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

Abstract

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.

Distribution of Aromatic Amino Acid Residues in Substrate-Binding Regions Modulates Substrate Specificity of Microbial Debranching Enzymes

“…The crystal structures of SsGDE (PDB ID: 7EAV), PaISO (PDB ID: 1BF2), and CrISO (PDB ID: 4OKD) were previously determined. According to the Carbohydrate Active Enzymes database (CAZy; ), SsGDE, PaISO, and CrISO are classified as members of the glycoside hydrolase family 13, subfamily 11 (GH13_11) .…”

“…21,22 In contrast, isoamylase shows substrate preference for highmolecular-mass amylopectin. 23,24 Apart from pullulanase and isoamylase, a glycogen-debranching enzyme from Saccharolobus solfataricus (SsGDE) has been characterized, and it exhibits substrate preference for glycogen and maltodextrin, 25,26 with a significantly different substrate specificity than those of pullulanase and isoamylase. SsGDE exists in a dimeric form at its optimum pH and exhibits hydrolytic activity on α-1,6glucosidic linkages in starch-derived substrates.…”

“…SsGDE exists in a dimeric form at its optimum pH and exhibits hydrolytic activity on α-1,6glucosidic linkages in starch-derived substrates. 25,27 Based on the substrate specificity, SsGDE could be an attractive alternative to hydrolyze the α-1,6-glucosidic linkages in liquefied starch, facilitating further hydrolysis with other amylases for improving starch utilization. SsGDE can specifically hydrolyze the α-1,6-glucosidic linkages in maltodextrin and share a similar hydrolysis mode with isoamylase.…”

“…Therein, pullulanase (EC 3.2.1.41) and isoamylase (EC 3.2.1.68), as members of DBEs, have been intensively studied. , These DBEs can act on α-1,6-glucosidic linkages in substrates but display different substrate specificities . For example, the type I pullulanase preferentially acts on α-1,6-linkages in pullulan and limits dextrins. , In contrast, isoamylase shows substrate preference for high-molecular-mass amylopectin. , Apart from pullulanase and isoamylase, a glycogen-debranching enzyme from Saccharolobus solfataricus (SsGDE) has been characterized, and it exhibits substrate preference for glycogen and maltodextrin, , with a significantly different substrate specificity than those of pullulanase and isoamylase. SsGDE exists in a dimeric form at its optimum pH and exhibits hydrolytic activity on α-1,6-glucosidic linkages in starch-derived substrates. , Based on the substrate specificity, SsGDE could be an attractive alternative to hydrolyze the α-1,6-glucosidic linkages in liquefied starch, facilitating further hydrolysis with other amylases for improving starch utilization.…”

“…The crystal structures of Pseudomonas amyloderamosa isoamylase (PaISO), Chlamydomonas reinhardtii isoamylase (CrISO), and the glycogen-debranching enzyme from Saccharolobus solfataricus (SsGDE) have been determined and are available in the RCSB Protein Data Bank (RCSB PDB, ). ,, Structural analysis reveals that SsGDE shares a high degree of structural similarity with PaISO and CrISO. Therefore, the distinct substrate specificities observed between SsGDE and isoamylases may be attributed to their different substrate-binding modes.…”