Broader than expected tolerance for substitutions in the WCGPCK catalytic motif of yeast thioredoxin 2.

Shayna L Vicker, Eran N Maina, Abigail K Showalter, Nghi Tran, Emma E Davidson, Morgan R Bailey, Stephen W McGarry, Wilson M Freije, James D West

Free radical biology & medicine 2022 Jan

filter terms:

Thioredoxins constitute a key class of oxidant defense enzymes that facilitate disulfide bond reduction in oxidized substrate proteins. While thioredoxin's WCGPCK active site motif is highly conserved in traditional model organisms, predicted thioredoxins from newly sequenced genomes show variability in this motif, making ascertaining which genes encode functional thioredoxins with robust activity a challenge. To address this problem, we generated a semi-saturation mutagenesis library of approximately 70 thioredoxin variants harboring mutations adjacent to their catalytic cysteines, making substitutions in the Saccharomyces cerevisiae thioredoxin Trx2. Using this library, we determined how such substitutions impact oxidant defense in yeast along with how they influence disulfide reduction and interaction with binding partners in vivo. The majority of thioredoxin variants screened rescued the slow growth phenotype that accompanies deletion of the yeast cytosolic thioredoxins; however, the ability of these mutant proteins to protect against H2O2-mediated toxicity, facilitate disulfide reduction, and interact with redox partners varied widely, depending on the site being mutated and the substitution made. We report that thioredoxin is less tolerant of substitutions at its conserved tryptophan and proline in the active site motif, while it is more amenable to substitutions at the conserved glycine and lysine. Our work highlights a noteworthy plasticity within the active site of this critical oxidant defense enzyme. Copyright © 2021 Elsevier Inc. All rights reserved.

Citation

Shayna L Vicker, Eran N Maina, Abigail K Showalter, Nghi Tran, Emma E Davidson, Morgan R Bailey, Stephen W McGarry, Wilson M Freije, James D West. Broader than expected tolerance for substitutions in the WCGPCK catalytic motif of yeast thioredoxin 2. Free radical biology & medicine. 2022 Jan;178:308-313