Fig 1.
Molecular interactions and peroxide decomposition reactions of 2-Cys Prx.
The thiolate nucleophilicity of peroxidatic cysteine (CysP) is increased by hydrogen bonds with the catalytic triad Arg and Thr residues (i). The hydroperoxide (Hpx) is trapped by the Arg hydrogen bond which is able to target the Hpx to the Prx active site (ii). The shift of the Arg and Thr hydrogen bonds from the CysP thiolate to the substrate stabilizes the Hpx and increase the thiolate reactivity enabling the SN2 mechanism (iii). After the catalytic reaction, the CysP is oxidized to cysteine sulfenic acid (CysP-SOH) and the release of the leaving group (R-OH, meaning a water molecule in the case of hydrogen peroxide, or the alcohol derivative, in the case of organic hydroperoxides) is assisted by polar interaction with Arg (iv). The cysteine sulfenic acid formation triggers structural changes which allow the condensation among CysP-SOH with CysR-SH (v) resulting in the disulfide formation and concomitant release of a water molecule (vi). The asterisk (*) in CysR, denotes the adjacent subunit of the homodimer.
Table 1.
Molar extinction coefficients, molecular weight and Uniprot code of the proteins used in this work.
Fig 2.
Chemical structures of compounds C1 and C2, isolated from branches of P. crassinervium.
The α, β-unsaturated carbonyl system are represented in red.
Fig 3.
NADPH oxidation assay of PaAhpC and SeAhpC after treatment with compounds C1 and C2.
To perform the assays PaAhpC and SeAhpC samples were previously reduced with DTT (5 mM/1h). The DTT excess was removed, and the enzymes were treated with 40 molar equivalents of C1 (A and C, red square) or C2 (B and D, red square) for 1hour at room temperature. The compounds excess was removed, and the peroxidase activity was accessed by the NADPH oxidation in reactions containing: 3 μM of PaAhpC or SeAhpC, 6 μM EcTrx, 0.9 μM EcTrxR and 150 μM NADPH in buffer 50 mM HEPES (pH = 7.4), 100 μM DTPA and 1 mM sodium azide. Reactions were incubated at 37°C for 5 minutes before being initiated by the addition of H2O2 (500 μM) and monitored spectrophotometrically at 340 nm, 37°C, for 5 minutes. Reactions containing PaAhpC or SeAhpC without natural compounds treatment (green square) were used as positive controls for peroxidase activity, and reactions without AhpC (black square) were used as negative controls. PaAhpC or SeAhpC samples previously treated with NEM were used as control of protein inhibition (blue square). All experiments were performed at least three times in triplicate.
Table 2.
Initial rates of hydrogen peroxide decomposition by PaAhpC or SeAhpC after treatment with compounds C1 or C2.
Fig 4.
IC50 determination of compound C2 for PaAhpC.
(A) Aliquots of PaAhpC enzyme were reduced with 5 mM DTT for 1 hour and then desalted to remove excess of reductant. Aliquots containing 10 μM of the reduced and desalted enzymes were treated varying the concentration of compound C2 (5, 10, 25, 50 and 100 μM for 1 hour at room temperature and subsequently desalted again. Next, 3μM of the treated proteins were added to reactions containing 6 μM EcTrx, 0.9 μM EcTrxR, 150 μM NADPH, 50 mM HEPES (pH 7.4), 100 μM DTPA and 1 mM sodium azide. The reactions were incubated at 37°C for 5 minutes before being initiated by the addition of 500 μM H2O2 and monitored spectrophotometrically at 340 nm, 37°C, for 5 minutes. Reactions without treatment with compound C2 were used as positive controls (green square), and reactions without the addition of AhpC were used as negative controls (black square). (B) The initial rates (v0) of each reaction were calculated, transformed into percentual values, and plotted to calculate the IC50 values of compound C2 for PaAhpC. All experiments were performed at least three times in triplicate.
Fig 5.
Evaluation of inhibition properties of compound C2 over HsPrx2 and the bacterial thiol proteins EcTrx and EcTrxR.
The enzymes HsPrx2, EcTrx and EcTrxR were reduced with DTT and desalted, then treated with 50 molar equivalents of compound C2 for one hour at RT (red square). The proteins were desalted again to remove excess compound and their peroxidase activity was analyzed by NADPH oxidation assay. (A) For Prx2, reactions were performed containing 5 μM of Prx2, 10 μM of ScTrx1, 0.3 μM of ScTrxR1, 150 μM of NADPH, 50 mM of HEPES (pH = 7.0), 100 μM of DTPA and 1 mM of sodium were used. For EcTrx (B) and EcTrxR (C) reactions consisted of 3 μM of PaAhpC, 6 μM of EcTrx, 0.9 μM of EcTrxR, 150 μM of NADPH, 50 mM of HEPES (pH = 7.4), 100 μM of DTPA and 1 mM sodium azide were used. Prior to experiments the reactions were incubated at 37°C/5 min and reactions were started by the addition of H2O2 (500 μM) and monitored spectrophotometrically at 340 nm, 37°C, for 5 minutes. The positive controls contained the proteins HsPrx2, EcTrx or EcTrxR without prior treatment with compound C2 (green square) and the negative control without the addition of PaAhpC (EcTrx or EcTrxR) (black square). The enzymes samples previously treated with NEM were used as control of protein inhibition (blue square). The experiments were performed three times using triplicates.
