9 research outputs found

    Molecular mechanisms underlying phenotypic degeneration in Cordyceps militaris: insights from transcriptome reanalysis and osmotic stress studies

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    Abstract Phenotypic degeneration in Cordyceps militaris poses a significant concern for producers, yet the mechanisms underlying this phenomenon remain elusive. To address this concern, we isolated two strains that differ in their abilities to form fruiting bodies. Our observations revealed that the degenerated strain lost the capacity to develop fruiting bodies, exhibited limited radial expansion, increased spore density, and elevated intracellular glycerol levels. Transcriptome reanalysis uncovered dysregulation of genes involved in the MAPK signaling pathway in the degenerate strain. Our RT-qPCR results demonstrated reduced expression of sexual development genes, along with upregulation of genes involved in asexual sporulation, glycerol synthesis, and MAPK regulation, when compared to the wild-type strain. Additionally, we discovered that osmotic stress reduced radial growth but increased conidia sporulation and glycerol accumulation in all strains. Furthermore, hyperosmotic stress inhibited fruiting body formation in all neutralized strains. These findings indicate dysregulation of the MAPK signaling pathway, the possibility of the activation of the high-osmolarity glycerol and spore formation modules, as well as the downregulation of the pheromone response and filamentous growth cascades in the degenerate strain. Overall, our study sheds light on the mechanisms underlying Cordyceps militaris degeneration and identifies potential targets for improving cultivation practices

    Distal Hydrophobic Loop Modulates the Copper Active Site and Reaction of AA13 Polysaccharide Monooxygenases

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    Polysaccharide monooxygenases (PMOs) use a type-2 copper center to activate O2 for the selective hydroxylation of one of the two C–H bonds of glycosidic linkages. Our electron paramagnetic resonance (EPR) analysis and molecular dynamics (MD) simulations suggest the unprecedented dynamic roles of the loop containing the residue G89 (G89 loop) on the active site structure and reaction cycle of starch-active PMOs (AA13 PMOs). In the Cu(II) state, the G89 loop could switch between an “open” and “closed” conformation, which is associated with the binding and dissociation of an aqueous ligand in the distal site, respectively. The conformation of the G89 loop influences the positioning of the copper center on the preferred substrate of AA13 PMOs. The dissociation of the distal ligand results in the bending of the T-shaped core of the Cu(II) active site, which could help facilitate its reduction to the active Cu(I) state. In the Cu(I) state, the G89 loop is in the “closed” conformation with a confined copper center, which could allow for efficient O2 binding. In addition, the G89 loop remains in the “closed” conformation in the Cu(II)-superoxo intermediate, which could prevent off-pathway superoxide release via exchange with the distal aqueous ligand. Finally, at the end of the reaction cycle, aqueous ligand binding to the distal site could switch the G89 loop to the “open” conformation and facilitate product release

    Distal Hydrophobic Loop Modulates the Copper Active Site and Reaction of AA13 Polysaccharide Monooxygenases

    No full text
    Polysaccharide monooxygenases (PMOs) use a type-2 copper center to activate O2 for the selective hydroxylation of one of the two C–H bonds of glycosidic linkages. Our electron paramagnetic resonance (EPR) analysis and molecular dynamics (MD) simulations suggest the unprecedented dynamic roles of the loop containing the residue G89 (G89 loop) on the active site structure and reaction cycle of starch-active PMOs (AA13 PMOs). In the Cu(II) state, the G89 loop could switch between an “open” and “closed” conformation, which is associated with the binding and dissociation of an aqueous ligand in the distal site, respectively. The conformation of the G89 loop influences the positioning of the copper center on the preferred substrate of AA13 PMOs. The dissociation of the distal ligand results in the bending of the T-shaped core of the Cu(II) active site, which could help facilitate its reduction to the active Cu(I) state. In the Cu(I) state, the G89 loop is in the “closed” conformation with a confined copper center, which could allow for efficient O2 binding. In addition, the G89 loop remains in the “closed” conformation in the Cu(II)-superoxo intermediate, which could prevent off-pathway superoxide release via exchange with the distal aqueous ligand. Finally, at the end of the reaction cycle, aqueous ligand binding to the distal site could switch the G89 loop to the “open” conformation and facilitate product release
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