Expression and biochemical characterization of beta-primeverosidase and application of beta-primeverosylamidine to affinity purification.
Beta-primeverosidase (PD) is a family 1 glycosidase catalyzing the hydrolysis of beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides) to release a disaccharide primeverose. To investigate how PD recognizes the disaccharide moiety of beta-primeverosides, the recombinant PD was expressed by a baculovirus-insect cell system. The recombinant PD was secreted from High Five cells and was properly modified with N-glycosylation and correct cleavage at the N-terminal signal peptide. The recombinant PD exhibited high substrate specificity to beta-primeverosides in terms of the glycone moiety, consistently with the substrate specificity of native PD from Camellia sinensis. Next, beta-glycosylamidines were synthesized as substrate analog inhibitors. Beta-primeverosylamidine strongly inhibited PD activity, but beta-glucosylamidine did not. Hence beta-primeverosylamidine is an ideal chemical tool for probing disaccharide recognition in the active site of PD. An affinity adsorbent for PD was prepared using beta-primeverosylamidine as a ligand. Affinity chromatography gave large amounts of PD with high purity, permitting crystallographic study.
Saino
Hiromichi
H
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Mizutani
Masaharu
M
Hiratake
Jun
J
Sakata
Kanzo
K
D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D017931:DNA Primers; D018076:DNA, Complementary; D004187:Disaccharides / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D007313:Insecta; D007700:Kinetics; D012323:RNA Processing, Post-Transcriptional; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D004187:Disaccharides; D011994:Recombinant Proteins; C530637:beta-primeverosylamidine; D006026:Glycoside Hydrolases; C460087:beta-primeverosidase