Crystal Structure and Biochemical Characterization of a New GH42 β-Galactosidase Family Member from a Marine Microbial Metagenome

Many microorganisms produce glycoside hydrolases to degrade environmental polysaccharides, enabling them to utilize these abundant polymers as carbon sources. β-Galactosidases are glycoside hydrolases that catalyze the cleavage of β-galactosidic bonds. Here, we report the identification and characterization of Bgal117, a GH42 β-galactosidase encoded in a marine microbial metagenomic library. Its crystal structure, determined at 3.01 Å resolution, displays the characteristic features of glycoside hydrolase family 42 (GH42). Bgal117 forms a flowerpot-shaped homotrimer in which the active-site pockets, located at the C-terminal ends of the (β/α)8-barrel domains, open toward the central cavity of the trimer, and a putative metal ion is coordinated by four cysteine residues at the periphery of the catalytic domain. Biochemical characterization showed that Bgal117 hydrolyzes oNPG, X-Gal, and lactose and retains activity across broad pH and temperature ranges, with highest activity under moderately acidic, low-salinity conditions. Its catalytic efficiency toward the artificial substrate oNPG is lower than that of many characterized β-galactosidases. These findings extend the structural and functional diversity of GH42 β-galactosidases to a marine metagenomic lineage and provide, to our knowledge, the first crystal structure of a GH42 enzyme from the phylum Verrucomicrobiota.