Sometimes space is a good thing: uncovering the structural determinants of cold adaptation in cellulases

Posted by daviskd2 on Wednesday, October 29, 2025 in News.

By Cameron I. Cohen

Cellulases are enzymes responsible for the hydrolysis of glycosidic bonds in cellulose, a polysaccharide which serves as the main structural material in plant cell walls. The efficient breakdown of cellulose is therefore necessary in industries such as paper and pulp manufacturing, food and juice production, textile processing and biorefining. By 2026, the cellulase market is projected to account for 20% of the total enzyme market. One of the biggest issues facing cellulase design is the high reaction temperature required for saccharification, or the degradation of cellulose into fermentable monosaccharides.

Developing cellulases capable of operating at lower temperatures would  greatly reduce energy costs by eliminating the need for excess heating. Cold-adapted cellulases have been isolated from bacteria, earthworms, and fungi with the most extensively studied being the enzyme Cel5G from Pseudoalteromonas haloplanktis. Cel5G consists of three structural components: a catalytic domain (CD) for catalysis, a carbohydrate-binding module (CBM) for substrate recruitment, and a peptide linker connecting both domains. The cold adaptive behavior of Cel5G is known in part to rely on the peptide linker length, as shortening of this linker greatly reduces catalytic activity at lower temperatures, but little else is understood about the relationship between cellulase structure and cold activity.

This study, spearheaded by a former undergraduate student, Robbie Ge, in the Yang lab and supported by two postdoctoral fellows, Ning Ding and Yaoyukun Jiang, set out to investigate that exact connection. Using a series of molecular dynamics (MD) simulations on Cel5G and its variants, the researchers set out to identify the structural underpinnings of cellulase cold adaptation.

Cel5G and three variants were initially tested, each with identical CD and CBM domains, but variable linker regions. In wild-type Cel5G, the linker contains 3 circular loops which are stabilized by disulfide bonds. The three variants contained progressively shorter linker regions, and accordingly, progressively fewer loops with the shortest linker having no loops at all. MD simulations indicated that Cel5G displayed the greatest degree of flexibility as indicated by the largest change in RMSD from the starting structure. Building off this conclusion, the researchers next determined a domain separation index (DSI) for each variant and found that a positive correlation existed between DSI and catalytic activity at 10°C.

To further understand the mechanics of the linker domain, cysteine-to-alanine mutants of the previous variants were generated which eliminated the loops while keeping linker length the same. Interestingly, MD simulations of these enzymes demonstrated that while linker length was consistent, the “loop-less” constructs had a much lower DSI. In fact, there was no correlation between linker length alone and domain separation. Rather, the authors’ findings indicate that presence of disulfide-bond loops are vital for domain spacing and therefore flexibility of motion.

In examining the MD structures more closely, the researchers observed that constructs with greater DSI exhibited fewer interdomain hydrogen bonds. Interestingly, these changes in interface hydrogen bonding were shown to propagate all the way to the active site and affect the conformational landscape of the entire enzyme. All together, these data suggest that cold adaptive behavior in cellulases is modulated by enzyme flexibility, which is mediated primarily by the spacing between domains. These insights into the structural determinants of cellulase activity provide an exciting foundation for future enzyme design and has the potential to greatly impact industrial cellulose processing.

Make sure to check out Protein Science for the full story!