THE CRYSTALLIZATION AND STRUCTURAL ANALYSIS OF CELLULASES (AND OTHER GLYCOSIDE HYDROLASES): STRATEGIES AND TACTICS

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Publication details

Title of host publicationCELLULASES
DatePublished - 2012
Pages141-168
Number of pages28
PublisherELSEVIER ACADEMIC PRESS INC
Place of PublicationSAN DIEGO
EditorsHJ Gilbert
Original languageEnglish
ISBN (Print)978-0-12-415931-0

Publication series

NameMethods in Enzymology
PublisherELSEVIER ACADEMIC PRESS INC
Volume510
ISSN (Print)0076-6879

Abstract

The three-dimensional (3-D) structures of cellulases, and other glycoside hydrolases, are a central feature of research in carbohydrate chemistry and biochemistry. 3-D structure is used to inform protein engineering campaigns, both academic and industrial, which are typically used to improve the stability or activity of an enzyme. Examples of classical protein engineering goals include higher thermal stability, reduced metal-ion dependency, detergent and protease resistance, decreased product inhibition, and altered specificity. 3-D structure may also be used to interpret the behavior of enzyme variants that are derived from screening or random mutagenesis approaches, with a view to establishing an iterative design process. In other areas, 3-D structure is used as one of the many tools to probe enzymatic catalysis, typically dovetailing with physical organic chemistry approaches to provide complete reaction mechanisms for enzymes by visualizing catalytic site interactions at different stages of the reaction. Such mechanistic insight is not only fundamentally important, impacting on inhibitor and drug design approaches with ramifications way beyond cellulose hydrolysis, but also provides the framework for the design of enzyme variants to use as biocatalysts for the synthesis of bespoke oligosaccharides. Here we review some of the strategies and tactics that may be applied to the X-ray structure solution of cellulases (and other carbohydrate-active enzymes). The general approach is first to decide why you are doing the work, then to establish correct domain boundaries for truncated constructs (typically the catalytic domain only), and finally to pursue crystallization of pure, homogeneous, and monodisperse protein with appropriate ligand and additive combinations. Cellulase-specific strategies are important for the delineation of domain boundaries, while glycoside hydrolases generally also present challenges and opportunities for the selection and optimization of ligands to both aid crystallization, and also provide structural and mechanistic insight. As the many roles for plant cell wall degrading enzymes increase, so does the need for rapid high-quality structure determination to provide a sound structural foundation for understanding mechanism and specificity, and for future protein engineering strategies.

    Research areas

  • MACROMOLECULAR CRYSTALLIZATION, PROTEIN ISOELECTRIC POINT, CARBOHYDRATE-BINDING MODULES, X-RAY CRYSTALLOGRAPHY, ANGSTROM RESOLUTION, TRICHODERMA-REESEI, CELLOBIOHYDROLASE CEL6A, CRYSTAL-STRUCTURE, NONHYDROLYZABLE SUBSTRATE-ANALOG, BETA-GLUCOSIDASES

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