| Autor: |
Loberg MA; Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry , The College of Wooster , Wooster , Ohio 44691 , United States., Hurtig JE; Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry , The College of Wooster , Wooster , Ohio 44691 , United States.; Department of Microbiology & Molecular Genetics, McGovern Medical School , The University of Texas Health Science Center at Houston , Houston , Texas 77030 , United States., Graff AH; Department of Biochemistry and Center for Structural Biology , Wake Forest School of Medicine , Winston-Salem , North Carolina 27101 , United States., Allan KM; Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry , The College of Wooster , Wooster , Ohio 44691 , United States., Buchan JA; Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry , The College of Wooster , Wooster , Ohio 44691 , United States., Spencer MK; Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry , The College of Wooster , Wooster , Ohio 44691 , United States., Kelly JE; Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry , The College of Wooster , Wooster , Ohio 44691 , United States., Clodfelter JE; Department of Biochemistry and Center for Structural Biology , Wake Forest School of Medicine , Winston-Salem , North Carolina 27101 , United States., Morano KA; Department of Microbiology & Molecular Genetics, McGovern Medical School , The University of Texas Health Science Center at Houston , Houston , Texas 77030 , United States., Lowther WT; Department of Biochemistry and Center for Structural Biology , Wake Forest School of Medicine , Winston-Salem , North Carolina 27101 , United States., West JD; Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry , The College of Wooster , Wooster , Ohio 44691 , United States. |
| Abstrakt: |
To prevent the accumulation of reactive oxygen species and limit associated damage to biological macromolecules, cells express a variety of oxidant-detoxifying enzymes, including peroxiredoxins. In Saccharomyces cerevisiae, the peroxiredoxin Tsa1 plays a key role in peroxide clearance and maintenance of genome stability. Five homodimers of Tsa1 can assemble into a toroid-shaped decamer, with the active sites in the enzyme being shared between individual dimers in the decamer. Here, we have examined whether two conserved aromatic residues at the decamer-building interface promote Tsa1 oligomerization, enzymatic activity, and biological function. When substituting either or both of these aromatic residues at the decamer-building interface with either alanine or leucine, we found that the Tsa1 decamer is destabilized, favoring dimeric species instead. These proteins exhibit varying abilities to rescue the phenotypes of oxidant sensitivity and genomic instability in yeast lacking Tsa1 and Tsa2, with the individual leucine substitutions at this interface partially complementing the deletion phenotypes. The ability of Tsa1 decamer interface variants to partially rescue peroxidase function in deletion strains is temperature-dependent and correlates with their relative rate of reactivity with hydrogen peroxide and their ability to interact with thioredoxin. Based on the combined results of in vitro and in vivo assays, our findings indicate that multiple steps in the catalytic cycle of Tsa1 may be impaired by introducing substitutions at its decamer-building interface, suggesting a multifaceted biological basis for its assembly into decamers. |
