Modulation of Flexible Loops in Catalytic Cavities Reveals the Thermal Activation Mechanism of a Glycogen-Debranching Enzyme.

Some thermophilic enzymes not only exhibit high thermostability at high temperatures but also have an activation effect by thermal incubation. However, the correlations between temperature-induced structural modulation and thermal activation are still unclear. In this study, we selected a thermophilic glycogen-debranching enzyme from Saccharolobus solfataricus STB09 (SsGDE), which was a promising starch-debranching enzyme with a thermal activation property at temperatures ranging from 50 to 70 °C, to explore the thermal activation mechanism. Molecular dynamics simulations were performed for SsGDE at 30, 50, or 70 °C to reveal the temperature dependence of structure modulation and catalytic function. The results revealed that four loops (loop1 313-337, loop2 399-418, loop3 481-513, and loop4 540-574) in SsGDE were reshaped, which made the catalytic cavity more open. The internal residues, including the catalytic triad Asp3631, Glu399, and Asp471, could be exposed, due to the structural modulation, to exert catalytic functions. We proposed that the thermal activation effect of SsGDE was closely associated with the temperature-induced modulation of the catalytic cavity, which paved the way for further engineering enzymes to achieve higher catalytic performance and stability.