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.Amongthe 51 describedGH5subfamilies, 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 from Saccharophagus 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 of SdGluc5-26A adopts a stable trimeric quaternary structure also observable in solution. The N-terminal region of SdGluc5-26A 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 of SdGluc5-26A and of a mutant truncated at the N terminus. Ligand complex structures and kinetic analyses reveal that the N terminus governs the substrate specificity of SdGluc5-26A. 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.