A Push-Pull Loop Motif Controls Product Distribution in GH5 Endocellulases.

Area of Science:

• Biochemistry and Molecular Biology
• Enzymology
• Carbohydrate Chemistry

Background:

• Endocellulases are crucial for breaking down cellulose, but their action is often unpredictable.
• Controlling the products of cellulose deconstruction is challenging due to the stochastic nature of these enzymes.

Purpose of the Study:

• To elucidate the mechanism governing cleavage-site selection in GH5 endocellulases.
• To demonstrate rational programming of endocellulase product profiles through protein engineering.

Main Methods:

• Comparative analysis of two related GH5 endocellulases with differing product profiles.
• Molecular dynamics simulations to visualize substrate-enzyme interactions.
• Site-directed mutagenesis to alter loop structures and assess functional impact.

Main Results:

• A specific loop structure near the binding cleft dictates substrate positioning and hydrolysis outcomes.
• A triaspartate (DDD) loop promotes deep binding and central cleavage, while a DND loop leads to nonspecific hydrolysis.
• Minimal, reversible loop substitutions switched product distributions without affecting the catalytic core.

Conclusions:

• Endocellulase product profiles can be rationally controlled via loop engineering.
• This controlled hydrolysis is relevant for applications like phosphorylated sugar biosynthesis requiring specific oligosaccharide inputs.