Sandbox Reserved 1846

This Sandbox is Reserved from March 18 through September 1, 2025 for use in the course CH462 Biochemistry II taught by R. Jeremy Johnson and Mark Macbeth at the Butler University, Indianapolis, USA. This reservation includes Sandbox Reserved 1828 through Sandbox Reserved 1846.

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Leaf Branch Compost Cutinase

spinqualitylabelsall models Leaf Branch Compost Cutinase (PDB: 4EB0) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Introduction Leaf branch compost cutinase (LCC) is a versatile enzyme that can break down both natural plant polymers and synthetic plastics.. Its biological function is to degrade cutin, the waxy biopolymer on plant surfaces that prevents water loss and protects the plant from environmental stressors. LCC was discovered in a compost heap, where degradative enzymes are often found. Because cutinases are members of the α/β-hydrolase superfamily and can hydrolyze polymer esters, LCC has shown high efficiency in hydrolyzing polyethylene terephthalate (PET), which is a widely used plastic that contributes to pollution. Unlike many other PET-degrading enzymes, LCC has a high catalytic efficiency and is thermostable, which means it can function at temperatures that are optimal for industrial recycling processes. By breaking PET into its monomers, LCC promotes closed-loop recycling of plastic waste and reduces environmental accumulation. 4EB0 is the primary PDB file used throughout this page. The protein is an LCC mutant that has been optimized for thermostability. The model substrate is 2-HE(MHET)₃, a trimer of MHET (mono-(2-hydroxy-ethyl) terephthalate). MHET is an intermediate in the depolymerization of PET. A binding model of the substrate 2-HE(MHET)3 in wild-type LLC (4eb0.pdb) was constructed and refined to mimic the 3D structure illustrated in Figure 2 of reference “xx”. The software Maestro (Schrödinger, Inc; version 14.2.118) was used to construct the initial binding structure, followed by energy minimization in the context of the rigid protein that had previously been processed to add/refine all hydrogen atoms. The ligand model was then used without further modification to identify and illustrate the cited active-site residues. Function LCC catalyzes the hydrolysis of the ester bonds in polymers of PET, breaking them down into their constituent monomers: terephthalic acid and ethylene glycol. The enzyme operates through a catalytic triad that consists of Ser165, Asp210, and His242, where a reaction initiated by Ser165 leads to the hydrolysis of ester bonds in PET. During catalysis, the substrate binds in an elongated, predominantly hydrophobic groove present in the enzyme's structure. LCC functions best at elevated temperatures (around 65–72°C), which approaches the glass transition temperature of PET. This temperature range maximizes PET chain mobility and makes the polymer more accessible to enzymatic action. The enzyme has higher thermostability compared to other PET hydrolases, with a melting temperature of 84.7°C. This property allows it to remain functional under these high-temperature conditions. Unlike other PET hydrolases such as Is-PETase, BTA1, BTA2, and FsC, LCC also has substantially higher catalytic efficiency. Specifically, LCC has an initial PET-specific depolymerization rate of 93.2 mg TAeq·h⁻¹·mg⁻¹ enzyme at 65°C with amorphous PET. This means that it is at least 33 times more efficient than other tested enzymes. LCC's function is limited by PET crystallinity, as the enzyme can more effectively hydrolyze amorphous regions of the polymer. As PET crystallinity increases during the depolymerization reaction (due to exposure to elevated temperatures), the enzyme's efficiency decreases. This limits complete depolymerization unless optimal conditions and enzyme variants are used. Relevance With global plastic production reaching approximately 299 million tons annually, the need for effective waste management solutions is urgent. Enzymatic degradation is an alternative to conventional recycling methods that are often inefficient and taxing on the environment. One of the primary challenges in plastic waste management is the volume of mismanaged plastic entering marine environments. In 2010 alone, an estimated 31.9 million metric tons of plastic waste were classified as mismanaged, with a substantial portion ending up in the ocean. This causes harm to marine ecosystems, physical injury to wildlife, and disruption of food chains. Integrating LCC into existing waste management systems could substantially reduce the PET waste that enters the environment. Research suggests that a 77% reduction in mismanaged plastic waste could lower the annual input of plastic into the ocean to between 2.4 and 6.4 million metric tons by 2025. LCC hydrolyzes PET into its constituent monomers, which also supports the principles of a circular economy, where materials are reused rather than discarded. Enzymatic degradation allows for the production of biologically recycled PET with properties that are comparable to virgin materials. Structural Overview LCC consists of one α/β-hydrolase domain, typical of cutinases. In the α/β-hydrolase fold, 9 beta sheets (yellow) form a stable central core with x tilt. This is surrounded by 10 alpha helices (magenta) that contribute to the overall folding. The enzyme does not have a lid domain that covers its active site. Catalytic Triad Figure 1: Ser, His, Asp catalytic triad non-covalent stabilizing interactions with oxyanion hole. LCC catalyzes the breakdown of PET using a serine hydrolase mechanism with a catalytic triad of Ser165, His242, and Asp210. (Figure 1) (1) The reaction begins when His242 deprotonates Ser165, which activates it as a nucleophile. (2) Ser165 then attacks the carbonyl carbon of an ester bond in the PET polymer to form a tetrahedral transition state. This transition state is stabilized by an oxyanion hole formed by the backbone amides of Met166 and Tyr95. (3) Proton transfer then leads to formation of semi-stable