Structural and functional studies of the disulfide oxidorecdutases DsbA from Xylella fastidiosa

Author(s)

Fabio Cupri Rinaldi

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Física Gleb Wataghin
• Defense date: 2008-03-26

Examining board members

Beatriz Gomes Guimarães; Luis Eduardo Soares Netto; Igor Polikarpov; Iris Concepcion Linares de Torriani; Eduardo Granado Monteiro da Silva

• Advisor: José Antonio Brum; Beatriz Gomes Guimarães

Abstract

Disulfide oxidoreductase DsbA catalyzes disulfide-bond formation in proteins secreted to the periplasm and has been related to the folding process of virulence factors in many organisms. It is the most oxidizing of the thioredoxin-like proteins and DsbA redox power is understood in terms of the electrostatic interactions involving the active site motif CPHC. The plant pathogen Xylella fastidiosa has two chromosomal genes encoding two oxidoreductases belonging to the DsbA family and, in one of them, the canonical motif CPHC is replaced by CPAC. Aiming at the structural and functional characterization of X. fastidiosa DsbAs, the crystal structure of XfDsbA was solved at 1.9 Å resolution and the XfDsbA2 homology model was calculated. We also determined the redox potential of both enzymes by means of fluorescence experiments. The crystal structure of the XfDsbA revealed an electron density corresponding to an 8-mer peptide interacting with the hydrophobic groove on the surface of the monomer C next to the active site. This modeled peptide shows at first time in a high-resolution crystal structure the probable mode of interaction between DsbA and a substrate. Furthermore, the results presented in this work surprisingly show that, despite the absence of the active site histidine in XfDsbA2, both proteins have similar redox potentials. In addition, the structure of XfDsbA revealed critical differences in the interactions involving the active site residues. Biochemical assays with XfDsbA mutants were performed in order to investigate the residues which may be responsible for compensate for the lack of the conserved histidine in XfDsbA2. The results presented contribute to the understanding of DsbA molecular mechanism (AU)