Structure of <i>Lm</i>TK.

<p>(A) The protomer in complex with dThd and phosphate, ligands as spheres coloured by atom type. (B) Superposition using SSM [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003781#pntd.0003781.ref055" target="_blank">55</a>] showing the loop region corresponding to <i>Lm</i>TK residues 42–59 of <i>Lm</i>TK-dThd in lemon, <i>Lm</i>TK-dTTP in green, <i>Lm</i>TK-AP₅dT in ice blue, <i>Bc</i>TK (2ja1) in orange and hTK1 (1w4r) in pale crimson. The overall fold of <i>Lm</i>TK-dThd is shown in white. (C) The dimer in the asymmetric unit in the dThd-phosphate complex with the ligands shown as spheres coloured by atom type. (D) The tetramer generated using PISA [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003781#pntd.0003781.ref056" target="_blank">56</a>]. Figure made using CCP4mg [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003781#pntd.0003781.ref057" target="_blank">57</a>].</p

Stereo views of the ligand sites after superposition with SSM [55] in CCP4mg [57].

<p>(A) The dThd binding site of <i>h</i>TK1 (lemon), <i>Lm</i>TK-dThd (light blue) and <i>Lm</i>TK-dTTP (ice blue). (B) <i>Lm</i>TK-dThd (dark cyan), <i>Lm</i>TK-dTTP (ice blue), <i>Lm</i>TK-AP₅dT (light blue) and <i>Tm</i>TK-dThd-AppNHp (tan). The P-loop of <i>Lm</i>TK-AP₅dT is shown as a ribbon. Amino acid residues and ligands are shown as cylinders coloured by atom type with C and P atoms coloured by model. Hydrogen bonds are shown as dashed lines.</p

Analytical ultracentrifugation of C-terminally His-tagged <i>Lm</i>TK.

<p>Superposition of the c(s) distributions of the unliganded <i>Lm</i>TK and <i>Lm</i>TK-dThd-AppNHp samples are shown at concentrations in the range of 1–8 S.</p

Kinetic constants and substrate specificity of recombinant <i>Lm</i>TK and <i>h</i>TK1.

<p>Kinetic parameters for <i>Lm</i>TK are means of two or more independent experiments.</p><p>N.D. Not determined.</p><p>^a Activity measurements at 10 mM substrate concentration.</p><p>^b Kinetic constants obtained at 37°C by Munch-Peterson et al. [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003781#pntd.0003781.ref043" target="_blank">43</a>].</p><p>Kinetic constants and substrate specificity of recombinant <i>Lm</i>TK and <i>h</i>TK1.</p

Crystallographic data and statistics.

<p>¹ Ramachandran Plot generated with COOT (Emsley <i>et al</i>., 2010).</p><p>² Poor Rotamer analysis performed using <i>Molprobity</i> (Chen <i>et al</i>., 2010).</p><p>Crystallographic data and statistics.</p

Kinetic analysis of <i>Lm</i>TK.

<p>(A) ATP substrate dependent specific activity. Experiments were performed at 25 μM dThd and kinetic constants obtained by a least-squares best fit of the experimental data to the Hill equation. (B) Inhibition of enzyme activity by dTTP. Experiments were performed at 25 μM dThd and 500 μM ATP. The IC₅₀ value was calculated by non-linear regression analysis of the data. (C) The modulating effect of ATP on the inhibition of <i>Lm</i>TK by dTTP. dTMP formation was monitored at variable ATP concentrations and 25 μM dThd; in the absence of dTTP and in the presence of 3.5 μM and 7 μM dTTP. (D) Inhibition of enzyme activity by AP₅dT. Experiments were performed at 25 μM dThd and 500 μM ATP. The IC₅₀ value was calculated by non-linear regression analysis. Each point represents the average of three determinations, and error bars represent the standard deviation.</p

Purification of human carboxylesterases.

<p>(a) Size exclusion profiles of purified hCES1 (blue trace) hCES1 N79Q (green trace) and hCES1 S221A (red trace) enzymes from media of transfected HEK cells. Samples were run on a HiLoad 16/60Superdex 200 column (GE Healthcare) in 200 mM NaCl, 20 mM Tris-HCl, pH 7.5. The peak corresponds to a molecular weight of approximately 160 kDa as estimated from the elution volumes of globular proteins of known molecular weight: Aprotinin (6.5 kDa) Ribonuclease A (13.7 kDa) Carbonic Anhydrase (29 kDa), Ovalbumin (44 kDa), Conalbumin (75 kDa) Aldolase (158 kDa) Ferritin (440 kDa) and Blue Dextran 2000. (b) SDS-polyacrylamide gel of purified CES1 (lanes 1 and 2) and CES1 N79Q (lanes 3 and 4) untreated (lanes 1 and 3) and treated with PNGaseF (lanes 2 and 4). (c) The sedimentation velocity distribution for hCES1 N79Q. Data for hCES1 and hCES1 S221A gave the same profiles.</p

Enzyme activity of human carboxylesterases.

<p>Plots of initial rates of reaction against enzyme concentration assayed as described in the Methods section at a substrate concentration of 750 μM 4-NPA (a) CES1 (b) CES1 N79Q. Plots of initial reaction rates against substrate concentration for the hydrolysis of 4-NPA by (c) hCES1 and (d) hCES1 N79Q (3.4 nM each enzyme). The molarity of the enzyme was calculated assuming 100% trimer with a molecular weight of 182.7 kDa.</p

Binding of (5<i>S</i>)‑Penicilloic Acid to Penicillin Binding Protein 3

β-Lactam antibiotics react with penicillin binding proteins (PBPs) to form relatively stable acyl-enzyme complexes. We describe structures derived from the reaction of piperacillin with PBP3 (<i>Pseudomonas aeruginosa</i>) including not only the anticipated acyl-enzyme complex but also an unprecedented complex with (5<i>S</i>)-penicilloic acid, which was formed by C-5 epimerization of the nascent (5<i>R</i>)-penicilloic acid product. Formation of the complex was confirmed by solution studies, including NMR. Together, these results will be useful in the design of new PBP inhibitors and raise the possibility that noncovalent PBP inhibition by penicilloic acids may be of clinical relevance