Investigation on the requirements for YbbN/CnoX displaying thiol-disulfide oxidoreductase and chaperone activities

YbbN/CnoX are proteins that display a Trx domain linked to a tetratricopeptide (TPR) domain, which are involved in protein-protein interactions and protein folding processes. YbbN from Escherichia coli (EcYbbN) displays a co-chaperone (holdase) activity that is induced by HOCl (bleach). EcYbbN contains a SQHC motif within the Trx domain and displays no thiol-disulfide oxidoreductase activity. EcYbbN also presents a second Cys residue at Trx domain (Cys63) 24 residues away from SQHF motif that can form mixed disulfides with substrates. Here, we compared EcYbbN with two other YbbN proteins: from Xylella fastidiosa (XfYbbN) and from Pseudomonas aeruginosa (PaYbbN). While EcYbbN displays two Cys residues along a SQHC[N24]C motif; XfYbbN and PaYbbN present two and three Cys residues in the CAPC[N24]V and CAPC[N24]C motifs, respectively. These three proteins are representatives of evolutionary conserved YbbN subfamilies. In contrast to EcYbbN, both XfYbbN and PaYbbN: (1) reduced an artificial disulfide (5,5'-dithiobis-(2-nitrobenzoic acid) = DTNB); and (2) supported the peroxidase activity of Peroxiredoxin Q from X. fastidiosa, suggesting that in vivo 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), like the canonical XfTrx (XfTsnC). Furthermore, EcYbbN (as described before) 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 C40A mutant lost the disulfide reductase activity, while kept its HOCl-induced chaperone function. Finally, we generated a P. aeruginosa strain with the ybbN gene deleted, which did not present increased sensitivity to heat shock or to oxidants or to reductants. Altogether, our results suggest that different YbbN/CnoX proteins display distinct properties and activities, depending on the presence of the three conserved Cys residues. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/034579v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@6970f0org.highwire.dtl.DTLVardef@75da52org.highwire.dtl.DTLVardef@15067c9org.highwire.dtl.DTLVardef@1cdd97d_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- CXXC motif is required for the thiol-disulfide reductase activity of YbbN proteins. - XfYbbN and PaYbbN display thiol-disulfide oxidoreductase activity - The affinities of XfTrxR for XfYbbN and XfTsnC (canonical Trx) are comparable - XfYbbN and EcYbbN, but not PaYbbN, display holdase activity induced by hypochlorous acid - Engineering EcYbbN/CnoX by inserting a Cys residue in the SQHC motif resulted in a gain of function (thiol-disulfide oxidoreductase activity) and abolished the HOCl-induced holdase activity.