Table 3.
Initial rates of peroxide decomposition after treatment with compound C2 of HsPrx2, EcTrx and EcTrxR.
Fig 6.
Inhibition characteristics of compound C2 over PaAhpC with growing concentrations of organic hydroperoxide.
(A) NADPH oxidation assay of PaAhpC samples previously reduced with DTT and desalted, then treated with 20 μM of compound C2 for one hour at RT previously treated. The reactions contained with 3 μM of PaAhpC, 6 μM of EcTrx, 0.9 μM of EcTrxR, 150 μM of NADPH, 50 mM of HEPES (pH = 7.4), 100 μM of DTPA and 1 mM sodium azide were used. Prior to experiments the reactions were incubated at 37°C/5 min and reactions were started by the addition of growing concentration of cumene hydroperoxide—CHP (50–750 μM) and monitored spectrophotometrically at 340 nm, 37°C, for 5 minutes. As negative control was used reactions without the enzyme (black square) and as positive control were used PaAhpC samples without prior treatment with compound C2 (malibu square). The experiments were performed three times using triplicates. (B) Plot of the rates of NADPH oxidation by samples of pretreated with compound C2 (PaAhpC/C2; red dots and line) or without inhibitor treatment (PaAhpC/C2; black dots and line).
Table 4.
Enzymatic parameters determined to cumene hydroperoxide (CHP) to PaAhpC pretreated without treatment or pre-treated with compound C2.
Fig 7.
Evaluation of AhpC intermolecular disulfide formation by non-reducing SDS-PAGE.
The molecular weight marker (Sigma Marker) was applied in the lane 1. Samples with 10 μM of PaAhpC were reduced with DTT 5 mM/1 h/RT (lanes 2), the excess was removed (lanes 3) and proteins were treated with 2 molar equivalents of H2O2/30 min/RT (lanes 4). As a positive control of the disulfide formation inhibition, PaAhpC samples previously reduced by DTT and desalted were treated with NEM (40 molar equivalents/1 h/RT) and then oxidized with hydrogen peroxide (30 eq.) (lanes 5). The DTT reduced and desalted samples were treated 40 and 100 molar equivalents of compound C2 and oxidized with hydrogen peroxide (30 eq.) (lanes 6 and 7). The experiments were performed three times presenting similar results.
Fig 8.
Alignment of the amino acid sequences of AhpCs from S. typhimurium and P. aeruginosa.
The alignment of AA sequences was performed using Clustal Ω and the figure was generated with the Jalview. The abbreviations used and access codes for Uniprot (www.uniprot.org) are: AhpC of S. typhimurium = StAhpC (Uniprot: P0A251) and AhpC of P. aeruginosa = PaAhpC (Uniprot: Q02UU0).
Fig 9.
Decameric structure of AhpC and docking results and best pose selection based in peroxiredoxin ligands found in pdb database.
(A) S. typhimurium AhpC (PDB = 4MA9) in reduced decameric state. The enzyme is composed by five obligate homodimers and the two upper homodimers are represented by molecular surface and colored in purple/beige and green/pink. The other dimers of the decamer are represented in cartoon and colored in light gray. The black box denotes the active site microenvironment located at the dimers interface. The catalytic triad residues Thr, CysP and Arg are colored in red, orange and blue, respectively. (B) Molecular docking results for compound C2. The molecules cluster of the compound C2 are represented by lines and colored in pink. (C) Superposition of the compound C2 best hit to StAhpC with ligands found in Prx structures deposited in the pdb database: H2O2 (PDB code = 3A2V; color = red), benzoate (2V32; 1HD2; 1OC3; 2V41; 1H4O; yellow) and tert-butylbenzene-diol (4K7O; white). The C2 is colored in pink with the atoms of α,β-unsaturated carbonyl system highlighted in white. The other molecules are represented in lines with different colors. The oxygens are in red. The catalytic triad (Thr, CysP and Arg) is represented by sticks. (D) Diagram of interacting residues of AhpC with compound C2 docked in the StAhpC active site. The dashed green lines represent hydrogen bonds and hydrophobic contacts are represented by red arcs with radiating spikes. The binding analysis was performed using the LigPLot+.