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

ABSTRACT: ?-Primeverosidase (PD) is a disaccharide-specific ?-glycosidase in tea leaves. This enzyme is involved in aroma formation during the manufacturing process of oolong tea and black tea. PD hydrolyzes ?-primeveroside (6-O-?-d-xylopyranosyl-?-d-glucopyranoside) at the ?-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-?-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-?-d-xylopyranosyl, subsite -1 well conserved among ?-glucosidases and specific for ?-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-?-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 ?-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-?-d-xylopyranosyl together. PD recognizes the ?-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 ?-primeveroside.

SUBMITTER: Saino H 

PROVIDER: S-EPMC4059125 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature