Characterisation of the Organophosphate Hydrolase Catalytic Activity of Ssopox

SUPPLEMENTARY INFORMATION FOR

Characterisation of the organophosphate hydrolase catalytic activity of SsoPox

Julien HIBLOT, Guillaume GOTTHARD, Eric CHABRIERE, Mikael ELIAS

FigureS1. Chemical structure of SDS (A) and DOC (B).

Text S1.

Investigation of the putative effect of SDS on the oligomeric state and on the global conformation of SsoPox

Results

Effect of SDS on SsoPox oligomerisation state. Gel filtration experiments in presence of 0%, 0.1% and 0.01% SDS were performed at room temperature. The elution profile of SsoPox in all conditions highlights an apparent size of 47kDa (Fig.S2A): an intermediate size between monomeric and dimeric forms (monomer size: 35.5kDa). This result is consistent with that was previously obtained (i.e. 50kDa)10 and tends to suggest that SDS has no apparent effect on oligomerisation state of SsoPox in size exclusion chromatography. In addition, DLS experiments were performed in presence of 0%, 0.1% and 0.01% of SDS. Particles in solution was at respective sizes of 80.0 ± 3.9 kDa, 89.5 ± 3.5 kDa and 82.0 ± 3.0 in presence of 0%, 0.1% and 0.01% of SDS (Fig.S2B). These results suggest that in solution SsoPox is dimeric in all the tested conditions.

Effect of SDS on the local protein conformation. Another possible effect of SDS would be to modulate the local flexibility of the protein. In an attempt of getting some insights, we performed tryptophan fluorescence monitoring in the presence (0.1% and 0.01%) and absence of SDS (Fig.S2C). SsoPox monomer contains 3 tryptophans (positions 36, 263, 278), one is buried into the protein (W278), another is at the dimer interface (W36) and the third is a key active site residue (W263). All of them are in a hydrophobic environment, referring to the crystal structure. The fluorescence spectrum performed without SDS, exhibiting a maximum fluorescence pick at about 330 nm comprises a typical spectrum for tryptophan in hydrophobic environment. No shifts of maximum fluorescence or major differences were observed in fluorescence spectrum recorded in the presence of 0.1% and 0.01% SDS (Fig.S2C). These results suggest that SDS doesn’t influence the local protein conformation of the protein.

FigureS2. Conformational analysis of SsoPox in presence of SDS.

A.Size exclusion chromatography profiles of SsoPox at room temperature and different SDS concentrations (0%, 0.1% and 0.01%). Shown is protein absorbance (OD280nm) versus elution volume for SsoPox. In each condition SsoPox exhibit an elution profile corresponding to 47kDa molecules. B.Intensity (%) of the signal versus molecular weight of particles of SsoPox at different SDS concentration by dynamic light scattering at room temperature. C.Fluorescence intensity in function of the wavelength after SsoPox tryptophan excitation at 280nm in presence of various concentration of SDS (0%, 0.1% and 0.01%). In all tested conditions, the maximum fluorescence is recorded at 330nm reflecting a hydrophobic environment of the tryptophans.

FigureS3. SsoPox inhibition by fensulfothion.

The inverse of catalytic efficiency (V) of SsoPox against paraoxon for each concentration of fensulfothion ([I]) is represented. The determined inhibition constant (KI) of fensulfothion is 7.78 ± 1.23mM.

FigureS4. Stereo figure of SsoPox’s active site bound with fensulfothion. The active site metals and water molecules are shown as spheres. The putative catalytic water is presented as W1. The 2Fobs-Fcalc electronic density map is contoured at 1σ.

Table SI. Paraoxonase activity comparison between SsoPox and SacPox at 70°C.

SsoPox / SacPoxc
kcat (s-1) / KM (µM) / kcat/KM (s-1M-1) / kcat (s-1) / KM (µM) / kcat/KM (s-1M-1)
Paraoxon / 0.24 ± 0.01a / 60 ± 9a / 4.00(±0.75).103a / ND / ND / 0.91 ± 0.06
2.29b / 2360b / 9.7.102b
3.98 ± 0.23d / 3270 ± 380d / 1.22(± 0.21).103d

a from Merone et. al (2005); b from Ng et al. (2010); c from Porzio et al. (2007), data were obtained at 75°C. d from this study.

Data obtained with cobalt as cofactor. ND corresponds to not determined values.

TableSII. Paraoxonase activity comparison between GsP, DrOPH and SsoPox at 25°C.

