The mechanism of substrate (aglycone) specificity in beta-glucosidases is revealed by crystal structures of mutant maize beta-glucosidase-DIMBOA, -DIMBOAGlc, and -dhurrin complexes

The mechanism and the site of substrate (i.e., aglycone) recognition and sp ecificity were investigated in maize beta -glucosidase (Glu1) by x-ray crys tallography by using crystals of a catalytically inactive mutant (Glu1E191D ) in complex with the natural substrate 2-O-beta -D-glucopyranosyl-4-hydrox y-7-methoxy-1,4-benzoxazin-3-one (DIMBOAGlc), the free aglycone DIMBOA, and competitive inhibitor para-hydroxy-S-mandelonitrile beta -glucoside (dhurr in). The structures of these complexes and of the free enzyme were solved a t 2.1-, 2.1-, 2.0-, and 2.2-Angstrom resolution. respectively. The structur al data from the complexes allowed us to visualize an intact substrate, fre e aglycone, or a competitive inhibitor in the slot-like active site of a be ta -glucosidase. These data show that the aglycone moiety of the substrate is sandwiched between W378 on one side and F198, F205, and F466 on the othe r. Thus, specific conformations of these four hydrophobic amino acids and t he shape of the aglycone-binding site they form determine aglycone recognit ion and substrate specificity in Glu1. In addition to these four residues. A467 interacts with the 7-methoxy group of DIMBOA. All residues but W378 ar e variable among beta -glucosidases that differ in substrate specificity. s upporting the conclusion that these sites are the basis of aglycone recogni tion and binding (i.e., substrate specificity) in -glucosidases. The data a lso provide a plausible explanation for the competitive binding of dhurrin to maize beta -glucosidases with high affinity without being hydrolyzed.