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

Ruminant animals rely on the activities of fl-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 fl-glucosidase from the bovine rumen metagenome. Br2 folds into a classical (fl/a)8-TIM barrel domain but displays unique structural features at loop fl5 -> a5 and a-helix 5, resulting in different positive subsites from those of other GH1 enzymes. Br2 exhibited the highest specificity toward laminaritriose, suggesting its involvement in fl-glucan hydrolysis in digested feed. We then substituted the residues at subsites +1 and + 2 of Br2 with those of Halothermothrix orenii fl-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 fl-glucosidases and may provide a basis for future enzyme engineering applications.