Seeding subfamilies in glycoside hydrolase family 116: New insights into the function and inhibitor sensitivity of subfamily 2.

Among glycoside hydrolase (GH) families and characterized enzymes reported in CAZy, GHs from Archaea remain largely underrepresented. Recent 'omics' studies have uncovered numerous uncharacterized GH sequences from extremophilic Archaea, providing unprecedented opportunities to study their diversity, structure, function, and properties. The GH family 116 (GH116) was originally built around a characterized archaeal member, revealing homologous sequences in Eukaryota and Bacteria. Although previous studies proposed or attempted GH116 subdivision into distinct subfamilies associated with different substrate specificity and inhibitor sensitivity, such a classification has never been formally established, mainly because few GH116 enzymes have been biochemically characterized. As a result, its functional landscape remains poorly understood. Here, we formally establish GH116 subfamilies, starting from the discovery of nine novel archaeal GH116 sequences identified from metagenomics samples collected from two mud/water pools in the extreme environment of Pisciarelli solfatara. These sequences were assigned to subfamilies 2 and 3, mainly hyperthermophilic archaeal enzymes. By combining phylogenetic analyses, structural modeling, and biochemical studies, we identified subfamily-specific structural domains that may serve as structural markers and suggest that these subfamilies represent functionally specialized groups. Focusing on subfamily 2, previously with only one characterized member, we biochemically characterized two novel enzymes. Our results expand GH116 functional diversity, reporting for the first time β-galactosidase activity within this family and showing β-xylosidase activity in subfamily 2, previously observed only in subfamily 3. We also examined active-site inhibition profiles and, through active-site mapping and mutational analysis, elucidated the molecular basis of inhibitor sensitivity in one enzyme of this subgroup.