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J Biol Chem. 2014 Jun 13;289(24):16826-34. doi: 10.1074/jbc.M114.553271. Epub 2014 Apr 21.

Crystal structures of β-primeverosidase in complex with disaccharide amidine inhibitors.

Author information

  • 1From the College of Science and Engineering, Aoyama Gakuin University, Sagamihara-shi, Kanagawa 252-5258,
  • 2the Faculty of Science, Okayama University, Okayama-shi, Okayama 700-8530.
  • 3the Institute for Chemical Research, Kyoto University, Uji-shi, Kyoto 611-0011.
  • 4the Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, and.
  • 5the Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan.


β-Primeverosidase (PD) is a disaccharide-specific β-glycosidase in tea leaves. This enzyme is involved in aroma formation during the manufacturing process of oolong tea and black tea. PD hydrolyzes β-primeveroside (6-O-β-d-xylopyranosyl-β-d-glucopyranoside) at the β-glycosidic bond of primeverose to aglycone, and releases aromatic alcoholic volatiles of aglycones. PD only accepts primeverose as the glycone substrate, but broadly accepts various aglycones, including 2-phenylethanol, benzyl alcohol, linalool, and geraniol. We determined the crystal structure of PD complexes using highly specific disaccharide amidine inhibitors, N-β-primeverosylamidines, and revealed the architecture of the active site responsible for substrate specificity. We identified three subsites in the active site: subsite -2 specific for 6-O-β-d-xylopyranosyl, subsite -1 well conserved among β-glucosidases and specific for β-d-glucopyranosyl, and wide subsite +1 for hydrophobic aglycone. Glu-470, Ser-473, and Gln-477 act as the specific hydrogen bond donors for 6-O-β-d-xylopyranosyl in subsite -2. On the other hand, subsite +1 was a large hydrophobic cavity that accommodates various aromatic aglycones. Compared with aglycone-specific β-glucosidases of the glycoside hydrolase family 1, PD lacks the Trp crucial for aglycone recognition, and the resultant large cavity accepts aglycone and 6-O-β-d-xylopyranosyl together. PD recognizes the β-primeverosides in subsites -1 and -2 by hydrogen bonds, whereas the large subsite +1 loosely accommodates various aglycones. The glycone-specific activity of PD for broad aglycone substrates results in selective and multiple release of temporally stored alcoholic volatile aglycones of β-primeveroside.

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.


Disaccharide; Enzyme Structure; Glycoconjugate; Glycoside Hydrolase; Glycosylamidine; Oligosaccharide; Plant Defense; Tea; Volatile Emission; β-Primeverosidase

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