The use of cellulases remains a major cost in the production of renewable fuels and chemicals from lignocellulosic biomass. Fungi secrete copper-dependent polysaccharide monooxygenases (PMOs) that ...oxidatively cleave crystalline cellulose and improve the effectiveness of cellulases. However, the means by which PMOs recognize and cleave their substrates in the plant cell wall remain unclear. Here, we present structures of Neurospora crassa PMO-2 and PMO-3 at 1.10 and 1.37 Å resolution, respectively. In the structures, dioxygen species are found in the active sites, consistent with the proposed cleavage mechanism. Structural and sequence comparisons between PMOs also reveal that the enzyme substrate-binding surfaces contain highly varied aromatic amino acid and glycosylation positions. The structures reported here provide evidence for a wide range of PMO substrate recognition patterns in the plant cell wall, including binding modes that traverse multiple glucan chains.
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► 1.1/1.37 Å polysaccharide monooxygenases structures have active-site dioxygen species ► The structures reveal a large variance in substrate binding surfaces ► Proposed electron transport pathways in polysaccharide monooxygenases are conserved ► Polysaccharide monooxygenases and CBM33 enzymes likely bind substrates differently
Fusion of intracellular transport vesicles requires soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18-family (SM) proteins. Membrane-bridging SNARE ...complexes are critical for fusion, but their spontaneous assembly is inefficient and may require SM proteins in vivo. We report x-ray structures of Vps33, the SM subunit of the yeast homotypic fusion and vacuole protein–sorting (HOPS) complex, bound to two individual SNAREs. The two SNAREs, one from each membrane, are held in the correct orientation and register for subsequent complex assembly. Vps33 and potentially other SM proteins could thus act as templates for generating partially zipped SNARE assembly intermediates. HOPS was essential to mediate SNARE complex assembly at physiological SNARE concentrations. Thus, Vps33 appears to catalyze SNARE complex assembly through specific SNARE motif recognition.
Polysaccharide monooxygenases (PMOs), also known as lytic PMOs (LPMOs), enhance the depolymerization of recalcitrant polysaccharides by hydrolytic enzymes and are found in the majority of ...cellulolytic fungi and actinomycete bacteria. For more than a decade, PMOs were incorrectly annotated as family 61 glycoside hydrolases (GH61s) or family 33 carbohydrate-binding modules (CBM33s). PMOs have an unusual surface-exposed active site with a tightly bound Cu(II) ion that catalyzes the regioselective hydroxylation of crystalline cellulose, leading to glycosidic bond cleavage. The genomes of some cellulolytic fungi contain more than 20 genes encoding cellulose-active PMOs, suggesting a diversity of biological activities. PMOs show great promise in reducing the cost of conversion of lignocellulosic biomass to fermentable sugars; however, many questions remain about their reaction mechanism and biological function. This review addresses, in depth, the structural and mechanistic aspects of oxidative depolymerization of cellulose by PMOs and considers their biological function and phylogenetic diversity.
Markerless motion capture using deep learning approaches have potential to revolutionize the field of biomechanics by allowing researchers to collect data outside of the laboratory environment, yet ...there remain questions regarding the accuracy and ease of use of these approaches. The purpose of this study was to apply a markerless motion capture approach to extract lower limb angles in the sagittal plane during the vertical jump and to evaluate agreement between the custom trained model and gold standard motion capture. We performed this study using a large open source data set (N = 84) that included synchronized commercial video and gold standard motion capture. We split these data into a training set for model development (n = 69) and test set to evaluate capture performance relative to gold standard motion capture using coefficient of multiple correlations (CMC) (n = 15). We found very strong agreement between the custom trained markerless approach and marker-based motion capture within the test set across the entire movement (CMC > 0.991, RMSE < 3.22°), with at least strong CMC values across all trials for the hip (0.853 ± 0.23), knee (0.963 ± 0.471), and ankle (0.970 ± 0.055). The strong agreement between markerless and marker-based motion capture provides evidence that markerless motion capture is a viable tool to extend data collection to outside of the laboratory. As biomechanical research struggles with representative sampling practices, markerless motion capture has potential to transform biomechanical research away from traditional laboratory settings into venues convenient to populations that are under sampled without sacrificing measurement fidelity.
This article assesses the effects of Tulsa, Oklahoma's early childhood education programs on social-emotional outcomes, examining teacher ratings of children's behavior from the Adjustment Scales for ...Preschool Intervention and a measure of attentiveness using fixed effects regressions with propensity score matching. The sample includes 2,832 kindergarten students in 2006, of whom 1,318 participated in the Tulsa Public Schools (TPS) pre-K program and 363 participated in the CAP of Tulsa County Head Start program the previous year. Program participation was associated with lower timidity and higher attentiveness for TPS pre-K alumni and a marginally significant reduction in timidity for Head Start alumni. Results were similar for the free luncheligible subsample. We conclude that high-quality, school-based preschool programs can enhance social-emotional development.
As states have upgraded their commitment to pre-K education over the past two decades, questions have arisen. Critics argue that program effects are likely to fade out or disappear over time, while ...supporters contend that program effects are likely to persist under certain conditions. Using data from Tulsa Public Schools, three neighboring school districts, and the state of Oklahoma, and propensity score weighting, we estimate the effects of Tulsa's universal, school-based pre-K program on multiple measures of academic progress for middle school students. We find enduring effects on math achievement test scores, enrollment in honors courses, and grade retention for students as a whole, and similar effects for certain subgroups. We conclude that some positive effects of a high-quality pre-K program are discernible as late as middle school.
The ubiquitous fungal polysaccharide monooxygenases (PMOs) (also known as GH61 proteins, LPMOs, and AA9 proteins) are structurally related but have significant variation in sequence. A ...heterologous expression method in Neurospora crassa was developed as a step toward connecting regioselectivity of the chemistry to PMO phylogeny. Activity assays, as well as sequence and phylogenetic analyses, showed that the majority of fungal PMOs fall into three major groups with distinctive active site surface features. PMO1s and PMO2s hydroxylate glycosidic positions C1 and C4, respectively. PMO3s hydroxylate both C1 and C4. A subgroup of PMO3s (PMO3*) hydroxylate C1. Mutagenesis studies showed that an extra subdomain of about 12 amino acids contribute to C4 oxidation in the PMO3 family.
Fungal-derived, copper-dependent polysaccharide monooxygenases (PMOs), formerly known as GH61 proteins, have recently been shown to catalyze the O2-dependent oxidative cleavage of recalcitrant ...polysaccharides. Different PMOs isolated from Neurospora crassa were found to generate oxidized cellodextrins modified at the reducing or nonreducing ends upon incubation with cellulose and cellobiose dehydrogenase. Here we show that the nonreducing end product formed by an N. crassa PMO is a 4-ketoaldose. Together with isotope labeling experiments, further support is provided for a mechanism involving oxygen insertion and subsequent elimination to break glycosidic bonds in crystalline cellulose.
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