Structural and enzymatic characterization of Os3BGlu6, a rice beta-glucosidase hydrolyzing hydrophobic glycosides and (1->3)- and (1->2)-linked disaccharides

Plant Physiol. 2009 Sep;151(1):47-58. doi: 10.1104/pp.109.139436. Epub 2009 Jul 8.

Abstract

Glycoside hydrolase family 1 (GH1) beta-glucosidases play roles in many processes in plants, such as chemical defense, alkaloid metabolism, hydrolysis of cell wall-derived oligosaccharides, phytohormone regulation, and lignification. However, the functions of most of the 34 GH1 gene products in rice (Oryza sativa) are unknown. Os3BGlu6, a rice beta-glucosidase representing a previously uncharacterized phylogenetic cluster of GH1, was produced in recombinant Escherichia coli. Os3BGlu6 hydrolyzed p-nitrophenyl (pNP)-beta-d-fucoside (k(cat)/K(m) = 67 mm(-1) s(-1)), pNP-beta-d-glucoside (k(cat)/K(m) = 6.2 mm(-1) s(-1)), and pNP-beta-d-galactoside (k(cat)/K(m) = 1.6 mm(-1)s(-1)) efficiently but had little activity toward other pNP glycosides. It also had high activity toward n-octyl-beta-d-glucoside and beta-(1-->3)- and beta-(1-->2)-linked disaccharides and was able to hydrolyze apigenin beta-glucoside and several other natural glycosides. Crystal structures of Os3BGlu6 and its complexes with a covalent intermediate, 2-deoxy-2-fluoroglucoside, and a nonhydrolyzable substrate analog, n-octyl-beta-d-thioglucopyranoside, were solved at 1.83, 1.81, and 1.80 A resolution, respectively. The position of the covalently trapped 2-F-glucosyl residue in the enzyme was similar to that in a 2-F-glucosyl intermediate complex of Os3BGlu7 (rice BGlu1). The side chain of methionine-251 in the mouth of the active site appeared to block the binding of extended beta-(1-->4)-linked oligosaccharides and interact with the hydrophobic aglycone of n-octyl-beta-d-thioglucopyranoside. This correlates with the preference of Os3BGlu6 for short oligosaccharides and hydrophobic glycosides.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Catalytic Domain
  • Cloning, Molecular
  • Disaccharides / metabolism*
  • Gene Expression Regulation, Plant / physiology
  • Glycosides / metabolism*
  • Models, Molecular
  • Oryza / genetics*
  • Oryza / metabolism*
  • Plant Proteins / chemistry
  • Plant Proteins / genetics
  • Plant Proteins / metabolism*
  • Protein Binding
  • Protein Conformation
  • Substrate Specificity
  • beta-Glucosidase / genetics
  • beta-Glucosidase / metabolism*

Substances

  • Disaccharides
  • Glycosides
  • Plant Proteins
  • beta-Glucosidase