Structural and Biochemical Characterization of Xylella fastidiosa DsbA Family Members: New Insights into the Enzyme−Substrate Interaction

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 among 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. Biochemical assays showed that both X. fastidiosa homologues have similar redox properties and the determination of the crystal structure of XfDsbA revealed substitutions in the active site of X. fastidiosa enzymes, which are proposed to compensate for the lack of the conserved histidine in XfDsbA2. In addition, electron density maps showed a ligand bound to the XfDsbA active site, allowing the characterization of the enzyme interaction with an 8-mer peptide. Finally, surface analysis of XfDsbA and XfDsbA2 suggests that X. fastidiosa enzymes may have different substrate specificities.

Structural and Mechanistic Insights into Unusual Thiol Disulfide Oxidoreductase

“…5C). Thioredoxin and DsbA proteins generally contain the conserved residues Asp 26 and Glu 24 , respectively, which have been described as involved in the activation of the second cysteine (Fig. 5D).…”

“…One hypothesis is that an aspartic acid conserved in all canonical Trx is responsible for this deprotonation (32). It is to be noticed that a double alanine mutation of the conserved Asp 26 and Lys 57 in Trx and Glu 24 and Lys 58 in DsbA did not change the pK a of cysteines in both enzymes. These data support the hypothesis that these residues are not involved in the properties of the dithiol active center (33).…”

Chitosan does not inhibit enzymatic action of human pancreatic lipase in Langmuir monolayers of 1,2-didecanoyl-glycerol (DDG)

“…5B, which are assigned to amide I of HPL. Therefore, even though HPL was injected in the subphase, it went to the interface and took a preferential orientation, with the peak position of the amide I band indicating that the enzyme predominantly adopts ␣-helix and turns structures during its action in hydrolyzing DDG [86,87].…”

Backbone and side chain 1H, 15N and 13C assignments for the oxidised and reduced forms of the oxidoreductase protein DsbA from Staphylococcus aureus

“…The structure of the prototype enzyme in this class, DsbA from E. coli, has been solved and its catalytic mechanisms studied extensively (Guddat et al 1998;Martin et al 1993). The structures of DsbA homologues from other Gram-negative bacteria including Vibrio cholerae (Hu et al 1997, Neisseria meningitidis (Vivian et al 2008), Wolbachia pipientis and Xylella fastidiosa (Rinaldi et al 2009) have also been determined, and DsbA enzymes of Gram-negative bacteria have been implicated in functional aspects including motility and expression of virulence factors (Kadokura et al 2003).…”