Site-directed mutagenesis of rat thioltransferase: effects of essential cysteine residues for the protection against oxidative stress.
A cDNA of rat liver thioltransferase was cloned and then expressed using pMAL-c expression vector in Escherichia coli. Recombinant rat liver thioltransferase was expressed as a fusion protein with maltose-binding protein and then purified by amylose resin column chromatography to be homogeneity on 12.5% SDS-polyacrylamide gel electrophoretic analysis. The expressed proteins were shown as two bands at around 53 and 41 kDa, suggesting that the high molecular one was a fusion protein of recombinant thioltransferase (11.7 plus 41 kDa) and the other (smaller one) was a maltose-binding protein (41 kDa). A recombinant thioltransferase catalyzed a thiol/disulfide exchange reaction in the same way as thioltransferases purified from various sources. Compared with wild type, the mutants C23A, C26A, C79A, and C83A showed 0%, 17%, 82%, and 86% in the enzymatic activity, respectively. In addition, wild-type-transfected bacteria expressed in bacterial cells showed a strong resistance to H(2)O(2) treatment as well as the case of active mutants (C79A and C83A), but inactive mutants (C23A and C26A) showed no resistance to H(2)O(2) treatment as same as mocktransfection. Thioltransferase can be important for survival of bacterial cells under oxidative stress.
Terada
Tomoyuki
T
Laboratory of Biochemistry, Faculty of Pharmacy, Osaka Ohtani University, Tondabayashi, Osaka 584-8540, Japan. teradat@osaka-ohtani.ac.jp
Okamoto
Kei-ichi
K
Nishikawa
Jun-ichi
J
Miura
Takeshi
T
Nishinaka
Toru
T
Nishihara
Tsutomu
T
D000595:Amino Acid Sequence; D000818:Animals; D002470:Cell Survival / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D015723:Gene Library; D054477:Glutaredoxins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000633:toxicity; D008099:Liver; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018384:Oxidative Stress / Q000235:genetics*; D016133:Polymerase Chain Reaction; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D014169:Transformation, Bacterial
D054477:Glutaredoxins; D011993:Recombinant Fusion Proteins; D006861:Hydrogen Peroxide; D003545:Cysteine