Heterologous expression and characterization of a MoAA16 polysaccharide monooxygenase from the rice blast fungus Magnaporthe oryzae

Background: Cellulose is an organic carbon source that can be used as a sustainable alternative for energy, materials, and chemicals. However, the substantial challenge of converting it into soluble sugars remains a major obstacle in its use as a biofuel and chemical feedstock. A new class of enzymes knowns as copper-dependent polysaccharide monooxygenases (PMOs) or lytic polysaccharide monooxygenases (LPMOs) can break down polysaccharides such as cellulose, chitin, and starch through oxidation. This process enhances the efficiency of cellulose degradation by cellulase. Results: The genome of the fungus Magnaporthe oryzae, the causal agent of rice blast disease, contains the MGG_00245 gene, which encodes a putative PMO referred to as MoAA16. MoAA16 has been found to be highly expressed in planta during the early stages of fungal infection. The gene was optimized for heterologous expression in Pichia pastoris, and its oxidative cleavage activity on cellulose was characterized by analyzing soluble oligosaccharide products using highperformance anion exchange chromatography (HPAEC-PAD). The reaction catalyzed by MoAA16 requires 2 electrons from an electron donor, such as ascorbic acid, and aerobic conditions. It primarily produces Glc1 to Glc4 oligosaccharides, as well as oxidized cellobionic and cellotrionic acids. MoAA16 has been observed to enhance cellulase hydrolysis on phosphoric acid swollen cellulose (PASC) substrate, resulting in the production of more monosaccharide products. Conclusions: Our findings reveal the successful heterologous expression of MoAA16 in P. pastoris and its cellulose-active PMO properties. These results highlight the potential of MoAA16 as a promising candidate for applications in biofuel production and chemical synthesis. How to cite: Nguyen HM, Le LQ, Sella L, et al. Heterologous expression and characterization of a MoAA16 polysaccharide monooxygenase from the rice blast fungus Magnaporthe oryzae. Electron J Biotechnol 2023. https://doi.org/10.1016/j.ejbt.2023.06.002

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

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

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

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

Longitudinal changes in pancreatic and adipocyte hormones following Roux-en-Y gastric bypass surgery

AIMS/HYPOTHESIS: Bariatric surgery is an effective treatment for severe obesity, as in addition to dramatic weight loss, co-morbidities such as type 2 diabetes are frequently resolved. Although altered gastrointestinal peptide hormone secretion and its relationship with post-surgical improvements in insulin sensitivity has been studied, much less is known about long-term changes in pancreatic and adipose tissue-derived hormones. Our objective was to conduct a comprehensive longitudinal investigation of the endocrine changes following Roux-en-Y gastric bypass surgery (RYGBP), focusing on pancreatic and adipocyte hormones and systemic markers of inflammation. METHODS: Nineteen severely obese women (BMI 45.6± 1.6 kg/m(2)) were studied prior to RYGBP, and at 1, 3, 6, and 12 months after RYGBP. Body composition was assessed before surgery and at 1 and 12 months. RESULTS: Pre-surgical adiposity was correlated with circulating adipocyte hormones (leptin, visfatin) and inflammatory molecules (IL-6, high sensitivity C-reactive protein [hsCRP], monocyte chemoattractant protein-1). As expected, RYGBP reduced fat mass and fasting insulin and glucose concentrations. In addition, reductions of fasting pancreatic polypeptide (PP) and glucagon concentrations were observed at 1 and 3 months, respectively. In the 12 months following RYGBP, concentrations of most adipocyte hormones (leptin, acylation-stimulating hormone and visfatin, but not retinol-binding hormone-4) and inflammatory molecules (IL-6, hsCRP and soluble intracellular adhesion molecule-1) were significantly reduced. Reductions of insulin resistance (measured by homeostasis model assessment of insulin resistance) were independently associated with changes of glucagon, visfatin and PP. Pre-surgical HMW adiponectin concentrations independently predicted losses of body weight and fat mass. CONCLUSIONS/INTERPRETATION: These results suggest that pancreatic and adipocyte hormones may contribute to the long-term resolution of insulin resistance after RYGBP