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

Abstract

The maize (Zea mays) ␤-glucosidase Zm-p60.1 has been implicated in regulation of plant development by the targeted release of free cytokinins from cytokinin-O-glucosides, their inactive storage forms. The crystal structure of the wild-type enzyme was solved at 2.05-Å resolution, allowing molecular docking analysis to be conducted. This indicated that the enzyme specificity toward substrates with aryl aglycones is determined by aglycone aromatic system stacking with W373, and interactions with edges of F193, F200, and F461 located opposite W373 in a slot-like aglycone-binding site. These aglyconeactive site interactions recently were hypothesized to determine substrate specificity in inactive enzyme substrate complexes of ZM-Glu1, an allozyme of Zm-p60.1. Here, we test this hypothesis by kinetic analysis of F193I/Y/W mutants. The decreased K m of all mutants confirmed the involvement of F193 in determining enzyme affinity toward substrates with an aromatic aglycone. It was unexpected that a 30-fold decrease in k cat was found in F193I mutant compared with the wild type. Kinetic analysis and computer modeling demonstrated that the F193-aglycone-W373 interaction not only contributes to aglycone recognition as hypothesized previously but also codetermines catalytic rate by fixing the glucosidic bond in an orientation favorable for attack by the catalytic pair, E186 and E401. The catalytic pair, assigned initially by their location in the structure, was confirmed by kinetic analysis of E186D/Q and E401D/Q mutants. It was unexpected that the E401D as well as C205S and C211S mutations dramatically impaired the assembly of a catalysis-competent homodimer, suggesting novel links between the active site structure and dimer formation.␤-Glucosidases (␤-glucoside glucohydrolases, EC 3.2.1.21) are a widespread group of enzymes hydrolyzing a broad variety of aryl-and alkyl-␤-dglucosides as well as glucosides with only a carbohydrate moiety. Interest in ␤-glucosidase research reflects essential functions of ␤-glucosidases in a variety of basic biological processes ranging from developmental regulation to chemical defense against pathogen attack, and in a number of industrial applications such as biomass conversion. In plants, ␤-glucosidases have been implicated in regulating various aspects of development, e.g. phytohormone activation (Smith and van Staden, 1978; Brzobohatý et al., 1994), cell wall degradation in the endosperm during germination (Leah et al., 1995), and pathogen defense reactions (Poulton, 1990).Plant ␤-glucosidases are classified as family 1 of retaining glycosyl hydrolases according to their primary structure (Henrissat and Bairoch, 1993;Henrissat et al., 1995). Hydrolysis of a glycosidic bond involves two essential carboxylates, one acting as a general acid/base catalyst and the other as a nucleophile (Sinnott, 1990). The hydrolysis is initiated by the nucleophilic attack at the anomeric carbon (C1) of the substrate that results in the formation of glycosyl enzyme intermediate followed by the release of the...

Molecular and Structural Characterization of Hexameric β-d-Glucosidases in Wheat and Rye

“…However, the k cat of ScGlu for DIBOA-Glc and DIMBOA-Glc was enhanced by this mutation, and the k cat of TaGlu1a-F198A toward DIMBOA-Glc was about 30% that of the wild-type protein. These results are somewhat different from those for the maize glucosidase, where replacement of Phe-198 by smaller amino acids caused a more drastic reduction in k cat (Zouhar et al, 2001;Verdoucq et al, 2003). The crystal structure of the ZmGlu1 mutants revealed that the F198V mutation changes the orientation of the side chain of Phe-466, one of the Phe residues constituting the hydrophobic binding pocket, leading to complete loss of activity against pNP-Glc (Verdoucq et al, 2003).…”

“…This may have been due to interference caused by several glycerol molecules bound in the active site. The binding of a glycerol molecule at the active site was reported for the maize glucosidase Zm-p60.1 (Zouhar et al, 2001). A sulfate ion derived from LiSO 4 in the crystallization buffer was also observed, which was fixed by Ser-366 and Asp-271, and Arg-434 of an adjacent subunit as well.…”

“…The two residues in ZmGlu1 (Arg-295 and Asp-342) that were shown to form the intermonomer salt bridges (Czjzek et al, 2001) are conserved in TaGlu1b (Arg-296 and Asp-343). The intramolecular disulfide bond responsible for maintaining the dimeric form of Zm-p60.1 (Zouhar et al, 2001) is also conserved in the wheat glucosidase. Furthermore, the other amino acids mapping to the monomer-monomer interface of ZmGlu1 show high similarity to the corresponding regions of the wheat and rye glucosidases.…”

“…The aglycone moiety of DIMBOAGlc was shown to be stabilized by four aromatic side chains: Trp-378 on one side and three Phe residues on the other side (Czjzek et al, 2001;Zouhar et al, 2001). Although are conserved among the five monocot b-D-glucosidases (glucosidases from wheat, rye, maize, sorghum, and oats), Phe-205 and Phe-466 are substituted by a His and Ser, respectively, in TaGlu1 and His and Gly, respectively, in ScGlu.…”

“…The primary structure showed about 70% similarity to ZmGlu1 for which the preferred substrate is DIMBOA-Glc. Four hydrophobic amino acids of the maize glucosidase sandwich the aromatic aglycone moiety of the substrate (Czjzek et al, 2000(Czjzek et al, , 2001Zouhar et al, 2001) and are thought to be essential for the recognition of the aglycone moiety. Furthermore, Ala-467 was demonstrated to make contact with the methoxy group of DIMBOAGlc.…”