Replacement of threonine-55 with glycine decreases the reduction rate of OsTrx20 by glutathione

Thioredoxins (Trxs) are small ubiquitous oxidoreductase proteins with two redox-active Cys residues in a conserved active site (WCG/PPC) that regulate numerous target proteins via thiol/disulfide exchanges in the cells of prokaryotes and eukaryotes. The isoforms OsTrx23 with a typical active site (WCGPC) and OsTrx20 with an atypical active site (WCTPC) are two Trx h- type isoforms in rice that were previously found to be reduced by NADPH-dependent thioredoxin reductase and GSH/Grx system, respectively. In the present work the reduction of mutants G41T(OsTrx23), T55G(OsTrx20), K48D(OsTrx20) and T55G-K48D (OsTrx20) as well as wild types OsTrx23 and OsTrx20 were tested in the reaction containing either NADPH/NTR or glutathione (GSH). The results revealed that reduction rate of T55G(OsTrx20) was remarkably decreased by GSH as compared to WtOsTrx20 highlighting the critical role of Thr-55 in interaction of OsTrx20 with GSH. On the other hand a significant decrease in the reduction rate of G41T(OsTrx23) was observed in reaction containing NADPH-dependent thioredoxin reductase as compared with readuction rate of WtOsTrx23. These results suggest that first residue after N-terminal active site Cys is one of the critical residue in determination of system that Trxs can be reduced in.