Activities of the wild-type and mutant enzymes.

<p>The data are the mean ± S.D. of three independent experiments. ND, not detectable.</p

Models of 4-HPPD in complex with 4-HPP substrate.

<p>Superimposition of the model for the wild-type enzyme and the X-ray crystal structure of human 4-HPPD (PDB code: 3ISQ) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069733#pone.0069733-Pilka1" target="_blank">[16]</a> (A), and the models of ΔG379 (B), and Q375N (C) mutant enzymes (stereo image). The protein is shown as a cartoon and colored grey and cyan for the wild-type model and crystal structure or mutant models, respectively. The metal and 4-HPP present as sphere and stick models and colored green and yellow for wild-type and mutant enzymes, respectively. Hydrogen bonding interactions are shown as dashed black lines in (B). The putative substrate binding cavity is shown as Jacks style and colored green and magenta for wild-type and Q375N mutant models, respectively in (C). (D–G) Presentation of the crystal structure (D), wild-type (E), ΔG379 (F), and Q375N (G) models as surface styles and colored by interpolated charge. The C-terminus and residues in the final helix are shown as a ribbon and stick model, respectively, and colored in cyan. The cavity for putative substrate binding cavity is colored yellow. The metal and 4-HPP present as sphere and stick models and colored green.</p

SDS-PAGE analyses of wild-type and mutant 4-HPPD enzymes.

<p>(A) Fractions are shown for the different purification steps of wild-type 4-HPPD. Lane 1, cell crude lysate; Lane 2, following Q-sepharose anion-exchange chromatography; Lane 3, following SOURCE 15PHE hydrophobic interaction chromatography; Lane 4, concentrated fractions after S-100 Sephacryl gel filtration chromatography. (B) Purified wild-type and mutant 4-HPPD enzymes. (C) Western blotting of (B).</p

Apparent kinetic parameters of wild-type and mutant 4-HPPD enzymes measured using the oxygraph assay.

<p>Reported data are mean ± S.D. of three independent experiments.</p

Alignment of amino acid sequences of the C-terminus of human 4-HPPD with enzymes from other species [37].

<p>Fully conservative sequences and residues in iron binding sphere are colored grey and dark grey, respectively. Abbreviations used: <i>h</i>4-HPPD, <i>Homo sapiens</i> (human) 4-HPPD; <i>r</i>4-HPPD, <i>Rattus norvegicus</i> (rat) 4-HPPD; <i>zm</i>4-HPPD, <i>Zea mays</i> 4-HPPD; <i>at</i>4-HPPD, <i>Arabidopsis thaliana</i> 4-HPPD; <i>sa</i>4-HPPD, <i>Streptomyces avermitilis</i> 4-HPPD; <i>pf</i>4-HPPD, <i>Pseudomonas fluorescens</i> 4-HPPD. The sequences for the <i>h</i>4-HPPD and <i>r</i>4-HPPD C-terminal tail (G379 to M393) are highlighted by the square frame.</p

The purification of recombinant human 4-HPPD.

a<p>Steps I to III indicate the pooled fractions after the Q-Sepharose column, after the SOURCE 15PHE column and concentrated fractions pooled after the S-100 Sephacryl column, respectively. The 4-HPPD activity was measured by the formation of HG in µmol/min (U) using the HPLC assay.</p

LPS analysis of <i>S</i>. <i>flexneri</i> expressing wild-type WzzB_SF or the WzzBSFA107P mutant.

<p>Whole-cell lysates of <i>S</i>. <i>flexneri</i> RMA4053 strains expressing either wild-type WzzB_SF or the WzzB_SF^A107P mutant proteins were (A) treated by proteinase-K and electrophoresed on a SDS 15% polyacrylamide gel, followed by detection of LPS by silver-staining (the first 20 Oag repeat units are indicated); or (B) electrophoresed on SDS 15% polyacrylamide gels and then subjected to western immunoblotting with WzzB_SF polyclonal antibodies. The size of the full-length WzzB_SF protein (~36 kDa) is indicated. Each lane corresponds to 5 x 10⁷ bacterial cells.</p

Crystallographic data.

