Crystal structures of forty- and seventy-one-substitution variants of hydroxynitrile lyase from rubber tree
Professor Romas Kazlauskas [1]
Molecular Tour
Beyond the Active Site: How Distant Residues Orchestrate Enzyme Function
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.
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. Remarkably, both variants 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.
These results reveal a sophisticated network where 'second-shell' residues, located beyond direct substrate contact, orchestrate catalytic efficiency through long-range effects. This finding suggests that creating truly efficient enzymes requires reengineering entire structural neighborhoods—a principle that could revolutionize approaches to enzyme design for biotechnology and medicine and help explain why natural enzymes maintain such complex architectures.
References
- ↑ doi: https://dx.doi.org/10.1107/S2059798325007065
