Functional diversity of YbbN/CnoX proteins: Insights from a comparative analysis of three thioredoxin-like oxidoreductases from Pseudomonas aeruginosa, Xylella fastidiosa and Escherichia coli

• Published in: Redox biology 2024-06, Vol.72, p.103128, Article 103128
• Main Authors: Meireles, Diogo de Abreu, Yokomizo, César Henrique, Silva, Francisnei Pedrosa, Venâncio, Thiago Motta, Degenhardt, Maximilia Frazão de Souza, Oliveira, Cristiano Luis Pinto de, Netto, Luis Eduardo Soares
• Format: Article
• Language: English
• Subjects: Chaperone/holdase activity; Oxidoreductase activity; Tetratricopeptide-repeat domain; Thioredoxin
• ISSN: 2213-2317; 2213-2317
• DOI: 10.1016/j.redox.2024.103128

YbbN/CnoX are proteins that display a Thioredoxin (Trx) domain linked to a tetratricopeptide domain. YbbN from Escherichia coli (EcYbbN) displays a co-chaperone (holdase) activity that is induced by HOCl. Here, we compared EcYbbN with YbbN proteins from Xylella fastidiosa (XfYbbN) and from Pseudomonas aeruginosa (PaYbbN). EcYbbN presents a redox active Cys residue at Trx domain (Cys63), 24 residues away from SQHC motif (SQHC[N24]C) that can form mixed disulfides with target proteins. In contrast, XfYbbN and PaYbbN present two Cys residues in the CXXC (CAPC) motif, while only PaYbbN shows the Cys residue equivalent to Cys63 of EcYbbN. Our phylogenetic analysis revealed that most of the YbbN proteins are in the bacteria domain of life and that their members can be divided into four groups according to the conserved Cys residues. EcYbbN (SQHC[N24]C), XfYbbN (CAPC[N24]V) and PaYbbN (CAPC[N24]C) are representatives of three sub-families. In contrast to EcYbbN, both XfYbbN and PaYbbN: (1) reduced an artificial disulfide (DTNB) and (2) supported the peroxidase activity of Peroxiredoxin Q from X. fastidiosa, suggesting that these proteins might function similarly to the canonical Trx enzymes. Indeed, XfYbbN was reduced by XfTrx reductase with a high catalytic efficiency (kcat/Km = 1.27 x 107 M−1 s−1), similar to the canonical XfTrx (XfTsnC). Furthermore, EcYbbN and XfYbbN, but not PaYbbN displayed HOCl-induced holdase activity. Remarkably, EcYbbN gained disulfide reductase activity while lost the HOCl-activated chaperone function, when the SQHC was replaced by CQHC. In contrast, the XfYbbN CAPA mutant lost the disulfide reductase activity, while kept its HOCl-induced chaperone function. In all cases, the induction of the holdase activity was accompanied by YbbN oligomerization. Finally, we showed that deletion of ybbN gene did not render in P. aeruginosa more sensitive stressful treatments. Therefore, YbbN/CnoX proteins display distinct properties, depending on the presence of the three conserved Cys residues. •CXXC motif is required for the thiol-disulfide reductase activity of YbbN proteins.•The affinities of XfTrxR for XfYbbN and XfTsnC (canonical Trx) are comparable.•XfYbbN and EcYbbN, but not PaYbbN, display holdase activity induced by HOCl.•Engineering EcYbbN/CnoX by inserting a single Cys residue into SQHC motif resulted in a gain of function (thiol-disulfide oxidoreductase activity) and abolished the HOCl-induced holdase activity.•The holdase activity indu