Ohr (Organic Hydroperoxide Resistance Protein) Possesses a Previously Undescribed Activity, Lipoyl-dependent Peroxidase

Abstract

The Ohr (organic hydroperoxide resistance) family of 15-kDa Cys-based, thiol-dependent peroxidases is central to the bacterial response to stress induced by organic hydroperoxides but not by hydrogen peroxide. Ohr has a unique three-dimensional structure and requires dithiols, but not monothiols, to support its activity. However, the physiological reducing system of Ohr has not yet been identified. Here we show that lipoylated enzymes present in the bacterial extracts of Xylella fastidiosa interacted physically and functionally with this Cys-based peroxidase, whereas thioredoxin and glutathione systems failed to support Ohr peroxidase activity. Furthermore, we could reconstitute in vitro three lipoyl-dependent systems as the Ohr physiological reducing systems. We also showed that OsmC from Escherichia coli, an orthologue of Ohr from Xylella fastidiosa, is specifically reduced by lipoyl-dependent systems. These results represent the first description of a Cys-based peroxidase that is directly reduced by lipoylated enzymes.

Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite

“…enzymes accept electrons from lipoyl groups of proteins (23,24) in contrast to Prx and Gpx, which are most commonly reduced by thioredoxin or glutaredoxin/glutathione (GSH) (1,2).…”

“…It is known that the Ohr from Xylella fastidiosa (XfOhr) is 10,000-fold more efficient in the removal of t-BHP compared with H 2 O 2 (23). However, because t-BHP and cumene hydroperoxide (CHP) are artificial compounds, the identity of the biological oxidizing substrates for Ohr remains to be identified.…”

Investigation on solubilization protocols in the refolding of the thioredoxin TsnC from Xylella fastidiosa by high hydrostatic pressure approach

“…To express recombinant TsnC, in this work a BL21(DE3) E. coli strain was transformed with the vector pET15b containing the TsnC gene cloned into the EcoRI/Hind III restriction sites [21,22]. A colony was randomly picked from transformants that were grown on LB plates (10 g L À1 tryptone, 5 g L À1 yeast extract, 10 g L À1 NaCl) containing 100 lg mL À1 ampicillin and inoculated in 0.5 L of 2-HKSII rich medium [23].…”

“…Thioredoxin activity was assayed on the basis of its ability to catalyze the reduction of the artificial disulfide 5,5 0 -dithiobis(2-nitrobenzoic acid) (DTNB) to 2-nitro-5-thiobenzoic acid (TNB), as previously described [22]. Briefly, the protein TsnC refolded at HHP at concentrations of 1, 0.75, 0.5 or 0.25 lM were incubated with 200 lM b-nicotinamide adenine dinucleotide 2 0 -phosphate reduced (NADPH), 1 lM thioredoxin reductase (TrxR), and 100 lM DTPA in 20 mM Tris-HCl, pH 8.0, in a 96-well plate, to give a total reaction volume of 370 lL.…”

“…TsnC displays the characteristic features of thioredoxins such as low molecular weight and the Trp-Cys-Gly-Pro-Cys motif in the active site. Remarkably, TsnC can reduce peroxiredoxin PrxQ [21] but not Ohr [22], two Cys-based, thiol-dependent peroxidases from X. fastidiosa. Because the enzymatic assay for TsnC is well established, this protein is suitable for application in refolding studies that consider not only solubility, but also activity for optimization purposes.…”