Effect of substitution of Cys11 by Ser on the activity and dimerization of one of rice thioredoxin isoforms (OsTrx23) using site-directed mutagenesis

Thioredoxins (Trxs) are low-molecular-mass proteins with two cysteins in their active site WC(G/P)PC which are involved in reversible reduction of disulfide bonds. In contrast to animals and prokaryotes that typically possess one or a few genes encoding Trxs, higher plants contain eight different Trx types: f, m, x, y, z, o, s, and h. The cytoplasmic type Trxs (htype) constitute a particularly large subgroup in higher plans. For instance, in the genome of rice (Oryza sativa), nine genes encoding putative Trx h were identified. Among these nine isoforms OsTrx20, OsTrx1, OsTrx18 and OsTrx23 have additional cys residue in N-terminal in comparison to the other OsTrxh isoforms. In order to study the critical role of this cysteine in OsTrx23, we replaced it with serine using site-directed mutagenesis. Both wild type and mutant proteins were heterologously expressed in Rosetta (DE3)- a strain of Escherichia coli-. The purification of these proteins enabled us to compare their activity in reaction with insulin as substrate. In addition, the dimerization of proteins were analysed under non- reducingand reducing condition with DTT. The results shows that whereas wild type OsTrx23 is present in monomer and dimer forms, the mutant OsTrx23(C11S) is dominantly in monomer form. The activity of mutant was almost similar to wild type. These results suggest that Cys11 at the Nterminal of OsTrx23 has a key role in dimerization of this protein by creating disulfide bonds.