Linker-Mediated Domain Separation Enhances Cold Adaptation in Cellulases

Cold-adapted cellulases are crucial for reducing energy demands in industrial processes by enabling efficient cellulose saccharification at lower temperatures. However, the structural basis of cold adaptation in bidomain cellulases remains poorly understood. Our prior studies on bidomain amylases suggest the hypothesis that a greater degree of domain separation is linked to improved cold adaptation. Here, we investigated the hypothesis in the bidomain cellulase Cel5G and its linker-modified variants through molecular dynamics simulations. Domain separation, quantified by domain separation index (DSI), positively correlates with catalytic efficiency at 10°C, meaning that Cel5G variants with a greater DSI demonstrates a higher activity. Structural analyses show that disulfide-bonded loops are pivotal to maintain a high population of extended conformations that involve a greater DSI and reduced interdomain hydrogen bonding (H-bonding). Besides serving as a spacer, linkers in bidomain enzymes can also modulate the active site through dynamic allostery, fine-tuning the frequency of H-bond interactions to influence catalytic residues’ capability of binding or reacting to the substrate. Overall, this study enhances our structural and dynamics-based understanding of cold adaptation of bidomain enzymes and provide a new strategy for cold-adapted enzyme engineering.