Introduction of a More Glutaredoxin-like Active Site to PDI Results in Competition between Protein Substrate and Glutathione Binding

Abstract

Proteins in the thioredoxin superfamily share a similar fold, contain a -CXXC- active site, and catalyze oxidoreductase reactions by dithiol-disulfide exchange mechanisms. Protein disulfide isomerase (PDI) has two -CGHC- active sites. For in vitro studies, oxidation/reduction of PDI during the catalytic cycle is accomplished with glutathione. Glutathione may act as electron donor/acceptor for PDI also in vivo, but at least for oxidation reactions, GSSG probably is not the major electron acceptor and PDI may not have evolved to react with glutathione with high affinity, but merely having adequate affinity for both glutathione and folding proteins/peptides. Glutaredoxins, on the other hand, have a high affinity for glutathione. They commonly have -CXFC- or -CXYC- active site, where the tyrosine residue forms part of the GSH binding groove. Mutating the active site of PDI to a more glutaredoxin-like motif increased its reactivity with glutathione. All such variants showed an increased rate in GSH-dependent reduction or GSSG-dependent oxidation of the active site, as well as a decreased rate of the native disulfide bond formation, with the magnitude of the effect increasing with glutathione concentration. This suggests that these variants lead to competition in binding between glutathione and folding protein substrates.

Komagataella phaffii Erp41 is a protein disulfide isomerase with unprecedented disulfide bond catalyzing activity when coupled to glutathione

“…Proteins with a similar domain architecture are also present in different ascomycete yeast and filamentous fungi, however, Erp41 differs from them, as the active site in the a’ domain includes a tyrosine in the motif CXYC. The presence of Y/F residues at this position between the catalytic cysteines is a characteristic trait of Grxs and is known to increase affinity for glutathione ( 12 , 45 ). We are not aware of prior studies on a PDI family member combining classical PDI-like and Grx-like active sites in a single protein.…”

“…The thioredoxin superfamily hosts a range of enzymes that can act as reductases, oxidases, isomerases, GSH S-transferases, peroxidases, or glutaredoxins (Grxs) ( 6 , 10 ). The redox potential, stability of the disulfide-dithiol active site states, and substrate specificity define their main activity ( 11 , 12 , 13 ). For instance, PDIs typically exhibit a -CGHC- motif, in which the histidine stabilizes spatially adjacent thiolate anions, causing instability to the disulfide bond, and thus favor catalysis of dithiol oxidation in substrate proteins ( 14 ).…”

Glutathione and Glutaredoxin—Key Players in Cellular Redox Homeostasis and Signaling

“…Although these two proteins have a similar 3D structure and thiol-disulfide catalytic mechanism, their cellular environments are quite different; thus, Grx catalyzes the reduction of glutathionyl mixed disulfides, whereas PDI catalyzes the formation of intramolecular disulfides. Ruddock and coworkers have been studying these two enzymes, and they contributed an original research article to this compendium [ 80 ] in which they reported the mutation of the PDI CXXC motif, converting histidine to tyrosine or phenylalanine, thereby changing the signature sequence to be more similar to that of class I glutaredoxins. These substitutions for histidine were found to change the binding affinity of PDI for its protein substrates and glutathione.…”

Characterization of a DsbA family protein reveals its crucial role in oxidative stress tolerance of Listeria monocytogenes

“…The TDORs in bacteria include the thioredoxin (Trx) system, glutaredoxin (Grx) system, and disulfide bond formation protein (Dsb) proteins, which are commonly referred to as the thioredoxin superfamily ( 13 , 14 ). The thioredoxin superfamily shares a similar fold and contains a -CXXC- active site ( 15 ). They are involved in scavenging peroxide-active -SH groups and activating transcription of genes related to anti-oxidative stress to protect damaged proteins from inactivation and prevent aggregation of misfolded proteins ( 16 ).…”