Functional and structural characterization of the mitochondrial thioredoxin system from Saccharomyces cerevisiae

Author(s)

Eduardo Hiroshi Nakamatsu

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB)
• Defense date: 2012-09-14

Examining board members

Luis Eduardo Soares Netto; Luciana Amaral Haddad; Oswaldo Keith Okamoto; Marcos Antonio de Oliveira

• Advisor: Luis Eduardo Soares Netto; Gisele Monteiro de Souza

Abstract

NADPH, thioredoxin and thioredoxin reductase, comprising the thioredoxin system, are involved in the reduction of specific disulfides linkages that play a large number of biological roles, such as: DNA synthesis, defense against oxidative stress, apoptosis and redox signaling. It has been shown that thioredoxin reductase-thioredoxin interactions are species-specific, therefore we have investigated here the substrate specificity of mitochondrial Thioredoxin reductase 2 (ScTrxR2) from Saccharomyces cerevisiae towards other thioredoxins. ScTrxR2 specifically reduced yeast thioredoxins (thioredoxin 1 = ScTrx1, thioredoxin 2 = ScTrx2 and thioredoxin = ScTrx3), but failed to reduce thioredoxin from Homo sapiens and from Escherichia coli. Furthermore, ScTrxR2 displayed similar catalytic efficiency towards ScTrx3, which is located in the mitochondria and ScTrx1 and ScTrx2 that are located in the cytosol. To understand the features of this phenomenon, we have solved the crystallographic structure of ScTrxR2 at 1,9Å resolution through molecular replacement using ScTrxR1 as search model (Oliveira et al., 2010)1. ScTrxR2 is a two-domain protein (NADPH and FAD binding domains). Low molecular weight thioredoxin reductases can adopt two conformations: flavin oxidized (FO) or flavin reduced (FR), the late one physically interacts with thioredoxins. Our ScTrxR2 crystal structure is in the FO conformation. Therefore, we have modeled the ScTrxR2 FR (Flavin reduced) conformation from our FO crystal structure and using the E. coli thioredoxin reductase crystallographic structure complexed with thioredoxin (PDB code 1F6M). Then, we have raised hypothesis that some amino acid residues that may be involved in the thioredoxin reductase-thioredoxin interactions. Next, site-directed mutants of yeast Trxs and ScTrx2 were generated. Through enzymatic and biochemical assays with these mutant proteins we are testing the hypothesis generated by structural analysis (AU)

• FAPESP's process: 07/06747-0 - Functional and structural characterization of mitochondrial thioredoxin system from Saccharomyces cerevisiae.
• Grantee: Eduardo Hiroshi Nakamatsu
• Support Opportunities: Scholarships in Brazil - Doctorate