Abstract
The mechanism and the site of substrate (i.e., aglycone)
recognition and specificity were investigated in maize β-glucosidase
(Glu1) by x-ray crystallography by using crystals of a catalytically
inactive mutant (Glu1E191D) in complex with the natural substrate
2-
O
-β-
d
-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one
(DIMBOAGlc), the free aglycone DIMBOA, and competitive inhibitor
para
-hydroxy-
S
-mandelonitrile
β-glucoside (dhurrin). The structures of these complexes and of the
free enzyme were solved at 2.1-, 2.1-, 2.0-, and 2.2-Å resolution,
respectively. The structural data from the complexes allowed us to
visualize an intact substrate, free aglycone, or a competitive
inhibitor in the slot-like active site of a β-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 other. Thus, specific
conformations of these four hydrophobic amino acids and the shape of
the aglycone-binding site they form determine aglycone recognition and
substrate specificity in Glu1. In addition to these four residues, A467
interacts with the 7-methoxy group of DIMBOA. All residues but W378 are
variable among β-glucosidases that differ in substrate specificity,
supporting the conclusion that these sites are the basis of aglycone
recognition and binding (i.e., substrate specificity) in
β-glucosidases. The data also provide a plausible explanation for the
competitive binding of dhurrin to maize β-glucosidases with high
affinity without being hydrolyzed.