Heterodimer formation between thioredoxin f and fructose 1,6‐bisphosphatase from spinach chloroplasts

Abstract

Chloroplast fructose 1,6-bisphosphatase (FBPase) is activated by reduction of a regulatory disulfide through thioredoxin f (Trx f). In the course of this reduction a transient mixed disulfide is formed linking covalently Trx f with FBPase, which possesses three Cys on a loop structure, two of them forming the redox-active disulfide bridge. The goal of this study was to identify the Cys involved in the transient mixed disulfide. To stabilize this reaction intermediate, mutant proteins with modified active sites were used. We identified Cys-155 of the FBPase as the one engaged in the formation of the mixed disulfide intermediate with Cys-46 of Trx f. ß

The Plastidic 2-Cysteine Peroxiredoxin Is a Target for a Thioredoxin Involved in the Protection of the Photosynthetic Apparatus against Oxidative Damage

“…Because the thioredoxin reduction catalytic mechanism proceeds via the formation of a transient covalent complex with the substrate, through a disulfide bridge between the first Cys of the thioredoxin active site and the substrate Cys (Yang and Wells, 1991), we used a mutation strategy to modify the CDSP32 active site. Replacing the second Cys of the active site with a Ser (a nonreactive structural analog) results in stabilization of the complex (Wynn et al, 1995;Goyer et al, 1999;Balmer and Schürmann, 2001). Using a CDSP32 active-site mutant, we show the formation of a CDSP32/ BAS1 mixed disulfide complex in vitro.…”

Plant Thioredoxin CDSP32 Regenerates 1-Cys Methionine Sulfoxide Reductase B Activity through the Direct Reduction of Sulfenic Acid

“…For this purpose, MSRB1, either reduced or substrate-oxidized, was incubated with reduced C222S CDSP32 in ratios of 1:1 and 1:4. A mutated form of the Trx lacking the resolving Cys was used to stabilize the complex formed through an intermolecular disulfide bridge as shown for many targets of Trxs (24,39). Then proteins were analyzed using nonreducing SDS-PAGE and Coomassie Blue staining (Fig.…”

Three thioredoxin targets in the inner envelope membrane of chloroplasts function in protein import and chlorophyll metabolism

“…PTC52 and PAO of barley produced by hybrid-selected translation were incubated with Pchlide a and pheophorbide a. As controls, in vitro-translated and purified PTC52-(His) 6 and PAO-(His) 6 of Arabidopsis thaliana were used. After incubation, the assays were subjected to a step of gel filtration to remove unbound pigments.…”