<p>Values in parentheses are for the highest resolution shell.</p><p>¹<i>R</i>_meas =.Σ_<i>hkl</i>{1/[1(<i>hkl</i>)-1]}^1/2Σ_<i>i</i> | I_<i>i</i>(<i>hkl</i>)-(I(<i>hkl</i>))|/Σ_<i>hkl</i>Σ_i I_i(<i>hkl</i>).</p><p>²<i>R</i>_p.i.m. = Σ_<i>hkl</i>{1/[N(<i>hkl</i>)-1]}^1/2Σ_<i>i</i> | I_<i>i</i>(<i>hkl</i>)-(I(<i>hkl</i>))|/Σ_<i>hkl</i>Σ_i I_i(<i>hkl</i>).</p><p>³<i>R</i>_work = Σ_<i>hkl</i>(||Fobs_<i>hkl</i>|-|Fcalc_<i>hkl</i>||)/|Fobs_<i>hkl</i>|, where |Fobs_<i>hkl</i>| and |Fcalc_<i>hkl</i>| represent the observed and calculated structure factor amplitudes. <i>R</i>_free is equivalent to <i>R</i>_work but calculated using 5% of the reflections not used in refinement.</p><p>⁴ Calculated using Molprobity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138266#pone.0138266.ref033" target="_blank">33</a>].</p><p>Crystallographic data.</p

VS-COPS binding measurements.

<p>(A) Raw data for the binding between the His-tagged periplasmic domain of WzzB_SF and VS-COPS. The experiment comprised 5 steps: (a) initial baseline stabilization (30 s); (b) loading of 2 μM of His-tagged WzzB_SF to the Ni-NTA biosensor (120 s); (c) stabilization of the baseline with the reaction buffer (30 s); (d) loading of 4.5 μM VS-COPS (association, purple curve) or the reaction buffer alone (control, black curve) (60 s); and (e) wash with reaction buffer (disassociation, 60 s). The increase of signal during the loading of His-tagged WzzB_SF to the Ni-NTA sensor (Step b) indicates that the binding was effective. 4.5 μM VS-COPS gave a significant binding signal at the association step (step d), compared to the control. (B) R_eq values, indicating the biosensor signal shift induced by the binding of WzzB_SF (solid line) and WzzB_SF^A107P (dashed line) to VS-COPS at equilibrium (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138266#sec002" target="_blank">Materials and Methods</a> section), were fitted with the Hill equation for cooperative binding (Y = X^h*R_max /(X^h+K_d)), yielding a Hill coefficient of 2.6, which demonstrates positive cooperativity. The binding of His-tagged WzzB_SF^A107P and VS-COPS could not be analysed at high VS-COPS concentrations due to non-specific binding of VS-COPS to the sensor (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138266#sec002" target="_blank">Materials and Methods</a> section).</p

Formaldehyde cross-linking analysis.

<p><i>S</i>. <i>flexneri</i> RMA4053 strains expressing either WzzB_SF or WzzB_SF^A107P were harvested, resuspended in potassium phosphate buffer, and treated with 0.5% formaldehyde (+) at 25°C; controls were incubated without formaldehyde (-). Cells were resuspended in the sample buffer and heated at 60°C for 5 min, electrophoresed on a SDS 12% polyacrylamide gel, followed by western immunoblotting with WzzB_SF antibodies. The black arrow indicates the extra band present in WzzB_SF^A107P non-cross-linked sample; the stars indicate the extra bands present in WzzB_SF^A107P cross-linked sample; and the diamonds indicate the ~30 kDa form of WzzB_SF that has been reported previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138266#pone.0138266.ref022" target="_blank">22</a>]. Each lane corresponds to ~5 x 10⁷ bacterial cells.</p