Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 819 Structural and Functional Studies of Glycoside Hydrolase Family 12 Enzymes from Trichoderma reesei and other Cellulolytic Microorganisms BY MATS SANDGREN ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2003 Dissertation for the Degree of Doctor of Philosophy in Molecular Biology presented at Uppsala University in 2003. ABSTRACT Sandgren, M. 2003. Structural and functional studies of glycoside hydrolase family 12 enzymes from Trichoderma reesei and other cellulolytic microorganisms. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the faculty of Science and Technology 819. 68 pp. Uppsala. ISBN 91-554-5562-X Cellulose is the most abundant organic compound on earth. A wide range of highly specialized microorganisms, have evolved that utilize cellulose as carbon and energy source. Enzymes called cellulases, produced by these cellulolytic organisms, perform the major part of cellulose degradation. In this study the three-dimensional structure of four homologous glycoside hydrolase family 12 cellulases will be presented, three fungal enzymes; Humicola grisea Cel12A, Hypocrea schweinitzii Cel12A, Trichoderma reesei Cel12A, and one bacterial; Streptomyces sp. 11AG8 Cel12A. The structural and biochemical information gathered from these and 15 other GH family 12 homologues has been used for the design of variants of these enzymes. These variants have biochemically been characterized, and thereby the positions and the types of mutations have been identified responsible for the biochemical differences between the homologous enzymes, e.g., thermal stability and activity. The three-dimensional structures of two T. reesei Cel12A variants, where the mutations have significant impact on the stability or the activity of the enzyme have been determined. Four ligand complex structures of the WT H. grisea Cel12A enzyme, that have made it possible to characterize the interactions between substrate and enzyme, have also been determined. The structural and biochemical studies of these closely related GH family 12 enzymes, and their variants, have provided insight on how specific residues contribute to protein thermal stability and enzyme activity. This knowledge can in the future serve as a structural toolbox, i.e., to design Cel12A enzymes with specific properties and features by introducing subtle changes in structural components of the enzymes. These can then be utilized to develop new industrial products or fine-tune enzymes in already existing applications. Key Words: Cellulase, endoglucanase, thermal stability, homologues, protein structure, ligand complex, X-ray crystallography Mats Sandgren, Department of Cell and Molecular Biology, Uppsala University, Biomedical Centre, Box 596, SE-751 24 Uppsala, Sweden. E-mail: [email protected] ©Mats Sandgren 2003 ISSN: 1104-232X ISBN: 91-554-5562-X Printed in Sweden by Uppsala University, Universitetstryckeriet, Uppsala 2003 To my lovely family TABLE OF CONTENTS PAPERS INCLUDED IN THE THESIS ...................................................................... 6 ABBREVIATIONS..................................................................................................... 7 1INTRODUCTION ................................................................................................... 8 2BACKGROUND .....................................................................................................10 2.1 Wood............................................................................................................10 2.1.1 Cellulose............................................................................................10 2.1.2 Other plant cell wall components......................................................13 2.2 Cellulolytic organisms .................................................................................13 2.2.1 Trichoderma reesei ...........................................................................14 2.2.2 Hypocrea schweinitzii .......................................................................14 2.2.3 Humicola grisea ................................................................................15 2.2.4 Streptomyces sp. 11AG8....................................................................15 2.2.5 Industrial applications of glycoside hydrolases.................................15 2.3 Cellulases.....................................................................................................15 2.3.1 Classification of cellulases................................................................16 2.3.2 Domain structure organization of cellulases.....................................17 2.3.3 Hydrolytic mechanism of cellulases..................................................18 2.4 The cellulolytic system of Trichoderma reesei............................................20 2.4.1 Induction of cellulases.......................................................................24 2.4.2 Synergy between cellulases...............................................................24 2.4.3 Three-dimensional Structures of T. reesei cellulases ........................26 3METHODS.............................................................................................................27 3.1 X-ray crystallography..................................................................................27 3.1.2 Phase determination ..........................................................................27 3.1.3 Model building and structure refinement ..........................................30 3.2 Protein crystallization ..................................................................................30 4RESULTS AND DISCUSSION................................................................................32 4.1 Aim of thesis................................................................................................32 4.2 The Trichoderma reesei Cel12A structure...................................................32 4.2.1 Crystallization and structure determination.......................................33 4.2.2 Protein structure................................................................................34 4.2.3 Substrate-binding cleft......................................................................35 4.3 Thermal stability and activity of GH family 12 enzymes ............................36 4.3.1 Thermal stability................................................................................38 4.3.2 Relative enzyme activity ...................................................................39 4.3.3 Structural features affecting stability.................................................40 4.3.4 Discussion.........................................................................................44 4.4 The Humicola grisea Cel12A structure, and stabilizing cysteines..............44 4.4.1 Thermal stability................................................................................44 4.4.2 Relative enzyme activity ...................................................................46 4.4.3 Protein structures...............................................................................46 4.4.4 Discussion.........................................................................................49 4.5 H. grisea Cel12A complex structures..........................................................49 4.5.1 Overall protein structures..................................................................49 4.5.2 Oligosaccharide complexes...............................................................49 4.5.3 Protein carbohydrate interactions......................................................52 4.5.4 Transglycosylation............................................................................54 5CONCLUDING REMARKS ...................................................................................56 6 ACKNOWLEDGEMENTS .....................................................................................57 7 REFERENCES .......................................................................................................58 8 APPENDICES ........................................................................................................67 PAPERS INCLUDED IN THE THESIS This thesis is based upon the following original publications and manuscripts, and will be referred to in the summary by their Roman numerals: I. Sandgren, M., Shaw, A., Ropp, T. H., Wu, S., Bott R., Cameron, A. D., Ståhlberg, J., Mitchinson, C. and Jones, T. A. (2001). The X-ray crystal structure of the Trichoderma reesei family 12 endoglucanase 3, Cel12A, at 1.9 Å resolution. J. Mol. Biol. 308: 295-210 II. Sandgren, M., Gualfetti, P. J., Shaw, A., Gross, L. S, Saldajeno, M., Day, A. G., Jones, T. A. and Mitchinson, C. (2003). Comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability. Protein Sci. 12: 848-866 III. Sandgren, M., Gualfetti, P. J., Paech, C., Paech, S., Shaw, A., Gross, L., Saldajeno, M., Berglund, G. I., Jones, T. A. and Mitchinson C. (2003). The Humicola grisea Cel12A enzyme structure at 1.2 Å resolution, and the recruitment of residues important for thermal stability of glycoside hydrolase family 12 enzymes. Submitted. IV. Sandgren, M., Shaw, A., Gualfetti, P. J., Gross, L. G., Berglund, G. I., Ståhlberg J., Kenne, L., Driguez H.,, Jones, T. A. and Mitchinson C. (2003). Crystal complex structures reveals how a cellulose chain is bound in the 35