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| | <StructureSection load='4eb0' size='350' frame='true' align='right' caption='Wild Type PET Hydrolase' scene='10/1075191/Wild_type_pet_hydrolase/1'> | | <StructureSection load='4eb0' size='350' frame='true' align='right' caption='Wild Type PET Hydrolase' scene='10/1075191/Wild_type_pet_hydrolase/1'> |
| | + | '''Wild-Type PET Hydrolase''' |
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| - | '''Introduction''' | + | ''Why is this important?'' |
| | + | PET plastic is a major pollutant. LCC can help break it down more efficiently. |
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| - | ''Environmental Issues'' | + | ''Enzyme structure'' |
| - | Pollution is one of the largest environmental issues facing our globe today. It is estimated that there is 359 million tons of plastic waste, excluding all other forms of material waste, produced annually around the world. Of this plastic waste, 150–200 million tons ends up sitting in landfills or as pollution in our natural environments. The most common form of plastic waste is poly-ethylene terephthalate (PET), generally used in soft drink containers and plastic water bottles. Only about 10% is recycled. The methods currently in use are not viable on a global scale.
| + | This enzyme is a serine hydrolase with a catalytic triad and a hydrophobic pocket. |
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| - | ''Current Plastic Recycling Methodology''
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| - | Strides have been made toward recycling PET plastics, but most current methods are not viable globally. Enzymes—especially cutinases and hydrolases—have been promising, but many suffer from low thermal stability. PET needs to be broken down at or above 70°C, which causes most enzymes to denature. Furthermore, many enzymes don’t fully break PET down to its monomers, making complete recycling impossible.
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| - | Leaf compost cutinase (LCC), in both its wild-type and mutated forms, is a promising enzyme that can function at higher temperatures and shows potential to help resolve global plastic recycling challenges.
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| - | '''Structure'''
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| - | ''Overall Topology''
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| - | LCC is a monomeric serine hydrolase with 258 amino acids and an amphipathic structure. It features a <scene name='10/1075191/Overall_topology/1'>secondary structure</scene> of alpha helices and beta turns, characteristic of the alpha-beta hydrolase family. The <scene name='10/1075191/Active_site_overall_topology/3'>catalytic triad</scene> in its active site is a hallmark of this family. LCC is 33× more efficient than other studied PET-degrading enzymes.
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| - | ''Alpha-Beta Hydrolase Family''
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| - | This enzyme contains an [https://en.wikipedia.org/wiki/Alpha/beta_hydrolase_superfamily alpha-beta hydrolase fold]. The fold includes a chymotrypsin-like catalytic triad, hydrophobic binding pocket, and an oxyanion hole. The motif G-X-Nu-X-G allows for a tight nucleophilic loop. Some members also contain HX₄D motifs, providing acyltransferase activity. Functions across this family include proteolysis, signal transduction, and lipid metabolism.
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| - | '''Active Site'''
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| - | ''Structure''
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| - | The <scene name='10/1075193/Hydrophobic_binding_pocket/3'>hydrophobic binding pocket</scene> stabilizes PET through aromatic π-stacking and Van der Waals interactions. No structure of LCC bound to PET exists, so ligand positions have been approximated.
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| - | <b>[Add table with residues + monomers]</b>
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| - | ''Mechanism''
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| - | The catalytic triad (insert scene) facilitates proton transfer, activating S165 for nucleophilic attack. It targets the carbonyl carbon of the PET polymer, forming a tetrahedral intermediate. The <scene name='10/1075193/Oxyanion_hole/1'>oxyanion hole</scene> (Y95 and M166) stabilizes the intermediate. H242 then protonates the leaving group oxygen, cleaving the scissile bond.
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| - | In a second step, a water molecule—activated by H242 and D210—attacks, forming a second tetrahedral intermediate. H242 protonates the leaving group again, restoring the carbonyl and freeing the product.
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| - | [[Image:PET_hydrolase_Mechanism.jpeg|400 px|left|thumb|Figure Legend]]
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| - | <b>[Upload higher-resolution version]</b>
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| - | '''Mutations'''
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| - | ''Tournier et al. Mutagenesis''
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| - | Tournier et al. studied mutations in the active site using a <scene name='10/1075190/Ligand/2'>PET substrate model</scene>. Out of 11 targeted residues, most mutations decreased activity—except F243I and F243W, which increased it.
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| - | <scene name='10/1075190/F243_wt/3'>WT F243 Residue</scene><br />
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| - | <scene name='10/1075190/F243w_mutant/4'>F243W with ligand</scene><br />
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| - | <scene name='10/1075190/F243i/1'>F243I</scene>
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| - | ''ICCG/WCCG Mutants''
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| - | The ICCG and WCCG variants increased catalytic efficiency and thermal stability. A disulfide bond was introduced by mutating <scene name='10/1075191/residues D238 and S283/2'>D238/S283 to cysteines</scene>, replacing a metal-binding motif (E208/D238/S283). This raised melting temperature from 84.7°C (WT) to 94.5°C, though activity slightly decreased.
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| - | <scene name='10/1075191/Wild_type_y127/1'>Wild type Y127</scene><br />
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| - | <scene name='10/1075191/Mutation_y127_to_g127/1'>Mutation G127</scene>
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| - | ''Glycosylation''
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| - | Three <scene name='10/1075191/All_glycosylation_sites/3'>glycosylation sites</scene> were introduced by Shirke et al. using N-linked modifications. These improved thermal stability and catalytic activity.
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| - | Patterns used:
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| - | - 197–199 (N-T-S)
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| - | - 239–241 (N-A-S) – no individual impact
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| - | - 266–268 (N-D-T)
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| - | <scene name='10/1075191/First_glycosylation_site/3'>Site 1</scene>
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| - | <scene name='10/1075191/Second_glycosylation_site/2'>Site 2</scene>
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| - | <scene name='10/1075191/Third_glycosylation_site/2'>Site 3</scene>
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| - | Glycosylated LCC was more stable (10°C higher Tm), unfolded more slowly, and retained 85% activity at 80°C (vs. 50% for WT).
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| - | '''Conclusions'''
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| - | ''Improved Thermal Stability''
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| - | Thermal stability is essential for PET degradation above 70°C. The disulfide mutation increased the enzyme’s melting point by ~10°C.
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| - | ''Depolymerization Efficiency of Mutant LCCs''
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| - | ICCG and WCCG reached ~90% depolymerization in 9.3–10.5 hours, compared to 53% in 20 hours for WT.
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| - | ''Implications for Plastic Recycling''
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| - | These discoveries suggest a path forward for more efficient enzymatic PET recycling—bringing us closer to scalable, environmentally sustainable plastic waste solutions.
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| | </StructureSection> | | </StructureSection> |
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| - | =References= | |
| - | <references/> | |
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| - | =Student Contributors= | |
| - | *Georgia Apple | |
| - | *Emily Hwang | |
| - | *Anjali Rabindran | |
