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.

The maize β-glucosidase isoenzymes ZMGlu1 and ZMGlu2 hydrolyse the abundant natural substrate DIMBOAGlc (2-O-β-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one), whose aglycone DIMBOA (2,4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) is the major defence chemical protecting seedlings and young plant parts against herbivores and other pests. The two isoenzymes hydrolyse DIMBOAGlc with similar kinetics but differ from each other and their sorghum homologues with respect to specificity towards other substrates. To gain insights into the mechanism of substrate (i.e. aglycone) specificity between the two maize isoenzymes and their sorghum homologues, ZMGlu1 was produced in Escherichia coli, purified, crystallized and its structure solved at 2.5 Å resolution by X-ray crystallography. In addition, the complex of ZMGlu1 with the non-hydrolysable inhibitor p-nitrophenyl β-d-thioglucoside was crystallized and, based on the partial electron density, a model for the inhibitor molecule within the active site is proposed. The inhibitor is located in a slot-like active site where its aromatic aglycone is held by stacking interactions with Trp-378. Whereas some of the atoms on the non-reducing end of the glucose moiety can be modelled on the basis of the electron density, most of the inhibitor atoms are highly disordered. This is attributed to the requirement of the enzyme to accommodate two different species, namely the substrate in its ground state and in its distorted conformation, for catalysis.

The maize β-glucosidase isoenzymes ZMGlu1 and ZMGlu2 hydrolyse the abundant natural substrate DIMBOAGlc (2-O-β-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one), whose aglycone DIMBOA (2,4-hydroxy-7-methoxy-1,4benzoxazin-3-one) is the major defence chemical protecting seedlings and young plant parts against herbivores and other pests. The two isoenzymes hydrolyse DIMBOAGlc with similar kinetics but differ from each other and their sorghum homologues with respect to specificity towards other substrates. To gain insights into the mechanism of substrate (i.e. aglycone) specificity between the two maize isoenzymes and their sorghum homologues, ZMGlu1 was produced in Escherichia coli, purified, crystallized and its structure solved at 2.5 A / resolution by X-ray crystallography. In addition, the complex of ZMGlu1 with the Abbreviations used : ZMGlu, isoenzyme of Zea mays β-glucosidase ; DIMBOAGlc, 2-O-β-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3one ; pNPTGlc, p-nitrophenyl β-D-thioglucoside ; TRCBGlu, Trifolium repens cyanogenic β-glucosidase ; SAMyr, Sinapis alba myrosinase ; BPBGlu, Bacillus polymyxa β-glucosidase ; LLBGlu, Lactococcus lactis phospho-β-galactosidase ; SSBGlu, Sulfolobus solfataricus β-glucosidase ; TABGlu, Thermosphaera aggregans β-glucosidase. 1 To whom correspondence should be addressed (e-mail czjzek!afmb.cnrs-mrs.fr and aevatan!vt.edu). non-hydrolysable inhibitor p-nitrophenyl β--thioglucoside was crystallized and, based on the partial electron density, a model for the inhibitor molecule within the active site is proposed. The inhibitor is located in a slot-like active site where its aromatic aglycone is held by stacking interactions with Trp-378. Whereas some of the atoms on the non-reducing end of the glucose moiety can be modelled on the basis of the electron density, most of the inhibitor atoms are highly disordered. This is attributed to the requirement of the enzyme to accommodate two different species, namely the substrate in its ground state and in its distorted conformation, for catalysis.