Structural and functional characterisation of TesA - a novel lysophospholipase A from Pseudomonas aeruginosa

Abstract

TesA from Pseudomonas aeruginosa belongs to the GDSL hydrolase family of serine esterases and lipases that possess a broad substrate- and regiospecificity. It shows high sequence homology to TAP, a multifunctional enzyme from Escherichia coli exhibiting thioesterase, lysophospholipase A, protease and arylesterase activities. Recently, we demonstrated high arylesterase activity for TesA, but only minor thioesterase and no protease activity. Here, we present a comparative analysis of TesA and TAP at the structural, biochemical and physiological levels. The crystal structure of TesA was determined at 1.9 Å and structural differences were identified, providing a possible explanation for the differences in substrate specificities. The comparison of TesA with other GDSL-hydrolase structures revealed that the flexibility of active-site loops significantly affects their substrate specificity. This assumption was tested using a rational approach: we have engineered the putative coenzyme A thioester binding site of E. coli TAP into TesA of P. aeruginosa by introducing mutations D17S and L162R. This TesA variant showed increased thioesterase activity comparable to that of TAP. TesA is the first lysophospholipase A described for the opportunistic human pathogen P. aeruginosa. The enzyme is localized in the periplasm and may exert important functions in the homeostasis of phospholipids or detoxification of lysophospholipids.

Figure 1. Enzyme kinetics and localization of TesA.

(A) Enzyme kinetics of TesA. The hydrolysis of 1-hexyl-glycerophosphocholine (C6-GPC), 1-lauryl-glycerophosphocholine (C12-GPC) and 1-palmitoyl-glycerophosphocholine (C16-GPC) follow Michaelis-Menten kinetics. Released fatty acids were quantified after 30 min incubation of 1 µg of purified TesA with 100 µl of lysoPL substrate at 37°C. (B) Subcellular localisation of TesA in P. aeruginosa PA01. Zymogram indicating esterase activity of cell compartments (S: supernatant, M: membrane fraction, Pp: periplasm fraction, Cy: cytoplasmic fraction) from P. aeruginosa PA01 transformed with pBBR-TesA (TesA) and pBBR1mcs-3 (EV, empty vector). Molecular weights of protein standard in kDa are indicated on the right. Esterase activity was monitored under UV light using the fluorescent substrate 4-methylumbelliferyl butyrate.

Figure 2. Crystal structure of TesA.

Superposition of the overall structures of TesA (light blue); TAP (PDB code 1IVN, transparent green, Q=0.731) and EstA (PDB code 3HP4, transparent red, Q=0.743). Secondary structural elements and loops are labelled according to the TesA sequence. Two conserved water molecules (in PDB file Wat502 rt and Wat506 lft) in the TesA structure are shown as magenta balls. The loops which are structurally different from TAP and EstA are indicated in dark blue in the TesA structure. Note the narrow entrance of the catalytic site (between loop_110-117 and loop_141-160) in TesA structure. The black arrow indicates the position of the switch loop.

Figure 3. The catalytic site.

(A) Superposition of TesA, TAP and EstA (colour labelling as in Figure 2). (B) Superposition of TesA and the catalytic domain of EstA of P. aeruginosa (PDB code 1KVN, transparent yellow). The catalytic residues (S9, H159, and D156) as well as residues forming the oxyanion hole (G46, N75) are shown as stick models. In the TesA structure, these residues are coloured by element with carbon in yellow, nitrogen in blue and oxygen in red. The dotted line represents the hydrogen-bond between the catalytic residues D156 and H159 in the TesA structure.

Figure 4. TesA reveals a compact and rigid structure.

(A) Comparison of the crystal structures of TesA (light blue) and TAP (green). Note the positioning of loop_110-117 and loop_141-160 (both loops shown in dark blue) in TesA at the entrance of the substrate binding cavity, and the shift of helix α7 towards the core. Dotted lines represent the interatomic distances between Cα atoms of residues Pro112 and Gly148 in TesA, and equivalent residues Pro110 and Leu146 in TAP that are 6.1 Å and 13.6 Å, respectively. (B) Plot of average temperature factor of the protein backbone versus residue of TesA and TAP (PDB ID: 1IVN). Switch loop_78-83, loop_110-117 and loop_141-160 are indicated as shaded regions.

Figure 5. Conserved waters and hydrogen bonds in the catalytic site.

(A) TesA, (B) TAP (PDB ID: 1IVN), (C) EstA (PDB ID: 3HP4), and (D) esterase domain of the autotransporter EstA (PDB ID: 3KVN). The hydrogen bonds mediated by the two conserved water molecules (S1 and S3 shown as magenta balls) are shown as dotted lines.

Figure 6. Rational mutagenesis of TesA.

(A) Electrostatic potential maps around TesA, TAP and variant TesA D17S/L162R showing the field that propagates into the solvent. Cutoff values of +1.8 kT/e and -1.8 kT/e were used to define the blue and red contours, respectively. Note the positive potential localized at R160 in TAP (structural equivalent L162 is indicated in TesA), and R162 in variant TesAD17S/L162R). The orientation of molecules is as in Figure 2. (B) Enhanced thioesterase activity of TesA. Thioesterase activity assays were performed using lauroyl-CoA (25 µM) as a substrate with purified his-tagged TesA (1 µg); activity of the wild type TesA (WT) was taken as 100%. The results are mean values of three independent measurements with standard deviation indicated by error bars.