Unique properties of a Dictyostelium discoideum carbohydrate-binding module expand our understanding of CBM-ligand interactions

Author(s)

Liberato, Marcelo Vizona ; Campos, Bruna Medeia ; Tomazetto, Geizecler ; Crouch, Lucy Isobel ; Garcia, Wanius ; de Mattos Zeri, Ana Carolina ; Bolam, David Nichol ; Squina, Fabio Marcio

• Document type: Journal article
• Source: Journal of Biological Chemistry; v. 298, n. 5, p. 10-pg., 2022-05-02.

Abstract

Deciphering how enzymes interact, modify, and recognize carbohydrates has long been a topic of interest in academic, pharmaceutical, and industrial research. Carbohydrate-binding modules (CBMs) are noncatalytic globular protein domains attached to carbohydrate-active enzymes that strengthen enzyme affinity to substrates and increase enzymatic efficiency via targeting and proximity effects. CBMs are considered auspicious for various biotechnological purposes in textile, food, and feed industries, representing valuable tools in basic science research and biomedicine. Here, we present the first crystallo-graphic structure of a CBM8 family member (CBM8), DdCBM8, from the slime mold Dictyostelium discoideum, which was identified attached to an endo-beta-1,4-glucanase (glycoside hy-drolase family 9). We show that the planar carbohydrate-binding site of DdCBM8, composed of aromatic residues, is similar to type A CBMs that are specific for crystalline (multichain) poly-saccharides. Accordingly, pull-down assays indicated that DdCBM8 was able to bind insoluble forms of cellulose. However, affinity gel electrophoresis demonstrated that DdCBM8 also bound to soluble (single chain) polysaccharides, especially glu-comannan, similar to type B CBMs, although it had no apparent affinity for oligosaccharides. Therefore, the structural charac-teristics and broad specificity of DdCBM8 represent exceptions to the canonical CBM classification. In addition, mutational analysis identified specific amino acid residues involved in ligand recognition, which are conserved throughout the CBM8 family. This advancement in the structural and functional character-ization of CBMs contributes to our understanding of carbohydrate-active enzymes and protein-carbohydrate in-teractions, pushing forward protein engineering strategies and enhancing the potential biotechnological applications of glyco-side hydrolase accessory modules. (AU)

• FAPESP's process: 15/50590-4 - Lignin valorization in cellulosic ethanol plants: biocatalytic conversion via ferulic acid to high value chemicals
• Grantee: Fábio Márcio Squina
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Thematic Grants
• FAPESP's process: 13/06336-0 - Study of the Molecular Basis of Carbohydrate Binding Modules (CBMs) from Metagenomic Screenings
• Grantee: Bruna Medéia de Campos
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 15/23279-6 - Metagenomic and metasecretomic analyses of an enrichment microbial consortium of anaerobic fungi to the degradation of sugarcane bagasse: prospection of cellulosomas and lignocellolytic enzymes
• Grantee: Geizecler Tomazetto
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 20/15595-3 - Functional and structural study of a cellulosomal cellulase from anaerobic fungus and its dockerin-scaffoldin interaction
• Grantee: Wanius José Garcia da Silva
• Support Opportunities: Regular Research Grants
• FAPESP's process: 14/04105-4 - Structural and functional characterization of new cellulases, focusing in the relation between catalytic domains and CBMs
• Grantee: Marcelo Vizoná Liberato
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 20/05784-3 - EMU approved in grant 15 / 50590-4: chromatographic system and detectors for analysis of sugars and lignocellulosic monolignols
• Grantee: Fábio Márcio Squina
• Support Opportunities: Multi-user Equipment Program
• FAPESP's process: 15/14009-5 - Study of the binding interactions of carbohydrate binding modules and carbohydrates
• Grantee: Bruna Medéia de Campos
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• FAPESP's process: 17/17275-3 - Studies of the mode of action of two lytic polysaccharide monooxygenases from insect (order: isoptera): molecular structure, bioinorganic chemistry and biotechnological applications
• Grantee: Wanius José Garcia da Silva
• Support Opportunities: Regular Research Grants