title: The Quaternary Structure of a Glycoside Hydrolase Dictates Specificity toward β-Glucans.
abstract: In the Carbohydrate-Active Enzyme (CAZy) database, glycoside hydrolase family 5 (GH5) is a large family with more than 6,000 sequences. Among the 51 described GH5 subfamilies, subfamily GH5_26 contains members that display either endo-β(1,4)-glucanase or β(1,3;1,4)-glucanase activities. In this study, we focused on the GH5_26 enzyme fromSaccharophagus degradans(SdGluc5_26A), a marine bacterium known for its capacity to degrade a wide diversity of complex polysaccharides.SdGluc5_26A displays lichenase activity toward β(1,3;1,4)-glucans with a side cellobiohydrolase activity toward β(1,4)-glucans. The three-dimensional structure ofSdGluc5_26A adopts a stable trimeric quaternary structure also observable in solution. The N-terminal region ofSdGluc5_26A protrudes into the active site of an adjacent monomer. To understand whether this occupation of the active site could influence its activity, we conducted a comprehensive enzymatic characterization ofSdGluc5_26A and of a mutant truncated at the N terminus. Ligand complex structures and kinetic analyses reveal that the N terminus governs the substrate specificity ofSdGluc5_26A. Its deletion opens the enzyme cleft at the -3 subsite and turns the enzyme into an endo-β(1,4)-glucanase. This study demonstrates that experimental approaches can reveal structure-function relationships out of reach of current bioinformatic predictions.
authors.affiliation: From the Institut des Sciences Moléculaires de Marseille-BiosCiences, UMR7313 CNRS, Aix-Marseille University, Pôle de l'Etoile, 13284 Marseille, France, the INRA, UMR1163, Biodiversité et Biotechnologie Fongiques, Aix-Marseille University, Polytech'Marseille, F-13288 Marseille, France.
authors.forename: Mickael
authors.initials: M
authors.lastname: Lafond
authors.affiliation: the Architecture et Fonction des Macromolécules Biologiques, UMR7257 CNRS, Aix-Marseille University, F-13288 Marseille, France, the INRA, USC1408 Architecture et Fonction des Macromolécules Biologiques, F-13288 Marseille, France, and.
authors.forename: Gerlind
authors.initials: G
authors.lastname: Sulzenbacher
authors.affiliation: the Architecture et Fonction des Macromolécules Biologiques, UMR7257 CNRS, Aix-Marseille University, F-13288 Marseille, France.
authors.forename: Thibaud
authors.initials: T
authors.lastname: Freyd
authors.affiliation: the Architecture et Fonction des Macromolécules Biologiques, UMR7257 CNRS, Aix-Marseille University, F-13288 Marseille, France, the INRA, USC1408 Architecture et Fonction des Macromolécules Biologiques, F-13288 Marseille, France, and the Department of Biological Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
authors.forename: Bernard
authors.initials: B
authors.lastname: Henrissat
authors.affiliation: the INRA, UMR1163, Biodiversité et Biotechnologie Fongiques, Aix-Marseille University, Polytech'Marseille, F-13288 Marseille, France, jean-guy.berrin@univ-amu.fr.
authors.forename: Jean-Guy
authors.initials: JG
authors.lastname: Berrin
authors.affiliation: the Architecture et Fonction des Macromolécules Biologiques, UMR7257 CNRS, Aix-Marseille University, F-13288 Marseille, France, the INRA, USC1408 Architecture et Fonction des Macromolécules Biologiques, F-13288 Marseille, France, and.
authors.forename: Marie-Line
authors.initials: ML
authors.lastname: Garron
mesh_terms: D000595:Amino Acid Sequence; D059001:Aquatic Organisms; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020563:Gammaproteobacteria / Q000737:chemistry* / Q000201:enzymology; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D047071:beta-Glucans / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D047071:beta-Glucans; D006026:Glycoside Hydrolases; C028020:licheninase; D043366:Cellulose 1,4-beta-Cellobiosidase
keywords: CAZyme; Saccharophagus degradans; crystallography; endoglucanase; enzyme; glycobiology; glycoside hydrolase; lichenase; oligomerization