Functional characterization and structural modeling of synthetic polyester-degrading hydrolases from Thermomonospora curvata

Abstract

Thermomonospora curvata is a thermophilic actinomycete phylogenetically related to Thermobifida fusca that produces extracellular hydrolases capable of degrading synthetic polyesters. Analysis of the genome of T. curvata DSM43183 revealed two genes coding for putative polyester hydrolases Tcur1278 and Tcur0390 sharing 61% sequence identity with the T. fusca enzymes. Mature proteins of Tcur1278 and Tcur0390 were cloned and expressed in Escherichia coli TOP10. Tcur1278 and Tcur0390 exhibited an optimal reaction temperature against p-nitrophenyl butyrate at 60°C and 55°C, respectively. The optimal pH for both enzymes was determined at pH 8.5. Tcur1278 retained more than 80% and Tcur0390 less than 10% of their initial activity following incubation for 60 min at 55°C. Tcur0390 showed a higher hydrolytic activity against poly(ε-caprolactone) and polyethylene terephthalate (PET) nanoparticles compared to Tcur1278 at reaction temperatures up to 50°C. At 55°C and 60°C, hydrolytic activity against PET nanoparticles was only detected with Tcur1278. In silico modeling of the polyester hydrolases and docking with a model substrate composed of two repeating units of PET revealed the typical fold of α/β serine hydrolases with an exposed catalytic triad. Molecular dynamics simulations confirmed the superior thermal stability of Tcur1278 considered as the main reason for its higher hydrolytic activity on PET.

Keywords: Polyester hydrolase; Polyethylene terephthalate (PET); Synthetic polyester; Thermomonospora curvata.

Figure 1

Effects of pH and temperature on the hydrolytic activity of Tcur1278 and Tcur0390. Activities of Tcur1278 and Tcur0390 against pNPB at different (A) pH and (B) reaction temperature conditions are shown as broken and solid lines, respectively. Error bars indicate the standard deviation of three determinations.

Figure 2

Thermal stability performance of Tcur1278 and Tcur0390. The residual hydrolytic activity was determined with (A) Tcur1278 and (B) Tcur0390 against pNPB over a period of 1 h at 50°C (solid line), 55°C (broken line) and 60°C (dotted line). Error bars indicate the standard deviation of three determinations.

Figure 3

Decomposition of polyester nanoparticles byT. curvatahydrolases. PCL hydrolysis by (A) Tcur1278 and (B) Tcur0390 at 49°C; PET hydrolysis by Tcur1278 at (C) 50°C, (D) 55°C and (E) 60°C, and by Tcur0390 at (F) 50°C. The initial rates of the square roots of turbidity decrease are plotted as a function of enzyme concentration (squares and diamonds). Error bars represent the standard deviation of duplicate determinations. Fitted data (solid lines) according to Eq. (1) are also shown.

Figure 4

Structural modeling of Tcur1278 and Tcur0390 polyester hydrolases. Homology modeling was performed with the Phyre2 web server (Kelley and Sternberg [2009]). The catalytic triad of (A) Tcur1278 and (B) Tcur0390 is formed by S130, D176 and H208. The 2PET model substrate was docked using GOLD 5.1 with its central ester bond constrained between 2.7 and 3.1 Å in the oxyanion hole formed by the main chain NH groups of F62 and M131 (broken yellow lines). The hydrogen bonds stabilizing the tetrahedral intermediate formed during the catalytic reaction are shown as broken lines in blue. The backbone structures are shown as gray cartoons. The electrostatic surface properties of Tcur1278 (C) and Tcur0390 (D) are shown with negatively charged residues in red, positively charged residues in blue and neutral residues in white/gray, respectively. The lipophilic surface properties of Tcur1278 (E) and Tcur0390 (F) are shown with hydrophilic residues in pink and hydrophobic residues in bright green, respectively. The docked 2PET model substrate is shown in cyan.

Figure 5

Molecular dynamics simulation of (A, C, E) Tcur1278 and (B, D, F) Tcur0390 polyester hydrolases. (A, B) Time courses of backbone RMSD changes during a simulation for 50 ns at 298 K (blue) and 353 K (red). (C, D) RMSF of C_α atoms per amino acid residue during a simulation for 50 ns at 298 K (blue) and 353 K (red). The purple spirals and arrows at the top of the RMSF graphs indicate α-helices and β-sheets, respectively. The catalytic triad residues are shown as solid spheres. (E, F) The distance of the catalytic H208 and S130 (H-S) during a simulation for 50 ns at 298 K (blue) and 353 K (red). For a clearer view, single simulation data from three simulations are shown.