Structural and biochemical studies of GH family 12 cellulases: improved thermal stability, and ligand complexes.
In this review we will describe how we have gathered structural and biochemical information from several homologous cellulases from one class of glycoside hydrolases (GH family 12), and used this information within the framework of a protein-engineering program for the design of new variants of these enzymes. These variants have been characterized to identify some of the positions and the types of mutations in the enzymes that are responsible for some of the biochemical differences in thermal stability and activity between the homologous enzymes. In this process we have solved the three-dimensional structure of four of these homologous GH 12 cellulases: Three fungal enzymes, Humicola grisea Cel12A, Hypocrea jecorina Cel12A and Hypocrea schweinitzii Cel12A, and one bacterial, Streptomyces sp. 11AG8 Cel12A. We have also determined the three-dimensional structures of the two most stable H. jecorina Cel12A variants. In addition, four ligand-complex structures of the wild-type H. grisea Cel12A enzyme have been solved and have made it possible to characterize some of the interactions between substrate and enzyme. The structural and biochemical studies of these related GH 12 enzymes, and their variants, have provided insight on how specific residues contribute to protein thermal stability and enzyme activity. This knowledge can serve as a structural toolbox for the design of Cel12A enzymes with specific properties and features suited to existing or new applications.
Sandgren
Mats
M
Department of Cell and Molecular Biology, Uppsala University, Biomedical Center, Husargatan 3, Box 596, SE-751 24 Uppsala, Sweden. mats@xray.bmc.uu.se
Ståhlberg
Jerry
J
Mitchinson
Colin
C
D000595:Amino Acid Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D002473:Cell Wall / Q000378:metabolism; D002480:Cellulase / Q000378:metabolism; D002482:Cellulose / Q000737:chemistry; D002942:Circular Dichroism; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000502:physiology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010944:Plants / Q000737:chemistry; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D008024:Ligands; D009844:Oligosaccharides; D011134:Polysaccharides; C007916:hemicellulose; D002482:Cellulose; D006026:Glycoside Hydrolases; D002480:Cellulase; D003545:Cysteine