Structural and mutational analysis of glycoside hydrolase family 1 Br2 β -glucosidase derived from bovine rumen metagenome.

Ruminant animals rely on the activities of β -glucosidases from residential microbes to convert feed fibers into glucose for further metabolic uses. In this report, we determined the structures of Br2, which is a glycoside hydrolase family 1 β -glucosidase from the bovine rumen metagenome. Br2 folds into a classical ( β / α )8 -TIM barrel domain but displays unique structural features at loop β 5→ α 5 and α -helix 5, resulting in different positive subsites from those of other GH1 enzymes. Br2 exhibited the highest specificity toward laminaritriose, suggesting its involvement in β -glucan hydrolysis in digested feed. We then substituted the residues at subsites +1 and + 2 of Br2 with those of Halothermothrix orenii β -glucosidase. The C170E and C221T mutations provided favorable interactions with glucooligosaccharide substrates at subsite +2, while the A219N mutation probably improved the substrate preference for cellobiose and gentiobiose relative to laminaribiose at subsite +1. The N407Y mutation increased the affinity toward cellooligosaccharides. These results give further insights into the molecular determinants responsible for substrate specificity in GH1 β -glucosidases and may provide a basis for future enzyme engineering applications.