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

Abstract

The mechanism and the site of substrate (i.e., aglycone) recognition and specificity were investigated in maize ␤-glucosidase (Glu1) by x-ray crystallography by using crystals of a catalytically inactive mutant (Glu1E191D) in complex with the natural substrate 2-O-␤-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOAGlc), the free aglycone DIMBOA, and competitive inhibitor para-hydroxy-S-mandelonitrile ␤-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 ␤-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 ␤-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 ␤-glucosidases. The data also provide a plausible explanation for the competitive binding of dhurrin to maize ␤-glucosidases with high affinity without being hydrolyzed.

Insights into the Functional Architecture of the Catalytic Center of a Maize beta -Glucosidase Zm-p60.1

“…Concomitantly with solving the Zm-p60.1 structure, structures of ZM-Glu1, an allozyme of Zmp60.1, and ZM-Glu1 inactive mutant substrate complex have been published by an independent group (Czjzek et al, 2000(Czjzek et al, , 2001. The Zm-p60.1 structure presented here is more appropriate for molecular docking and computer modeling because the ZMGlu1 structure was solved at lower resolution (2.5 Å).…”

“…The aglycone is sandwiched between W378 on one side and F198, F205, and F466 (equivalent to positions W373, F193, F200, and F461 in the sequence of Zm-p60.1) on the other side of the active center. The structure prompted the hypothesis that the specific conformation of these four hydrophobic amino acids and the shape of the aglycone-binding site they form determine aglycone recognition and substrate specificity in ZM-Glu1 (Czjzek et al, 2000).…”

Molecular basis of substrate specificity in family 1 glycoside hydrolases

“…The preponderance of hydrophobic interactions, which are less restrictive as to the orientation of the participants, suggests that the aglycone could have more freedom and diversity of positioning within the ZmGlu1 subsite þ 1 whereas in SbDhr1 the presence of hydrogen bonds, in which energy is strongly dependent on the orientation of the participants, would determine only one aglycone positioning favorable to catalysis (13,35).…”

“…Just two b-glycosidases were already crystallized in complex with a ligand occupying the aglycone binding region (13,35). Despite that, enzyme kinetics parameters (k cat /K m and K i ) for oligocellodextrins have been used to determine the number of subsites and binding energy of glucose residues in the aglycone binding region of several b-glycosidases.…”

“…This interaction may influence the positioning of this basal platform. Apart from that, the residues forming the subsite þ 1 ceiling are placed in a segment of the protein backbone that is stabilized by a disulfide bond between C210 and C216, which is exclusively found in plant b-glycosidases (35).…”