title: Effects of the linker region on the structure and function of modular GH5 cellulases.
abstract: The association of glycosyl hydrolases with catalytically inactive modules is a successful evolutionary strategy that is commonly used by biomass-degrading microorganisms to digest plant cell walls. The presence of accessory domains in these enzymes is associated with properties such as higher catalytic efficiency, extension of the catalytic interface and targeting of the enzyme to the proper substrate. However, the importance of the linker region in the synergistic action of the catalytic and accessory domains remains poorly understood. Thus, this study examined how the inter-domain region affects the structure and function of modular GH5 endoglucanases, by using cellulase 5A from Bacillus subtilis (BsCel5A) as a model. BsCel5A variants featuring linkers with different stiffnesses or sizes were designed and extensively characterized, revealing that changes in flexibility or rigidity in this region differentially affect kinetic behavior. Regarding the linker length, we found that precise inter-domain spacing is required to enable efficient hydrolysis because excessively long or short linkers were equally detrimental to catalysis. Together, these findings identify molecular and structural features that may contribute to the rational design of chimeric and multimodular glycosyl hydrolases.
authors.affiliation: Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas/SP, 13083-970, Brazil.
authors.forename: Diego M
authors.initials: DM
authors.lastname: Ruiz
authors.affiliation: Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas/SP, 13083-970, Brazil.
authors.forename: Valeria R
authors.initials: VR
authors.lastname: Turowski
authors.affiliation: Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas/SP, 13083-970, Brazil.
authors.forename: Mario T
authors.initials: MT
authors.lastname: Murakami
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D057075:Enzyme Assays; D007700:Kinetics; D016296:Mutagenesis; D000072417:Protein Domains; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D053838:Scattering, Small Angle; D013379:Substrate Specificity; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002480:Cellulase