Substrate Turnover Dynamics Guide Ketol-Acid Reductoisomerase Redesign for Increased Specific Activity

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Computational Chemistry

Background:

• Enzyme adaptation for industrial applications is limited by incomplete control over catalytic functions.
• Ketol-acid reductoisomerase (KARI) is crucial for industrial isobutanol production.
• Traditional enzyme redesign focuses on ground and transition states, potentially missing dynamic effects.

Purpose of the Study:

• To develop a rational computational approach to enhance enzyme catalytic activity.
• To identify mutations in KARI that increase its specific activity for isobutanol production.
• To investigate the role of enzyme dynamics in catalysis.

Main Methods:

• Utilized path sampling techniques and QM/MM simulations to model KARI substrate turnover dynamics.
• Employed machine learning to identify conformational features correlated with productive catalysis.
• Applied multistate protein redesign to select mutations stabilizing reactive conformations.

Main Results:

• Identified specific enzyme-substrate complex conformations promoting catalysis.
• Generated eight enzyme mutants with significantly improved calculated specific activity.
• Observed up to a (2 ± 1) × 10^4-fold increase in calculated kcat for the fastest variant.

Conclusions:

• Analyzing complete enzyme turnover dynamics provides insights for enzyme engineering.
• Stabilizing reaction-promoting conformations is an effective strategy for enhancing enzyme catalysts.
• This rational approach advances the design of enzymes for industrial applications like isobutanol synthesis.