Crystal structures of {beta}-primeverosidase in complex with disaccharide amidine inhibitors [Protein Structure and Folding]
April 21st, 2014 by Saino, H., Shimizu, T., Hiratake, J., Nakatsu, T., Kato, H., Sakata, K., Mizutani, M.
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 hydrolyses 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. Glu470, Ser473, and Gln477 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.