Cold-active enzymes exhibit high catalytic activity at low temperatures and an anomalous temperature optimum occurring before the onset of protein unfolding. This study investigates this peculiar property by comparing the functional and structural features of a cold-active glycoside hydrolase family 1 enzyme (M-GH1) with those of its mesophilic counterpart (Pp-GH1). Structural analysis and computational simulations reveal that the thermal profile of M-GH1 is due to a combination of local unfolding and weakened enzyme-substrate interactions. This behavior is attributed to the absence of an amino acid triad comprising M326-W412-F418, which stabilizes the loops surrounding the active site in the mesophilic enzyme. Introducing this triad into M-GH1 through rational mutagenesis yielded a more thermostable variant, whereas reciprocal mutations in Pp-GH1 resulted in a slight decrease of both the optimal temperature of catalysis and thermal stability. Phylogenetic analyses coupled with computational simulations suggest that this flexibility modulation mechanism is not universally conserved across the GH1 family but rather represents a targeted strategy for regulating the dynamics of the catalytic region. Overall, these findings identify a key structural determinant of temperature adaptation within the GH1 family.
Digiovanni et al. (2026) studied this question.