Enzymatic and structural characterization of hydrolysis of gibberellin A4 glucosyl ester by a rice β-D-glucosidase

In order to identify a rice gibberellin ester beta-n-glucosidase, gibberellin A4 beta-o-glucosyl ester (GA(4)-GE) was synthesized and used to screen rice beta-glucosidases. Os3BGlu6 was found to have the highest hydrolysis activity to GA(4)-GE among five recombinantly expressed rice glycoside hydrolase family GH1 enzymes from different phylogenic clusters. The kinetic parameters of Os3BGlu6 and its mutants E178Q E178A, E394D, E394Q and M251N for hydrolysis of p-nitrophenyl beta-D-glucopyranoside (pNPGlc) and GA(4)-GE confirmed the roles of the catalytic acid/base and nucleophile for hydrolysis of both substrates and suggested M251 contributes to binding hydrophobic aglycones. The activities of the Os3BGlu6 E178Q and E178A acid/base mutants were rescued by azide, which they transglucosylate to produce beta-D-glucopyranosyl azide, in a pH-dependent manner, while acetate also rescued Os3BGlu6 E178A at low pH. High concentrations of sodium azide (200-400 mM) inhibited Os3BGlu6 E178Q but not Os3BGlu6 E178A. The structures of Os3BGlu6 E178Q crystallized with either GA(4)-GE or pNPGlc had a native alpha-D-glucosyl moiety covalently linked to the catalytic nucleophile, E394, which showed the hydrogen bonding to the 2-hydroxyl in the covalent intermediate. These data suggest that a GH1 beta-glucosidase uses the same retaining catalytic mechanism to hydrolyze 1-O-acyl glucose ester and glucoside. (C) 2013 Elsevier Inc. All rights reserved.