Difference between revisions of "Part:BBa K5023002"
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<partinfo>BBa_K5023002 parameters</partinfo>
 
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[[Image:https://static.igem.wiki/teams/5023/wiki/registry/phl7lucas.jpeg|500px|left|thumb|'''Figure 22''': Illustration of ABTS oxidation by ECOL with time compared to the negative control. The increase in ABTS oxidized proves laccase activity.]]
 
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Figure 22 shows the illustration of ABTS oxidation by ECOL with time compared to the negative control. The increase in ABTS oxidized proves laccase activity even if a direct comparison with the original and not immobilized laccase solution was not possible due to the measuring methods.
 
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<html><img src="https://static.igem.wiki/teams/5023/wiki/registry/phl7lucas.jpeg" width="750px" /><br style="clear: both" /></html>
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<html><img src="https://static.igem.wiki/teams/5023/wiki/registry/phl7lucas.jpeg" width="500px" /><br style="clear: both" /></html>
  
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<i>Schematic of the Hifi cloning of D2</i>
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<i>Fig. 1 | Subsites I and II of polyester hydrolases.</i>
 
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<h4>a<h4> 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 stabi�lized 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.
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The catalytic triad and subsite I residues are conserved or conservatively sub�stituted. 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
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structure. Subsite II residues and the adjacent loop deviate more significantly,
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especially among thermophilic (type I) and mesophilic (type IIb) homologs. The
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loop-stabilizing disulfide bridge of IsPETase is not conserved in type I polyester
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hydrolases.
  
  
 
===References===
 
===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
 
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

Revision as of 02:29, 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<h4> 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 stabi�lized 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 sub�stituted. 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.

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