Structural basis for broad substrate specificity in higher plant beta-D-glucan glucohydrolases

Abstract

Family 3 β-D-glucan glucohydrolases are distributed widely in higher plants. The enzymes catalyze the hydrolytic removal of β-D-glucosyl residues from nonreducing termini of a range of β-D-glucans and β-D-oligoglucosides. Their broad specificity can be explained by x-ray crystallographic data obtained from a barley β-D-glucan glucohydrolase in complex with nonhydrolyzable S-glycoside substrate analogs and by molecular modeling of enzyme/substrate complexes. The glucosyl residue that occupies binding subsite -1 is locked tightly into a fixed position through extensive hydrogen bonding with six amino acid residues near the bottom of an active site pocket. In contrast, the glucosyl residue at subsite +1 is located between two Trp residues at the entrance of the pocket, where it is constrained less tightly. The relative flexibility of binding at subsite +1, coupled with the projection of the remainder of bound substrate away from the enzyme's surface, means that the overall active site can accommodate a range of substrates with variable spatial dispositions of adjacent β-D-glucosyl residues. The broad specificity for glycosidic linkage type enables the enzyme to perform diverse functions during plant development.