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.