Structural and functional studies of glycoside hydrolase family 12 enzymes from Trichoderma reesei and other cellulolytic microorganisms

Abstract: 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.