Alteration of enzyme specificity by computational loop remodeling and design

Area of Science:

• Enzyme engineering
• Computational biology
• Protein structure and function

Background:

• Enzyme specificity relies on precise positioning of active site functional groups.
• Altering enzyme specificity requires both sequence changes for new interactions and backbone remodeling for proper positioning.

Purpose of the Study:

• To develop a computational method for designing enzyme specificity by remodeling active site loops.
• To validate the method's ability to predict and remodel loop structures.
• To redesign human guanine deaminase for altered substrate specificity.

Main Methods:

• A computational design approach targeting loop regions near the enzyme's active site.
• Benchmarking the method against native protein-ligand complexes to assess accuracy in predicting loop length and conformation.
• Applying the method to redesign a loop in human guanine deaminase to interact with ammelide.

Main Results:

• The computational method accurately predicted native loop lengths and conformations in benchmark tests.
• The redesigned human guanine deaminase exhibited 100-fold increased activity on ammelide.
• The engineered enzyme showed a 2.5e4-fold decrease in activity on guanine, resulting in a 2.5e6-fold net specificity change.
• X-ray crystallography confirmed the remodeled loop adopted the computationally predicted conformation within 1-Å Cα RMSD.

Conclusions:

• The computational loop remodeling method is effective for altering enzyme specificity.
• This approach enables precise engineering of enzyme active sites for novel substrate interactions.
• The redesigned human guanine deaminase demonstrates significant shifts in substrate preference, validated by structural analysis.