title: Crystal structures of β-primeverosidase in complex with disaccharide amidine inhibitors.
abstract: 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.
authors.affiliation: From the College of Science and Engineering, Aoyama Gakuin University, Sagamihara-shi, Kanagawa 252-5258, saino@chem.aoyama.ac.jp.
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Saino
authors.affiliation: the Faculty of Science, Okayama University, Okayama-shi, Okayama 700-8530.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Shimizu
authors.affiliation: the Institute for Chemical Research, Kyoto University, Uji-shi, Kyoto 611-0011.
authors.forename: Jun
authors.initials: J
authors.lastname: Hiratake
authors.affiliation: the Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, and.
authors.forename: Toru
authors.initials: T
authors.lastname: Nakatsu
authors.affiliation: the Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, and.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Kato
authors.affiliation: the Institute for Chemical Research, Kyoto University, Uji-shi, Kyoto 611-0011.
authors.forename: Kanzo
authors.initials: K
authors.lastname: Sakata
authors.affiliation: the Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Mizutani
mesh_terms: D000595:Amino Acid Sequence; D028241:Camellia sinensis / Q000201:enzymology; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004187:Disaccharides / Q000737:chemistry* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry*; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D004187:Disaccharides; D010940:Plant Proteins; C530637:beta-primeverosylamidine; D006026:Glycoside Hydrolases; C460087:beta-primeverosidase
keywords: Disaccharide; Enzyme Structure; Glycoconjugate; Glycoside Hydrolase; Glycosylamidine; Oligosaccharide; Plant Defense; Tea; Volatile Emission; β-Primeverosidase
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