Site-directed mutagenesis reveals that N-terminal cysteines are involved in dimerization of OsTrx20 and OsTrx23

Abstract

Thioredoxins (Trxs) are small proteins containing two redox-active half-Cys residues with the sequence -Cys-X-X-Cys- that function as protein disulfide reductases in a variety of biological reactions. The cytoplasmic type Trxs (h-type) constitutes a particularly large sub-group in higher plants. OsTrx20 and OsTrx23 are two Trx h isoforms in rice which were heterologously expressed in E. coli previously and found to be partially dimer under non-reducing conditions. In the present study, we substituted two vicinal Cys (Cys3 and Cys4) in N-terminal of OsTrx20 as well as the single Cys (Cys11) in N-terminal of OsTrx23 with Ser and provided the recombinant forms of mutants C3SOsTrx20, C4SOsTrx20, C3S-C4SOsTrx20 and C11SOsTrx23. The mutants C11SOsTrx23 and C3S-C4SOsTrx20 appeared exclusively in monomeric form under non-reducing conditions, whereas the mutants C3SOsTrx20 and C4SOsTrx20 were appeared in both monomeric and dimeric forms. These results suggest that Cys11 in OsTrx23 and both Cys3 and Cys 4 in OsTrx20 are involved in dimer formation. DTT-dependent insulin assay shows that the N-terminal Cys residues in OsTrx23 and OsTrx20 are not essential for protein activity. By contrast, the replacement of Cys57 with Ser which is located in active site of OsTrx20 shows that Cys 57 is not involved in dimerization, but has a critical role in reduction activity. © 2014, Springer-Verlag Wien.