Journal:Acta Cryst D:S2059798325007065
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Converting one enzyme into another might seem straightforward—just swap out the residues that directly contact the substrate, and voilà, you have a new catalyst. However, new structural studies reveal that this intuitive approach falls dramatically short. Researchers attempting to transform hydroxynitrile lyase from rubber trees into an esterase discovered that modifying only the 16 residues directly surrounding the substrate produced disappointing catalytic activity. The real breakthrough came when they expanded their engineering to include 40 or 71 mutations, creating variants that not only matched but exceeded the performance of their target esterase. | Converting one enzyme into another might seem straightforward—just swap out the residues that directly contact the substrate, and voilà, you have a new catalyst. However, new structural studies reveal that this intuitive approach falls dramatically short. Researchers attempting to transform hydroxynitrile lyase from rubber trees into an esterase discovered that modifying only the 16 residues directly surrounding the substrate produced disappointing catalytic activity. The real breakthrough came when they expanded their engineering to include 40 or 71 mutations, creating variants that not only matched but exceeded the performance of their target esterase. | ||
| - | [[Image:Calpha_difference_vs_SABP2.jpg|thumb| | + | [[Image:Calpha_difference_vs_SABP2.jpg|thumb|left|300px|Displacement of Cα atoms (ΔCα) in HbHNL (PDB entry [[1yb6]]), HNL40 (PDB entry [[8sni]]), and HNL71 (PDB entry [[9clr]]) relative to wt SABP2]] |
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The crystal structures explain this dramatic difference in performance. The 40-mutation variant (HNL40) shows a partially restored oxyanion hole—a critical structural pocket that stabilizes the high-energy transition state during ester hydrolysis—while the 71-mutation variant (HNL71) achieves nearly perfect geometric matching with the target enzyme. Active-site & oxyanion hole of; <scene name='10/1087243/018_act_site_oxyanion_hole/5'>SABP2</scene>, superposition of <scene name='10/1087243/018_act_site_oxyanion_hole/6'>SABP2 vs HBHNL</scene>, superposition of <scene name='10/1087243/018_act_site_oxyanion_hole/7'>SABP2 vs HNL40</scene>, superposition of <scene name='10/1087243/018_act_site_oxyanion_hole/8'>SABP2 vs HNL71</scene>. Remarkably, both variants, ''i.e.'', HNL40 and HNL71, develop new tunnels connecting the active site to the protein surface, potentially providing escape routes for reaction products. These structural changes occurred with most mutations being located 6-14 Å away from the substrate-binding site. | The crystal structures explain this dramatic difference in performance. The 40-mutation variant (HNL40) shows a partially restored oxyanion hole—a critical structural pocket that stabilizes the high-energy transition state during ester hydrolysis—while the 71-mutation variant (HNL71) achieves nearly perfect geometric matching with the target enzyme. Active-site & oxyanion hole of; <scene name='10/1087243/018_act_site_oxyanion_hole/5'>SABP2</scene>, superposition of <scene name='10/1087243/018_act_site_oxyanion_hole/6'>SABP2 vs HBHNL</scene>, superposition of <scene name='10/1087243/018_act_site_oxyanion_hole/7'>SABP2 vs HNL40</scene>, superposition of <scene name='10/1087243/018_act_site_oxyanion_hole/8'>SABP2 vs HNL71</scene>. Remarkably, both variants, ''i.e.'', HNL40 and HNL71, develop new tunnels connecting the active site to the protein surface, potentially providing escape routes for reaction products. These structural changes occurred with most mutations being located 6-14 Å away from the substrate-binding site. | ||
Revision as of 15:43, 9 August 2025
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