Difference between revisions of "Part:BBa K5023002"

Latest revision as of 02:43, 11 October 2023

Polyester Hydrolase PHL7

PHL7 is a recently discovered metagenomic-derived polyester hydrolase. This enzyme is capable of efficiently degrading amorphous polyethylene terephthalate (PET) found in post-consumer plastic waste. It Is stable in a temperature range suitable for PET hydrolysis between 65°C to 70°C. It hydrolyzes PET most effectively around 70°C, which is close to the glass transition temperature (Tg) of the polymer. PHL7 interacts with the terephthalic acid (TPA) moiety of its substrate in a lock-and-key mechanism, rather than an induced fit. This is similar to the enzyme LCC but different from IsPETase. The ligand-induced opening of the substrate-binding groove in PHL7 is restricted due to decreased flexibility of subsite I, which is influenced by the residue H185. This prevents the aromatic residue W156 from moving away from the active site. The aromatic π-stacking clamp, comprised of residues F63 and W156, is crucial for the binding and hydrolysis of BHET. The overall folds of PHL7 and its cocrystal structure with TPA (PHL7×TPA) are nearly identical, with minor deviations in the orientations of active site amino acids.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 307
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 307
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 778
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 307
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 307
Illegal NgoMIV site found at 92
Illegal NgoMIV site found at 193
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 543

Fig. 1 | Subsites I and II of polyester hydrolases.

a)Surface representation of PHL7×TPA. TPA (gray) binds to subsite I (blue). Residues comprising the putative subsite II are conserved or semi-conserved (light green), cover a loop that is elongated in mesophilic homologs (dark green) or differ significantly between polyester hydrolases (red). b) Superimposition of PHL7×TPA (light blue) with IsPETase (salmon, PDB ID: 5XJH). An extended loop covering residues C239-Q247 of IsPETase deviates from its equivalent region (V211-D216) of PHL7 and is stabilized by a non-conserved disulfide bridge (C203-C239). Activity-regulating L210 lies upstream of that loop and spatially close to H130. c Comparison of structurally equivalent active site residues of PHL7 and its homologs LCC, TfCut2 and IsPETase. The catalytic triad and subsite I residues are conserved or conservatively substituted. Residues equivalent to L93, Q95, H185 and F189 in PHL7 can be considered as a part of subsite I although they do not directly interact with TPA in the cocrystal structure. Subsite II residues and the adjacent loop deviate more significantly, especially among thermophilic (type I) and mesophilic (type IIb) homologs. The loop-stabilizing disulfide bridge of IsPETase is not conserved in type I polyester hydrolases.

Fig. 3 | PET film degradation and thermal stability of PHL7 variants.

a)PET film weight loss after reaction times of 4 h and 8 h at 70 °C, data normalized to PHL7 WT. b) PET film weight loss (in weight %, after 16 h) of PHL7 WT compared to variants with reduced thermal stability at lower reaction temperatures. c) Analysis of PETfilm thickness reduction between 10 h to 15 h of reaction by impedance spectroscopy (n = 5 replicates). d) Melting temperatures of PHL7 WT and its variants. The primary y-axis shows the difference in Tm between the WT and variants, the secondary y-axis depicts the absolute melting temperature. If not other stated, mean values for n = 3 replicates ± SD are shown. * PHL7 variant S131A did not cause any PET weight loss after 24 h.

References

RICHTER, P. K. et al. Structure and function of the metagenomic plastic-degrading polyester hydrolase PHL7 bound to its product. Nature Communications, v. 14, n. 1, p. 1905, 5 abr. 2023. https://doi.org/10.1038/s41467-023-37415-x

@@ Line 28: / Line 28: @@
 <center>
-<html><img src="https://static.igem.wiki/teams/5023/wiki/registry/fig-3petfilm.png" width="500px" /><br style="clear: both" /></html>
+<html><img src="https://static.igem.wiki/teams/5023/wiki/registry/fig-3petfilm.png" width="350px" /><br style="clear: both" /></html>
 <i>Fig. 3 | PET film degradation and thermal stability of PHL7 variants.</i>
@@ Line 34: / Line 34: @@
 </center>
-<h5>a)</h5>PET film weight loss after reaction times of 4 h and 8 h at 70 °C, data normalized to PHL7 WT. <h5>b)</h5> PET film weight loss (in weight %, after 16 h) of PHL7 WT compared to variants with reduced thermal stability at lower reaction temperatures. <h5>c)</h5> Analysis of PETfilm thickness reduction between 10 h to 15 h of reaction by impedance spectroscopy (n = 5 replicates). <h5>d)</h5> Melting temperatures of PHL7 WT and its variants. The primary y-axis shows the difference in Tm between the WT and variants, the secondary y-axis depicts the absolute melting temperature. If not other stated, mean values for n = 3 replicates ± SD are shown. * PHL7 variant S131A did not cause any PET weight loss after 24 h.
+<strong>a)</strong>PET film weight loss after reaction times of 4 h and 8 h at 70 °C, data normalized to PHL7 WT. <strong>b)</strong> PET film weight loss (in weight %, after 16 h) of PHL7 WT compared to variants with reduced thermal stability at lower reaction temperatures. <strong>c)</strong> Analysis of PETfilm thickness reduction between 10 h to 15 h of reaction by impedance spectroscopy (n = 5 replicates). <strong>d)</strong> Melting temperatures of PHL7 WT and its variants. The primary y-axis shows the difference in Tm between the WT and variants, the secondary y-axis depicts the absolute melting temperature. If not other stated, mean values for n = 3 replicates ± SD are shown. * PHL7 variant S131A did not cause any PET weight loss after 24 h.