Functional and structural differences between isoflavonoid β-glycosidases from Dalbergia sp.

Abstract

Among isoflavonoid β-glucosidases from Dalbergia species, that from Dalbergia nigrescens hydrolyzes isoflavonoid-7-O-β-d-apiosyl-1,6-β-d-glucosides more efficiently, while Dalbergia cochinchinensis β-glucosidase (dalcochinase) hydrolyzes its rotenoid glycoside substrate, dalcochinin β-d-glucoside (I), more efficiently. A cDNA encoding a glycosylated β-glucosidase with 81% identity with dalcochinase was cloned from D. nigrescens seeds, and its protein (Dnbglu2) expressed in Pichia pastoris. Purified Dnbglu2 hydrolyzed the D. nigrescens natural substrates dalpatein 7-O-β-d-apiofuranosyl-(1 → 6)-β-d-glucopyranoside (II) and dalnigrein 7-O-β-d-apiofuranosyl-(1 → 6)-β-d-glucopyranoside (III) at 400- and 5000-fold higher catalytic efficiency (kcat/Km) than I. Dalcochinase was mutated at two amino acid residues, A454S and E455G, that are homologous to previously described substrate binding residues and differ from the corresponding residues in Dnbglu2. The double mutant showed 4- and 6.8-fold increases in relative activity toward II and III, respectively. However, this activity was only 3% that of Dnbglu2 β-glucosidase, indicating other determinants are important for isoflavonoid diglycoside hydrolysis. © 2007 Elsevier Inc. All rights reserved.