title: A dithiol glutaredoxin cDNA from sweet potato (Ipomoea batatas [L.] Lam): enzyme properties and kinetic studies.
abstract: Glutaredoxins (Grx) play an important role in reduction of protein glutathione mixed disulphides. An IbGrx cDNA (561 bp, EF362614) encoding a putative dithiol Grx was cloned from sweet potato (Ipomoea batatas [L.] Lam). The deduced amino acid sequence is conserved among the reported dithiol Grx, having a CGYC dithiol motif at the active site. A 3-D structural model was created based on the known crystal structure of a poplar Grx (GrxC1). To characterise the IbGrx protein, the coding region was subcloned into an expression vector and transformed into Escherichia coli. The recombinant His(6) -tagged IbGrx was expressed and purified by metal affinity chromatography. The purified enzyme showed a monomeric band, as demonstrated with 15% SDS-PAGE. The Michaelis constant (K(M) ) for ß-hydroxyethyl disulphide (HED) was 0.50 ± 0.08 Mm. The enzyme retained 60% activity at 80 °C for 16 min. The enzyme was active over a broad pH range from 6.0 to 11.0, and in the presence of imidazole up to 0.4 M. The enzyme was susceptible to protease.
authors.affiliation: Institute of Bioscience and Biotechnology and Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, 2 Pei-Ning Rd, Keelung, Taiwan.
authors.forename: X-W
authors.initials: XW
authors.lastname: Chi
authors.forename: C-T
authors.initials: CT
authors.lastname: Lin
authors.forename: Y-C
authors.initials: YC
authors.lastname: Jiang
authors.forename: L
authors.initials: L
authors.lastname: Wen
authors.forename: C-T
authors.initials: CT
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D054477:Glutaredoxins / Q000737:chemistry* / Q000235:genetics; D027723:Ipomoea batatas / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data
chemical_list: D018076:DNA, Complementary; D054477:Glutaredoxins