Linker-mediated domain separation enhances cold adaptation in cellulases

Area of Science:

• Biochemistry
• Structural Biology
• Enzyme Engineering

Background:

• Cold-adapted enzymes offer energy savings in industrial applications.
• Understanding the structural basis of cold adaptation in bidomain cellulases is crucial.
• Previous work suggested a 'linker spacer effect' in bidomain amylases.

Purpose of the Study:

• To investigate the 'linker spacer effect' in the bidomain cellulase Cel5G.
• To elucidate the structural and dynamic mechanisms of cold adaptation in Cel5G.
• To guide the engineering of novel cold-adapted enzymes.

Main Methods:

• Molecular dynamics simulations of Cel5G and its linker-modified variants.
• Quantification of domain separation using the domain separation index (DSI).
• Analysis of structural features like disulfide-bonded loops and hydrogen bonding.

Main Results:

• A positive correlation was found between DSI and catalytic turnover number at 10°C.
• Cel5G variants with higher DSI exhibited enhanced cold activity.
• Disulfide-bonded loops were identified as key to maintaining extended conformations and greater DSI.
• Reduced interdomain hydrogen bonding and modulated active site dynamics were observed.

Conclusions:

• The study provides structural and dynamic insights into cold adaptation of bidomain enzymes.
• Linkers play a dual role as spacers and modulators of active site dynamics.
• Findings guide the development of engineered cold-adapted cellulases for industrial use.