Abstract
β-Primeverosidase (PD) is a family 1 glycosidase catalyzing the hydrolysis of β-primeverosides (6-O-β-D-xylopyranosyl-β-D-glucopyranosides) to release a disaccharide primeverose. To investigate how PD recognizes the disaccharide moiety of β-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 β-primeverosides in terms of the glycone moiety, consistently with the substrate specificity of native PD from Camellia sinensis. Next, β-glycosylamidines were synthesized as substrate analog inhibitors. β-Primeverosylamidine strongly inhibited PD activity, but β-glucosylamidine did not. Hence β-primeverosylamidine is an ideal chemical tool for probing disaccharide recognition in the active site of PD. An affinity adsorbent for PD was prepared using β-primeverosylamidine as a ligand. Affinity chromatography gave large amounts of PD with high purity, permitting crystallographic study.
