Presentation Information
[1ENZ-01-KL]How Cooperatively-Acting Substitutions Outside the Active Site Enhance Catalysis
○Romas Kazlauskas1, Sharad Sarak1, Hong Yang1, Colin Pierce1, Panhavuth Tan1, Allison Cafferty1, Anne Dao1, Dana Junaidi1, Ke Shi1, Guillem Casadevall2, Silvia Osuna2,3, Robert Evans1 (1. University of Minnesota (USA), 2. Universitat de Girona (Spain), 3. ICREA (Spain))
Keywords:
esterase,protein engineering,catalytic activity,x-ray crystal structure,cooperativity
Engineering enzymes with substantially higher catalytic activity remains challenging. Although catalysis occurs in the active site, substitutions outside the active site frequently enhance activity, and the structural basis for these effects is often unclear.
Hydroxynitrile lyase from rubber tree, an α/β-hydrolase with poor esterase activity, was used as a model system. Homology-guided design introduced 15–71 substitutions to increase sequence similarity to esterases. Catalytic activity was measured, and X-ray crystal structures were determined to identify structural changes associated with increased activity.
The variants showed approximately 100-fold increases in esterase activity. Structural analysis revealed coordinated changes in backbone geometry and side-chain interactions, many of which reposition residues within the active site. Three structural changes account for the enhanced catalysis: enlargement of part of the active site to favor productive substrate binding, restoration of the oxyanion hole to enable ester hydrolysis, and formation of new tunnels that facilitate release of the alcohol product.
The substitutions are highly cooperative: removal of any one of fifteen key substitutions significantly decreases activity. This cooperativity arises because all three structural changes are required for efficient catalysis, and each structural change depends on multiple substitutions acting together.
These results demonstrate the essential role of indirectly acting, second-shell residues in α/β-hydrolase catalysis and show that coordinated structural remodeling outside the active site is a powerful strategy for engineering faster enzymes.
Hydroxynitrile lyase from rubber tree, an α/β-hydrolase with poor esterase activity, was used as a model system. Homology-guided design introduced 15–71 substitutions to increase sequence similarity to esterases. Catalytic activity was measured, and X-ray crystal structures were determined to identify structural changes associated with increased activity.
The variants showed approximately 100-fold increases in esterase activity. Structural analysis revealed coordinated changes in backbone geometry and side-chain interactions, many of which reposition residues within the active site. Three structural changes account for the enhanced catalysis: enlargement of part of the active site to favor productive substrate binding, restoration of the oxyanion hole to enable ester hydrolysis, and formation of new tunnels that facilitate release of the alcohol product.
The substitutions are highly cooperative: removal of any one of fifteen key substitutions significantly decreases activity. This cooperativity arises because all three structural changes are required for efficient catalysis, and each structural change depends on multiple substitutions acting together.
These results demonstrate the essential role of indirectly acting, second-shell residues in α/β-hydrolase catalysis and show that coordinated structural remodeling outside the active site is a powerful strategy for engineering faster enzymes.
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