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Novel β-galactosidase activity and first crystal structure of Glycoside Hydrolase family 154
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International audience. Polysaccharide Utilization Loci (PULs) are physically linked gene clusters conserved in the Gram-negative phylum of Bacteroidota and are valuable sources for Carbohydrate Active enZyme (CAZyme) discovery. This study focuses on BD-beta-Gal, an enzyme encoded in a metagenomic PUL and member of the Glycoside Hydrolase family 154 (GH154). BD-beta-Gal showed exo-beta-galactosidase activity with regiopreference for hydrolyzing beta-D-(1,6) glycosidic linkages. Notably, it exhibited a preference for D-glucopyranosyl (D-Glcp) over D-galactopyranosyl (DGalp) and D-fructofuranosyl (D-Fruf) at the reducing end of the investigated disaccharides. In addition, we determined the high resolution crystal structure of BD-beta-Gal, thus providing the first structural characterization of a GH154 enzyme. Surprisingly, this revealed an (alpha/alpha)(6) topology, which has not been observed before for beta-galactosidases. BD-beta-Gal displayed low structural homology with characterized CAZymes, but conservation analysis suggested that the active site was located in a central cavity, with conserved E73, R252, and D253 as putative catalytic residues. Interestingly, BD-beta-Gal has a tetrameric structure and a flexible loop from a neighboring protomer may contribute to its reaction specificity. Finally, we showed that the founding member of GH154, BT3677 from Bacteroides thetaiotaomicron, described as beta-glucuronidase, displayed exo-beta-galactosidase activity like BD-beta-Gal but lacked a tetrameric structure.
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