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

Wei R, Oeser T, Then J, Kühn N, Barth M, Schmidt J, Zimmermann W - AMB Express (2014)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, University of Leipzig, Johannisallee 21-23, Leipzig, D-04103, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: Similar to TfCut2 from T. fusca KW3 (Wei [2011]; Herrero Acero et al. [2011]), the catalytic triad of T. curvata polyester hydrolases formed by S130, D176 and H208 was found to be exposed to the solvent (Figure 4A-B). By docking of the 2PET model substrate, the substrate-binding pocket could be identified as a large groove on the surface of Tcur1278 and Tcur0390 (Figure 4C-F). The negative charge buried in this major groove was contributed by the deprotonated S130 (Figure 4C-D). As shown in Figure 4C-F, Tcur1278 and Tcur0390 displayed similar surface properties in the vicinity of the active site with extended hydrophobic regions around the substrate-binding groove.


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

Wei R, Oeser T, Then J, Kühn N, Barth M, Schmidt J, Zimmermann W - AMB Express (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231364&req=5

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.
Mentions: Similar to TfCut2 from T. fusca KW3 (Wei [2011]; Herrero Acero et al. [2011]), the catalytic triad of T. curvata polyester hydrolases formed by S130, D176 and H208 was found to be exposed to the solvent (Figure 4A-B). By docking of the 2PET model substrate, the substrate-binding pocket could be identified as a large groove on the surface of Tcur1278 and Tcur0390 (Figure 4C-F). The negative charge buried in this major groove was contributed by the deprotonated S130 (Figure 4C-D). As shown in Figure 4C-F, Tcur1278 and Tcur0390 displayed similar surface properties in the vicinity of the active site with extended hydrophobic regions around the substrate-binding groove.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, University of Leipzig, Johannisallee 21-23, Leipzig, D-04103, Germany.

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.

No MeSH data available.


Related in: MedlinePlus