Insights into the Specificity of Thioredoxin Reductase−Thioredoxin Interactions. A Structural and Functional Investigation of the Yeast Thioredoxin System

摘要

The enzymatic activity of thioredoxin reductase enzymes is endowed by at least two redox centers: anflavin and a dithiol/disulfide CXXC motif. The interaction between thioredoxin reductase and thioredoxin isngenerally species-specific, but the molecular aspects related to this phenomenon remain elusive. Here, weninvestigated the yeast cytosolic thioredoxin system, which is composed of NADPH, thioredoxin reductasen(ScTrxR1), and thioredoxin 1 (ScTrx1) or thioredoxin 2 (ScTrx2).We showed that ScTrxR1 was able to efficientlynreduce yeast thioredoxins (mitochondrial and cytosolic) but failed to reduce the human and Escherichia colinthioredoxin counterparts. To gain insights into this specificity, the crystallographic structure of oxidized ScTrxR1nwas solved at 2.4 A resolution. The protein topology of the redox centers indicated the necessity of a largenstructural rearrangement for FAD and thioredoxin reduction using NADPH. Therefore, we modeled a largenstructural rotation between the two ScTrxR1 domains (based on the previously described crystal structure,nPDB code 1F6M). Employing diverse approaches including enzymatic assays, site-directed mutagenesis,namino acid sequence alignment, and structure comparisons, insightswere obtained about the features involvednin the species-specificity phenomenon, such as complementary electronic parameters between the surfaces ofnScTrxR1 and yeast thioredoxin enzymes and loops and residues (such as Sern72nin ScTrx2). Finally, structuralncomparisons and amino acid alignments led us to propose a new classification that includes a larger number ofnenzymes with thioredoxin reductase activity, neglected in the low/high molecular weight classification.