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Role analysis of Thr44 of Tsa1 from Saccharomyces Cerevisiae in the hydroperoxide catalysis, interaction with Trx, oligomerization and overoxidation processes

Grant number: 13/16192-6
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): September 01, 2013
Effective date (End): December 31, 2014
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Marcos Antonio de Oliveira
Grantee:Melina Cardoso dos Santos
Home Institution: Universidade Estadual Paulista (UNESP). Campus Experimental do Litoral Paulista. São Vicente , SP, Brazil
Associated research grant:13/07937-8 - Redoxome - Redox Processes in Biomedicine, AP.CEPID

Abstract

Typical 2-Cys Peroxiredoxins (Prx) are antioxidant proteins universally distributed among the organisms and present in several cellular compartments. Despite their protective role to the cell, in multicellular eukaryotes it has been shown that these enzymes are related to various types of cancer, since they are able to maintain levels of hydroperoxides in sufficient amounts to allow cell growth, but not to allow apoptosis. Typical 2-Cys Prx are able to cleave a wide variety of hydroperoxides using a cysteine residue called peroxidase cysteine (CP) which, after substrate reduction, is oxidized to sulfenic acid (CP-SOH). The typical Cys Prx-2 are obligatory dimers and have a second cysteine residue involved in the catalytic cycle called resolving cysteine (CR-SH), which forms an intermolecular disulfide with CP of the adjacent monomer during the peroxides decomposition. These enzymes have a high reactivity over hydroperoxide (~ 107 M-1s-1) which is related to the maintenance of CP as thiolate (S-) for a Thr residue (or Ser, in some cases) and an Arg, which are believed to be able of stabilizing CP in the form of S-, reducing its pKa, and having a key role in the positioning and stabilization of the hydroperoxide in the active site, allowing CP to perform a SN2 substitution reaction in the catalyzis. Most Prx uses thioredoxin (Trx) as the reducing substrate. NADPH, in turn, reduces the Trx in a reaction catalyzed by thioredoxin reductase (TrxR). At high concentrations of hydroperoxides, the CP of eukaryotic 2-Cys Prx can be superoxidized to sulfinic acid cysteine (CP-SO2H), resulting in a structural rearrangement of the enzyme and causing a transition functional peroxidase - chaperone, involved in signal transduction by hydroperoxides. In Saccharomyces cerevisiae, the Prx Tsa1 is distinguished by its abundance (~ 380,000 molecules per cell in log phase growth) and, when hyperoxidized, may exhibit molecular chaperone activity. In previous project, we obtained mutants of the protein carrying a replacement of Thr44 residue in the catalytic triad of Tsa1 from S. cerevisiae by amino acids with distinct physicochemical characteristics (Ser, Ala and Val) and the reactivity of the protein over hydroperoxides and interaction with Trx have been evaluated. This project aims to give continuity to the previous project, evaluating the effects of substitutions on the pKa of CP in the process of hyperoxidation and subsequent functional transition peroxidase - chaperone, assessment of the mutations effects on the secondary and quaternary structure through methods involving circular dichroism spectroscopy (CD) size exclusion chromatography (SEC) and dynamic light scattering (DLS). Additionally, screening and refinements will be made to the Tsa1T44A crystallization conditions in order to determine the enzyme crystallographic structure. Overall, we believe that the results generated in this project will contribute very significantly to comprehension of the catalysis mechanisms, interaction with the reducing system and hyperoxidation of 2-Cys Prx typical