Sandbox Reserved 1846: Difference between revisions

The <scene name='10/1075248/4eb0_surface_w_stick_ligand/1'>molecular surface view of the enzyme-ligand interaction</scene> shows the overall shape and depth of the binding groove. The <scene name='10/1075246/4eb0_hydrophobicity_ligand/3'>cartoon representation of the enzyme-ligand interaction</scene>, 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.

<scene name='10/1075247/F243_original/1'>Phe243</scene> is located 3.6 Å from the ligand, <scene name='10/1075247/Original_15_mutation_structure/6'>Reference Phe243</scene>. Two mutations at this position, <scene name='10/1075247/F243i/1'>F243I</scene> and F243W, increase the catalytic activity of the enzyme. The larger tryptophan at position 243 stabilizes the ligand through stronger hydrophobic and π–π interactions, pulling it closer to the catalytic site despite its size. 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 [https://en.wikipedia.org/wiki/Pi-stacking#:~:text=In%20chemistry%2C%20pi%20stacking%20(also,interaction%22)%20is%20electrostatically%20repulsive. 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.<ref name="Tournier"/>

The mutation of Tyr to Gly at <scene name='10/1075247/Y127/4'>Tyr127</scene> also increases the thermostability, <scene name='10/1075247/Original_15_mutation_structure/6'>Reference Tyr127</scene>. 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 <scene name='10/1075247/Y127_spacefill/1'>Y127 representation</scene>. Gly is the smallest amino acid and lacks a side chain, providing greater flexibility. The mutation <scene name='10/1075247/Y127g/2'>Y127G</scene> melting point is increased to 87.0°C. The mutation reduces [https://en.wikipedia.org/wiki/Steric_effects#:~:text=Steric%20hindrance%20is%20the%20slowing,as%20slowing%20unwanted%20side%2Dreactions. steric hindrance] and relieves strain in the protein structure, demonstrated in <scene name='10/1075247/Y127g_space_fill/1'>Y127G representation</scene>. By increasing flexibility, the Y127G mutation helps the protein maintain its folded structure under heat stress.<ref name="Tournier"/>

Other stabilizing mutations, such as T96M, N246D, and N246M, were also tested, but excluded as they were not part of the top-performing mutant (ICCG), and therefore omitted for clarity.<ref name="Tournier"/>

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 <ref name="Tournier"/>. 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.<references/>