Fig 1.
Structural sequences alignment between PON1 and PON2 (A) and their N-terminal regions (B).
(A) Structural sequences alignment between PON1 (pdb:1v04) and PON2 3D model, with “h” and “s” indicating the alpha helix and the beta sheet regions respectively; the conserved residues are marked with asterisk. (B) Superposition between PON1 structure (cyan) and PON2 3D model (yellow), with highlighted by arrows regions 18–31 and 92–109 (red) mentioned in the text. (C) Sequences alignment of N-terminal regions of wild type PON1 and PON2 with the mutant version of crystallized PON1 (pdb:1v04) and the mutant version of PON2 that here was produced (rPON2); with “h” and “s” indicating the alpha helix and the beta sheet regions respectively. The His-tag added at the N-terminal of PON2 in place of the 49 N-terminal residues of the wild type, and the six residues mutagenized starting from the PON1 structure are indicated in grey.
Fig 2.
PON2 purification analysis by SDS-PAGE (A) and size-exclusion step on Superdex G-75 column (B).
(A) SDS-PAGE analysis of the recombinant PON2 purification steps. Line 1: molecular weight markers (ProSieve QuadColor Protein Marker; Lonza, Rockland ME, USA); line 2: 0.05 O.D.600nm of a whole BL21(DE3)/pT7.7-PON2 cells; line 3: 0.05 O.D.600nm of a whole IPTG-induced (1 mM, 3h) BL21(DE3)/pT7.7-PON2 cells; line 4: soluble fraction derived from a BL21(DE3)/pT7.7-PON2 cells (0.05 O.D.600nm) induced with IPTG (1 m, 3h); line 5: not soluble fraction derived from a BL21(DE3)/pT7.7-PON2 cells (0.05 O.D.600nm) induced with IPTG (1 m, 3h); line 6: 5 μg of total proteins present in a fraction eluted from Ni-NTA; line 7: 20 μl of the fraction 11 (F11) which represent peak 1 (continuous trace, Fig 2B); line 8: 20 μl of the fraction 14 (F14) which represent peak 2 (continuous trace, Fig 2B); line 9: 20 μl of the fraction 16 (F16) showing the secondary peak of activity (dot trace, Fig 2B); line 10: 20 μl of the fraction 21 (F21) showing the maximum of activity (dot trace, Fig 2B) eluted by the size-exclusion column corresponding to peak 3 (continuous trace, Fig 2B). (B) last purification step of recombinant PON2 on 16/60 Superdex G-75 column (120 ml). Protein was followed by absorbance reading at 280 nm (continuous trace) or by activity measurements with the substrate pNP-propionate (dot trace). Molecular weight markers used for the gel-filtration 16/60 Superdex G-75 column calibration were: blue dextran (V0); ovalbumin (66 kDa); soybean trypsin inhibitor (30 kDa); lysozyme (20 kDa); Citochrome C (13 kDa) (GE Healthcare, USA).
Table 1.
Substrate specificity of the purified recombinant human PON2.
Table 2.
Kinetic parameters of the purified rPON2 and K168 mutants.
Table 3.
Kinetic parameters of the purified rPON2 and K168 mutants.
Fig 3.
The PAO1 cells were grown o.n. and inoculated in a 96-well microplate in the presence and absence of rPON2 (50 and 100 μg/ml) and PON1 (100 μg/ml). PAO1 cells grown in presence of the enzyme storage buffer (20 mM pH 8.5 containing 0.2 mM Ca++) were used as control. After 48 h of incubation at 37°C, the medium was removed from the wells, and the biofilm formation was evaluated by crystal violet assay. The absorbance of each well was read at 540 nm. Biofilm formation was reported as a percentage in comparison with to the maximum amount of biofilm produced by POA1 cells grown in absence of enzymes (positive control). Each condition was made in six different wells and the experiment was carried out twice. Results are the average of duplicate and error bars show the range of the duplicate.
Fig 4.
Esterase activity (A) and western blot analysis (B) of rPON2 in HeLa cell extracts.
(A) Purified rPON2 was tested for its esterase activity in pure form, diluted in RIPA buffer, and mixed in HeLa cell extracts with or without 3OC12-HSL. The catalytic activity was followed in intervals ranging from 0 to 60 minutes. The assays were carried out with pNP-propionate as a substrate at 40°C. The activities were reported as residual activity (%) relative to the initial value measured. Results are the average of duplicate and error bars show the range of the duplicate. (B) Western blot analysis of rPON2 in HeLa cell extracts with 3OC12-HSL. Samples were withdrawn at the same time interval used to test the catalytic activity. By densitometric analyses (GelQuantNET, available at biochemlabsolution.com) the amount of rPON2 present in HeLa cell extracts was measured: at time t0 we considered the signal corresponding to the rPON2 as 100%, at 15 min the relative signal was 115%, at 30 min 108%, at 45 min 25% and after 60 min no signal was revealed.
Fig 5.
rPON2 and K168 mutants activity in HeLa cell extracts without (A) or with (B) 3OC12-HSL.
Purified enzymes were tested for their esterase activity in pure form or mixed with HeLa cell extracts, following the activity in intervals of time ranging from 0 to 60 minutes. The assays were carried out at 40°C using pNP-propionate as substrate. The activities were reported as residual relative activity (%) relative to the initial value measured. The rPON2 was reported in black, the mutant K168A in grey, and the mutant K168Rin white. (A) The incubation was made in HeLa cell extract not treated with 3OC12-HSL; (B) The incubation was made in HeLa cell extract treated with 3OC12-HSL (100 μM for 10 minutes). As controls, HeLa cell extracts supplemented with the rPON2 buffer only and rPON2 diluted in the RIPA buffer were used and no differences in activity were recorded up to 60 min incubation (data not show). Results are the average of duplicate and error bars show the range of the duplicates.