title: Functional and structural differences between isoflavonoid beta-glycosidases from Dalbergia sp.
abstract: Among isoflavonoid beta-glucosidases from Dalbergia species, that from Dalbergia nigrescens hydrolyzes isoflavonoid-7-O-beta-D-apiosyl-1,6-beta-D-glucosides more efficiently, while Dalbergia cochinchinensis beta-glucosidase (dalcochinase) hydrolyzes its rotenoid glycoside substrate, dalcochinin beta-d-glucoside (I), more efficiently. A cDNA encoding a glycosylated beta-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-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside (II) and dalnigrein 7-O-beta-d-apiofuranosyl-(1-->6)-beta-D-glucopyranoside (III) at 400- and 5000-fold higher catalytic efficiency (k(cat)/K(m)) 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 beta-glucosidase, indicating other determinants are important for isoflavonoid diglycoside hydrolysis.
authors.affiliation: Schools of Biochemistry and Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
authors.forename: Phimonphan
authors.initials: P
authors.lastname: Chuankhayan
authors.forename: Thipwarin
authors.initials: T
authors.lastname: Rimlumduan
authors.forename: Waraporn
authors.initials: W
authors.lastname: Tantanuch
authors.forename: Narumol
authors.initials: N
authors.lastname: Mothong
authors.forename: Prachumporn T
authors.initials: PT
authors.lastname: Kongsaeree
authors.forename: Pornphimon
authors.initials: P
authors.lastname: Metheenukul
authors.forename: Jisnuson
authors.initials: J
authors.lastname: Svasti
authors.forename: Ole N
authors.initials: ON
authors.lastname: Jensen
authors.forename: James R Ketudat
authors.initials: JR
authors.lastname: Cairns
mesh_terms: D000595:Amino Acid Sequence; D035782:Dalbergia / Q000201:enzymology*; D004789:Enzyme Activation; D006026:Glycoside Hydrolases / Q000737:chemistry*; D007529:Isoflavones / Q000737:chemistry*; D008969:Molecular Sequence Data; D012639:Seeds / Q000201:enzymology*; D013329:Structure-Activity Relationship
chemical_list: D007529:Isoflavones; C065753:isoflavanone; D006026:Glycoside Hydrolases