Structural studies of a glycoside hydrolase family 3 β-glucosidase from the model fungusNeurospora crassa

Abstract

The glycoside hydrolase family 3 (GH3) β-glucosidases are a structurally diverse family of enzymes. Cel3A fromNeurospora crassa(NcCel3A) belongs to a subfamily of key enzymes that are crucial for industrial biomass degradation. β-Glucosidases hydrolyse the β-1,4 bond at the nonreducing end of cellodextrins. The hydrolysis of cellobiose is of special importance as its accumulation inhibits other cellulases acting on crystalline cellulose. Here, the crystal structure of the biologically relevant dimeric form ofNcCel3A is reported. The structure has been refined to 2.25 Å resolution, with anRcrystandRfreeof 0.18 and 0.22, respectively.NcCel3A is an extensively N-glycosylated glycoprotein that shares 46% sequence identity withHypocrea jecorinaCel3A, the structure of which has recently been published, and 61% sequence identity with the thermophilic β-glucosidase fromRasamsonia emersonii.NcCel3A is a three-domain protein with a number of extended loops that deepen the active-site cleft of the enzyme. These structures characterize this subfamily of GH3 β-glucosidases and account for the high cellobiose specificity of this subfamily.