. Manuscript. V Laura von Schantz, Fredrika Gullfot, Sebastian Scheer, Lada Filonova, Lavinia Cicortas Gunnarsson, James E. Flint, Geoffrey Daniel, Eva Nordberg-Karlsson, Harry Brumer and Mats Ohlin (2009). Affinity maturation generates greatly improved xyloglucan- specific carbohydrate binding modules. BMC Biotechnology 9 (92). VI Fredrika Gullfot, Tien-Chye Tan, Laura von Schantz, Eva Nordberg Karlsson, Mats Ohlin, Harry Brumer and Christina Divne (2009). The crystal structure of XG-34, an evolved xyloglucan-specific carbohydrate-binding module. Proteins 78 (3): 785-789. vii The author’s contribution: Publication I: Experimental design and mathematical modelling, pH profiling and kinetic experiments with PttXET16-34 glycosynthases together with Maria Henriksson. Publication II: Design, cloning and expression of TmNXG1 glycosynthases, experimental design, characterisation including kinetics of all presented glycosynthases, synthesis of homoxyloglucans incl. product analysis by HPAEC-PAD and SEC-ELS. Writing of the manuscript including figures and tables. Publication III: Protein expression and purification, comparison of structural and MD simulation data and drawing of ligand plots. Publication IV: Design of study and all experimental work in silico and in vitro, writing of manuscript. Publication V: Binding studies with isothermal titration calorimetry on presented modules, writing of relevant sections including figures. Publication VI: Protein crystallisation and optimisation, drafting of manuscript (excluding data collection) and figures. Other contributions relevant to this thesis: Expression of TmNXG1, cloning and expression of TmNXG1-'YNIIG, assistance in drafting of the manuscript for: Baumann et al. (2007). Structural evidence for the evolution of xyloglucanase activity from xyloglucan endo-transglycosylases: biological implications for cell wall metabolism. Plant Cell 19(6):1947-1963. viii LIST OF ABBREVIATIONS AE Affinity electrophoresis CBM Carbohydrate binding module CNP Chloro nitrophenyl DMSO Dimethyl sulfoxide dNTP Deoxyribonucleotide triphosphate dsDNA Double-stranded DNA ELS Evaporative light scattering epPCR Error-prone PCR FITC Fluorescein isothiocyanate Fuc Fucose Gal Galactose GFC Gel filtration chromatography GH Glycoside hydrolase Glc Glucose GPC Gel permeation chromatography HPAEC High-performance anion-exchange chromatography HTS High-throughput screening ITC Isothermal titration calorimetry mAb Monoclonal antibody MD Molecular dynamics PAD Pulsed amperometric detection PCR Polymerase chain reaction SEC Size exclusion chromatography ssDNA Single-stranded DNA XET Xyloglucan endo-transglycosylase XEH Xyloglucan endo-hydrolase XGBM Xyloglucan binding module XGO Xylogluco-oligosaccharide Xyl Xylose ix x TABLE OF CONTENTS 1 Introduction .................................................................................................................... 1 1.1 Carbohydrate-active enzymes ...................................................................................................... 2 1.1.1 Glycoside hydrolases ............................................................................................................... 2 1.2 Xyloglucan ...................................................................................................................................... 4 1.2.1 Structure and nomenclature ................................................................................................... 5 1.2.2 Applications .............................................................................................................................
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