title: The mechanism of substrate (aglycone) specificity in beta -glucosidases is revealed by crystal structures of mutant maize beta -glucosidase-DIMBOA, -DIMBOAGlc, and -dhurrin complexes.
abstract: The mechanism and the site of substrate (i.e., aglycone) recognition and specificity were investigated in maize beta-glucosidase (Glu1) by x-ray crystallography by using crystals of a catalytically inactive mutant (Glu1E191D) in complex with the natural substrate 2-O-beta-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOAGlc), the free aglycone DIMBOA, and competitive inhibitor para-hydroxy-S-mandelonitrile beta-glucoside (dhurrin). The structures of these complexes and of the free enzyme were solved at 2.1-, 2.1-, 2.0-, and 2.2-A 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 beta-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 beta-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 beta-glucosidases. The data also provide a plausible explanation for the competitive binding of dhurrin to maize beta-glucosidases with high affinity without being hydrolyzed.
authors: Architecture et Fonction des Macromolecules Biologiques, Centre National de la Recherche Scientifique, 31 Chemin Joseph Aiguier, F13402 Marseille cedex 20, France.
authors: M
authors: M
authors: Czjzek
authors: M
authors: M
authors: Cicek
authors: V
authors: V
authors: Zamboni
authors: D R
authors: DR
authors: Bevan
authors: B
authors: B
authors: Henrissat
authors: A
authors: A
authors: Esen
mesh_terms: D000595:Amino Acid Sequence; D048588:Benzoxazines; D001665:Binding Sites; D005960:Glucosides / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009570:Nitriles / Q000378:metabolism*; D010078:Oxazines / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology / Q000378:metabolism*; D001617:beta-Glucosidase / Q000737:chemistry / Q000378:metabolism*
chemical_list: C418135:2-O-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one; D048588:Benzoxazines; D005960:Glucosides; D009570:Nitriles; D010078:Oxazines; D001617:beta-Glucosidase; C011220:dhurrin; C050733:2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one