Enhancing Cold Adaptation of Bidomain Amylases by High-Throughput Computational Engineering

Area of Science:

• Biochemistry
• Enzyme Engineering
• Structural Biology

Background:

• Cold-adapted enzymes offer industrial sustainability benefits by reducing energy use and emissions.
• The molecular mechanisms underlying enzyme cold adaptation are not well understood, limiting rational design.
• Bidomain enzymes, like amylases, present opportunities for engineering cold-active variants.

Purpose of the Study:

• To elucidate the molecular basis of cold adaptation in bidomain enzymes.
• To develop a strategy for engineering cold-adapted amylases.
• To investigate the role of interdomain separation in enzyme function at low temperatures.

Main Methods:

• Comparative analysis of cold-adapted Saccharophagus degradans amylase (sdA) and mesophilic Pseudomonas saccharophila amylase (psA) using molecular dynamics (MD) simulations.
• Biochemical assays to validate enzyme activity at low temperatures.
• In silico screening of enzyme variants using a novel domain separation index metric.

Main Results:

• Cold-adapted sdA shows greater interdomain separation between its catalytic domain (CD) and carbohydrate-binding module (CBM) at low temperatures compared to psA.
• A new metric, the domain separation index, was developed to guide enzyme variant screening.
• The engineered psA variant, psA121, exhibited a 3-fold increase in relative activity at 0 °C.
• MD simulations indicated that helical linkers in psA121 promote interdomain separation and dynamic allostery for enhanced low-temperature catalysis.

Conclusions:

• Interdomain separation is a critical factor contributing to cold adaptation in bidomain amylases.
• The domain separation index provides a valuable tool for in silico screening and engineering of cold-adapted enzymes.
• This study offers a framework for introducing cold adaptation into other enzyme systems, advancing sustainable industrial processes.