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

Abstract

Organic hydroperoxide resistance (Ohr) enzymes are unique Cysbased, lipoyl-dependent peroxidases. Here, we investigated the involvement of Ohr in bacterial responses toward distinct hydroperoxides. In silico results indicated that fatty acid (but not cholesterol) hydroperoxides docked well into the active site of Ohr from Xylella fastidiosa and were efficiently reduced by the recombinant enzyme as assessed by a lipoamide-lipoamide dehydrogenase-coupled assay. Indeed, the rate constants between Ohr and several fatty acid hydroperoxides were in the 10 7 -10 8 M −1 ·s −1 range as determined by a competition assay developed here. Reduction of peroxynitrite by Ohr was also determined to be in the order of 10 7 M −1 ·s −1 at pH 7.4 through two independent competition assays. A similar trend was observed when studying the sensitivities of a Δohr mutant of Pseudomonas aeruginosa toward different hydroperoxides. Fatty acid hydroperoxides, which are readily solubilized by bacterial surfactants, killed the Δohr strain most efficiently. In contrast, both wild-type and mutant strains deficient for peroxiredoxins and glutathione peroxidases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because the Δohr mutant was more sensitive than wild type to 3-morpholinosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In the case of H 2 O 2 insult, cells treated with 3-amino-1,2,4-triazole (a catalase inhibitor) were the most sensitive. Furthermore, fatty acid hydroperoxide and SIN-1 both induced Ohr expression in the wildtype strain. In conclusion, Ohr plays a central role in modulating the levels of fatty acid hydroperoxides and peroxynitrite, both of which are involved in host-pathogen interactions.hydroperoxides | thiols | Cys-based peroxidase | pathogenic bacteria | Pseudomonas aeruginosa

Functional and evolutionary characterization of Ohr proteins in eukaryotes reveals many active homologs among pathogenic fungi

“…In fact, we recently described that hydroperoxides derived from oleic, linoleic and arachidonic fatty acids are the biological substrates of bacterial Ohr, at least for enzymes from P. aeruginosa and X. fastidiosa [11]. Therefore, we determined the second order rate constant for the reaction between the reduced MfOhr del and linoleic acid hydroperoxide (LAOOH) by employing a competitive approach that follows redox dependent changes in the AhpE intrinsic fluorescence [11]. Like the bacterial enzymes, MfOhr del displayed an extraordinarily high rate constant (3.2 (±2.1)×10 8  M −1  s −1 ) for LAOOH reduction (Fig.…”

“…A. Reaction of MfOhr del with LAOOH was investigated by a competitive assay following the intrinsic tryptophan fluorescence emission (λ ex =290 nm and λ em =340 nm) of 2 µM AhpE [11]. The red and yellow lines represent the emission fluorescence of AhpE in its reduced (no LAOOH) and oxidized (added 1.8 µM LAOOH) states; in the absence of MfOhr del .…”

“…The rate constant for the reduction of LAOOH by MfOhr was calculated according to a competitive assay previously described [11] that takes advantage of the redox-dependent changes in the intrinsic fluorescence of AhpE, a Cys-based peroxidase from Mycobacterium tuberculosis (MtAhpE) [6]. Briefly, Ohr and AhpE were pre-reduced with 50 mM of DTT as described above.…”

“…The physiological role played by Ohr and OsmC has been linked to the defense against organic hydroperoxide insults [1], [5], [6], [7], [8], [9], [10], [11]. Ohr and OsmC are structurally distinct from peroxiredoxin (Prx) and glutathione-peroxidase (Gpx) enzymes [1], [2], although all are Cys-based, thiol dependent peroxidases.…”

“…Lipoyl groups covalently attached to some proteins are the biological reductants of these intramolecular disulfides [4]. Recently, we demonstrated that Ohr enzymes display high specificity for fatty acid hydroperoxides and peroxynitrite as oxidizing substrates [11].…”