An intracellular assay for activity screening and characterization of glutathione-dependent oxidoreductases.

Abstract

The thioredoxin fold superfamily is highly diverse and contains many enzymatically active glutathione-dependent thiol-disulfide oxidoreductases, for example glutaredoxins and protein disulfide isomerases. However, many thioredoxin fold proteins remain completely uncharacterized, their cellular function is unknown, and it is unclear if they have a redox-dependent enzymatic activity with glutathione or not. Classically, investigation of enzymatic activity involved time-consuming in vitro characterization of recombinant proteins, limiting the capacity to study novel mechanisms and structure-function relationships. Therefore, to accelerate our investigation of glutathione-dependent oxidoreductases, we have developed a high-throughput and semi-quantitative assay in yeast. We combined overexpression of the glutathione transporter OPT1 with genetic fusion constructs between glutathione-dependent oxidoreductases and redox-sensitive green fluorescent protein 2 (roGFP2) to allow the rapid characterization of enzymatic activity with physiological substrates. We show that the kinetics of roGFP2 oxidation by glutathione disulfide correlate well with the in vitro-determined activity of the genetically fused glutaredoxins or mutants thereof. Our assay thus allows direct screening of glutaredoxin activity and rapid investigation of structure-function relationships. We also demonstrate that our assay can be used to monitor roGFP2 oxidation by S-nitrosoglutathione (GSNO). We show that glutaredoxins efficiently catalyze oxidation of roGFP2 by GSNO in both live yeast cells and in vitro. In summary, we have established a novel assay for activity screening and characterization of glutathione-dependent oxidoreductases.