Kaenying W.Tagami T.Suwan E.Pitsanuwong C.Chomngam S.Okuyama M.Kongsaeree P.Kimura A.Kongsaeree P.T.Mahidol University2023-11-242023-11-242023-11-01Heliyon Vol.9 No.11 (2023)24058440https://repository.li.mahidol.ac.th/handle/123456789/91159Ruminant 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 β5→α5 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.MultidisciplinaryArticleSCOPUS10.1016/j.heliyon.2023.e219232-s2.0-85176773252