Substrate / SsoPox / GsPa / DrOPHb
Name / Structure / kcat (s-1) / KM (µM) / kcat/KM (s-1M-1) / kcat (s-1) / KM (µM) / kcat/KM (s-1M-1) / kcat (s-1) / KM (µM) / kcat/KM (s-1M-1)
Paraoxon / / 12.59 ± 1.29 / 24250 ± 3616 / 5.19(± 1.31).102 / 0.12 ± 0.02 / 2100 ± 320 / 5.47(±0.47).101 / 4.16 (±0.13).10-3 / 3000 ± 200 / 1.39 ± 0.11
Methyl-Paraoxon / / 2.71 ± 0.64 / 2142 ± 676 / 1.27(± 0.7).103 / 7.83(±0.33 ).10-3 / 270 ± 31 / 2.90(±0.13).101 / 7.67(±0.33).10-4 / 1300 ± 100 / 0.58 ± 0.05

a from Hawwa et al. (2009), data characterized at 35°C. b from Hawwa et al. (2009).

r corresponds to racemic solution. Data obtained with cobalt as cofactor.

Table SIII. Background hydrolysis rates in the conditions of the kinetics presented in Table I

[Substrat]
(M) / Nerve agent analogs / [Substrat] (M) / Paraoxon
70°C / [Substrat] (M) / Paraoxon / [Substrat] (M) / Me-paraoxon / Me-parathion / Malathion
CMP / CMP SDS 0.01% / IMP / 25°C / SDS 0.1% / SDS 0.01% / DOC 0.01% / DOC 0.05% / DOC 0.01%
0.00075 / 2.517 / 2.149 / - / 0.006 / 25.706 / 0.002 / -0.197 / -0.084 / -0.172 / 0.020 / -0.090 / -0.269 / 0.001 / -0.470 / -2.876 / -1.038
0.0005 / 2.502 / -0.908 / 1.526 / 0.005 / 24.794 / 0.0015 / -0.200 / 0.038 / -0.112 / 0.024 / -0.080 / -0.167 / 0.0008 / -0.271 / -0.131 / -0.118
0.00025 / 1.754 / 12.037 / 0.769 / 0.004 / 23.108 / 0.001 / -0.110 / -0.047 / -0.052 / 0.008 / 0.007 / -0.101 / 0.0006 / 0.078 / 0.265 / -0.516
0.0001 / 0.030 / 0.335 / 0.528 / 0.003 / 17.167 / 0.00075 / -0.077 / 0.025 / -0.087 / -0.032 / -0.006 / -0.087 / 0.0005 / 2.119 / 0.317 / 0.058
0.000075 / 0.488 / 0.382 / - / 0.002 / 10.961 / 0.0005 / -0.060 / -0.035 / -0.073 / 0.032 / 0.005 / -0.038 / 0.0004 / 0.218 / -1.388 / 0.042
0.00005 / -0.503 / 0.525 / 0.186 / 0.0015 / 6.923 / 0.00025 / -0.005 / -0.028 / -0.035 / 0 / -0.003 / -0.005 / 0.0002 / 0.096 / -0.599 / 0.658
0.000025 / -0.763 / 0 / - / 0.001 / 5.717 / 0.0001 / -0.005 / -0.003 / 0.013 / 0 / 0 / 0.022 / 0.0001 / -0.038 / -0.064 / 0.414
0.00001 / 0.427 / -0.239 / - / 0.0005 / 2.800 / 0.00005 / -0.025 / -0.025 / 0 / -0.003 / 0.027 / 0.006 / 0.00005 / -0.020 / -0.246 / -0.386

All reported velocities are in nmol/s.

Table SIV. Background hydrolysis rates in the conditions of the kinetics presented in Figure 2.

Condition / Background hydrolysis (nmol/s)
Paraoxon 1 mM / 1% Tween 20 / 0.055
0.1% Tween 20 / 0
0.01% Tween 20 / -0.025
10% DMSO / 0
1% DMSO / -0.129
800 mM Guanidinium Chloride / -0.041
80 mM Guanidinium Chloride / -0.079
8 mM Guanidinium Chloride / -0.102
800 µM Guanidinium Chloride / -0.088
80 µM Guanidinium Chloride / -0.131
8 µM Guanidinium Chloride / -0.128
800 mM Urea / 0.006
80 mM Urea / -0.102
8 mM Urea / -0.109
800 µM Urea / -0.134
80 µM Urea / -0.121
1% SDS / -0.017
0.5% SDS / 0.003
0.1% SDS / 0.002
0.05% SDS / 0.060
0.025% SDS / -0.069
0.01% SDS / -0.177
0.001% SDS / -0.148
0.0001% SDS / -0.140
Paraoxon 100 µM / 0.5% DOC / 0
0.25% DOC / 0.002
0.1% DOC / 0.036
0.05% DOC / 0.006
0.025% DOC / -0.009
0.01% DOC / 0.006
0.005 DOC / 0.006
0.001 DOC / 0.008