Designing Efficient Enzymes: Eight Predicted Mutations Convert a Hydroxynitrile Lyase into an Efficient Esterase

Area of Science:

• Biochemistry and Enzymology
• Protein Engineering
• Structural Biology

Background:

• Hydroxynitrile lyase (HbHNL) from rubber tree shares structural similarity with tobacco esterase (SABP2) but catalyzes different reactions.
• Differences in active site residues and structural features like the oxyanion hole and catalytic aspartate solvation affect HbHNL's esterase activity.
• Previous attempts to engineer esterase activity in HbHNL yielded limited success.

Approach:

• Utilized shortest path maps to identify amino acid residues with correlated movements to catalytic residues in both HbHNL and SABP2.
• Hypothesized that correcting specific structural differences, including oxyanion hole positioning and catalytic aspartate solvation, would enhance esterase activity.
• Introduced targeted amino acid substitutions based on shortest path map analysis to create HbHNL variants.

Key Points:

• A variant (HNL7TV) with four specific amino acid substitutions (C81L-N104T-V106F-G176S, plus H103V) exhibited esterase catalytic efficiency comparable to SABP2.
• An intermediate variant (HNL6V) showed partial correction of the oxyanion hole and altered catalytic aspartate solvation.
• Shortest path maps effectively predicted non-active site residues crucial for modulating catalytic activity.

Conclusions:

• Targeted protein engineering guided by computational methods like shortest path mapping can successfully alter enzyme function.
• Structural modifications outside the active site play a significant role in determining catalytic efficiency and substrate specificity.
• This study demonstrates a powerful approach for enzyme functional engineering and understanding structure-function relationships.