acyl-enzyme intermediate and the alcohol product. (4) A water molecule, activated by His242, then attacks the acyl-enzyme. This releases the second product and resets the enzyme’s active site. 5) Figure 2: LCC mechanism. LCC hydrolyzes PET using a catalytic triad (Ser165, His242, Asp210) to cleave its ester bonds via two tetrahedral transition states to an acyl-enzyme intermediate. Ligand Binding Pocket The substrate-binding site of LCC is a long, mainly hydrophobic groove that accommodates PET chains. This groove includes three subsites —designated −2, −1, and +1—that interact with specific PET units near the scissile ester bond. Hydrophobic residues such as F125, V212, M166, and F243 line the groove and facilitate substrate binding by interacting with the aromatic rings of the PET molecule. These interactions help align the substrate in the correct position for catalysis. The molecular surface view of the enzyme-ligand interaction shows the overall shape and depth of the binding groove. The cartoon representation of the enzyme-ligand interaction, the enzyme is shown as a ribbon diagram with the hydrophobic residues colored pink, to show how the PET chain fits snugly into the groove. Mutation Sites of Interest To improve the catalytic activity and thermostability of LCC, Tournier et al. (2020) used structure-guided enzyme engineering based on the crystal structure of LCC bound to a model PET substrate. Using molecular docking and enzyme–substrate contact analysis, the researchers identified 15 Residues in the first contact shell surrounding the substrate-binding groove. Of these, 11 positions were selected for saturation mutagenesis to determine how mutations could affect PET depolymerization. These sites were chosen for their interactions with the PET-like ligand or their proximity to the active site. Highly conserved residues essential for catalysis or structural stability​ were excluded. From this screen, two mutations at Phe243 (F243I and F243W) were shown to improve catalytic activity by optimizing substrate positioning within the groove. To increase thermostability, the authors targeted a region of the enzyme that is structurally analogous to known divalent metal binding sites in other cutinases. Instead of using stabilizing ions, which could complicate industrial degradation processes, they engineered a disulfide bridge by mutating Asp238 and Ser283 to Cys residues (D238C/S283C). Additional mutations were selected based on thermostability screening. Among these, Y127G improved the melting point without reducing activity. F243 F243 is located 3.6 Å from the ligand. Two mutations at this position, F243I and F243W, increase the catalytic activity of the enzyme. The F243I mutation inserts the smaller isoleucine whose side chain allows the ligand to sit closer. This reduces the ligand distance to 3.0 Å, improving substrate binding. The F243W mutation inserts the bulkier, nitrogen-containing aromatic aide chain. Trp brings the ligand slightly closer at 3.2 Å and introduces potential for new interactions, such as hydrogen bonding or π-stacking. Both mutations result in improved catalytic performance. The F243I mutant shows a 27.5% increase in activity, while the F243W mutant shows a 17.5% increase, compared to the wild-type enzyme. Tyr 127 The mutation of Tyr to Gly at Tyr127 also increases the thermostability. The melting point of Y127G is increased to 87.0°C from the WT melting point of 84.7°C. Tyr has a bulky, rigid aromatic side chain that can cause structural strain, shown in Y127 representation. Gly is the smallest amino acid and lacks a side chain, providing greater flexibility. The mutation Y127G melting point is increased to 87.0°C. The mutation reduces steric hindrance and relieves strain in the protein structure, demonstrated in Y127G representation. By increasing flexibility, the Y127G mutation helps the protein maintain its folded structure under heat stress. Ser 283 & Asp 238 Two wild-type residues, Ser283 and Asp238, were engineered to form a disulfide bond by replacing them with Cys. This decision was based on their spatial proximity in the 3D structure and their location in a region that resembles metal-binding sites in homologous PET-degrading enzymes. Unlike those metal-dependent sites, the LCC structure lacked coordinated ions. For that reason, the researchers engineered a covalent linkage instead to increase thermal stability without requiring additives like calcium. The wild-type protein has a melting point of 84.7°C, while the S283C and D238C mutant increased the melting point to 94.5°C, a 9.8°C improvement, which is higher than any other mutations. However, this increase in stability was accompanied by a 28% decrease in enzymatic activity compared to the wild-type. This trade-off between stability and activity shows the balance in enzyme engineering, as increasing structural integrity can sometimes restrict the flexibility needed for catalytic function. Group Mutations These mutations were combined to create multi-mutant LCC variants with improved activity and thermostability. The two most successful variants were: ICCG: F243I / D238C / S283C / Y127G WCCG: F243W / D238C / S283C / Y127G Other stabilizing mutations, such as T96M, N246D, and N246M, were also tested but are not included in this page's protein model. These were excluded because they were not part of the top-performing mutant (ICCG), and therefore omitted for clarity.

References

A binding model of the substrate 2-HE(MHET)3 in wild-type LLC (4eb0.pdb) was constructed and refined to mimic the 3D structure illustrated in Figure 2 of reference . The software Maestro (Schrödinger, Inc; version 14.2.118) was used to construct the initial binding structure, followed by energy minimization in the context of the rigid protein that had previously been processed to add/refine all hydrogen atoms. The ligand model was then used without further modification to identify and illustrate the cited active-site residues.

Student Contributors

Ashley Callaghan, Rebecca Hoff, & Simone McCowan