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A dithiol glutaredoxin cDNA from sweet potato ( Ipomoea batatas [L.] Lam): enzyme properties and kinetic studies
Author(s) -
Chi X.W.,
Lin C.T.,
Jiang Y.C.,
Wen L.,
Lin C.T.
Publication year - 2012
Publication title -
plant biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.871
H-Index - 87
eISSN - 1438-8677
pISSN - 1435-8603
DOI - 10.1111/j.1438-8677.2011.00544.x
Subject(s) - glutaredoxin , ipomoea , dithiol , biology , complementary dna , biochemistry , enzyme , recombinant dna , escherichia coli , enzyme assay , peptide sequence , open reading frame , microbiology and biotechnology , thioredoxin , gene , botany
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 M m . 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.