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Eco-friendly analogs of Trichogin GA IV, a short peptaibol produced by Trichoderma longibrachiatum, were assayed against Pyricularia oryzae, the causal agent of rice blast disease. In vitro and in vivo screenings allowed us to identify six peptides able to reduce by about 70% rice blast symptoms. One of the most active peptides was selected for further studies. Microscopy analyses highlighted that the treated fungal spores could not germinate and the fluorescein-labeled peptide localized on the spore cell wall and in the agglutinated cytoplasm. Transcriptomic analysis was carried out on P. oryzae mycelium 3 h after the peptide treatment. We identified 1,410 differentially expressed genes, two-thirds of which upregulated. Among these, we found genes involved in oxidative stress response, detoxification, autophagic cell death, cell wall biogenesis, degradation and remodeling, melanin and fatty acid biosynthesis, and ion efflux transporters. Molecular data suggest that the trichogin analogs cause cell wall and membrane damages and induce autophagic cell death. Ultrastructure observations on treated conidia and hyphae confirmed the molecular data. In conclusion, these selected peptides seem to be promising alternative molecules for developing effective bio-pesticides able to control rice blast disease.

Amyloid beta (Aβ) peptides are characterized as the major factors associated with neuron death in Alzheimer’s disease, which is listed as the most common form of neurodegeneration. Disordered Aβ peptides are released from proteolysis of the amyloid precursor protein. The Aβ self-assembly process roughly takes place via five steps: disordered forms → oligomers → photofibrils → mature fibrils → plaques. Although Aβ fibrils are often observed in patient brains, oligomers were recently indicated to be major neurotoxic elements. In this work, the neurotoxic compound S-shape Aβ11–42 tetramer (S4Aβ11–42) was investigated over 10 μs of unbiased MD simulations. In particular, the S4Aβ11–42 oligomer adopted a high dynamics structure, resulting in unsuccessful determination of their structures in experiments. The C-terminal was suggested as the possible nucleation of the Aβ42 aggregation. The sequences 27–35 and 39–40 formed rich β-content, whereas other residues mostly adopted coil structures. The mean value of the β-content over the equilibrium interval is ∼42 ± 3%. Furthermore, the dissociation free energy of the S4Aβ11–42 peptide was predicted using a biased sampling method. The obtained free energy is ΔGUS = −58.44 kcal/mol which is roughly the same level as the corresponding value of the U-shape Aβ17-42 peptide. We anticipate that the obtained S4Aβ11–42 structures could be used as targets for AD inhibitor screening over the in silico study.

By 2030, global rice production will need to increase to meet the demand of the growing world population. However, rice is severely affected by blast disease caused by the fungus Pyricularia oryzae, which accounts for 10–30% yield losses per year globally, thus posing a threat to the world’s most important food crop. The fungus can infect all parts of the rice plant, including leaves, nodes and panicles. In the early stages of the infection process, P. oryzae forms an infection structure called appressorium to break the plant cuticle. After an initial biotrophic phase, the fungus kills plant cells thus leading to visible symptoms. Currently, several methods for the management of rice blast disease, such as agronomic practices, use of resistant cultivars and synthetic fungicides, are available and can be exploited in an integrated management approach. However, despite some resistant rice varieties that have been developed by breeding programs, P. oryzae is able to rapidly develop new races that overcome resistance genes. Furthermore, the continuous use of fungicides may be harmful to humans and environment and can increase the risk of appearance of resistant fungal races. Biological control using microbial agents or plant extracts with antimicrobial activity is therefore considered a possible alternative and sustainable approach to control rice blast disease. However, biological protection is difficult to achieve in the field as its effectiveness is variable, highly depending on formulations and climatic conditions. Because of these drawbacks, durable control of rice blast still represents a substantial challenge. Innovative strategies for the identification of new molecules for the sustainable protection of rice are thus highly desirable.

African swine fever (ASF) is a highly infectious viral disease with high mortality. The most recent ASF outbreak in Vietnam began in 2019, posing a threat to spread to the neighbouring Asian countries. Without a commercial vaccine or efficient chemotherapeutics, rapid diagnosis and necessary biosecurity procedures are required to control the disease. While the diagnostic method of ASF recommended by the World Organization of Animal Health is real-time PCR, the ideal diagnosis procedure including master mix setup, template extraction and a high-cost qPCR equipment for many samples being tested simultaneously is not portable. In this study, a colorimetric loop-mediated isothermal amplification (LAMP) assay was modified and evaluated for ASF virus detection using crude serum samples collected from domestic pigs in Vietnam during the 2019 outbreak. The LAMP results can be readily visualized to the naked eye within 30 min without the requirement of DNA extraction and sophisticated equipment. The sensitivity, specificity and limit of detection of direct colorimetric LAMP assay were comparable to a commercial diagnostic real-time PCR kit. Results strongly indicate that the adapted colorimetric LAMP assay has a remarkable potential for the in-field diagnosis of ASF.

African Swine Fever (ASF) is a highly infectious viral disease with high mortality. The most recent ASF outbreak in Vietnam occurred in 2019, posing a threat to spread to the neighboring Asian countries. Without a commercial vaccine or efficient chemotherapeutics successfully developed, rapid diagnosis and necessary biosecurity procedures are required to control the disease. While the diagnosis method of ASF recommended by the World Organization of Animal Health is real-time PCR, it is not suitable for in-field detection of the disease. In this study, a colorimetric Loop-Mediated Isothermal Amplification (LAMP) assay was developed and evaluated for ASF virus detection using crude serum samples collected from domestic pigs in Vietnam during the 2019 outbreak. The LAMP results can be readily visualized to naked eyes within 30 minutes without the requirement of DNA extraction and sophisticated equipment. The sensitivity, specificity, and limit of detection of colorimetric LAMP assay were comparable to a commercial diagnostic real-time PCR kit. Results strongly indicate that the developed colorimetric LAMP assay is highly recommended for the in-field diagnosis of ASF.

The COVID-19, caused by the novel coronavirus SARS-CoV-2, has broken out of control all over the globe and put the majority of the world under lockdown. There have been no specific antiviral medications for SARS-CoV-2 while vaccines are still under development. Thus, rapid diagnosis and necessary public health measures are currently key parts to contain the pandemic. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the gold standard method for SARS-CoV-2 detection. However, this method is not suitable for point-of-care (POC) diagnosis because of the timeconsuming procedure, the requirements of biosafety conditions and expensive equipment. In this study, the colorimetric isothermal nucleic acid amplification tests (iNAATs) for SARS-CoV-2 based on loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and polymerase spiral reaction (PSR) were developed and compared. The three methods exhibited similar performance with the limit of detection (LOD) as low as just 1 copy per reaction when evaluated on the synthetic DNA fragments. The results can be read with naked eyes within 30 minutes without crossreactivity to closely related coronaviruses. When tested with SARS-CoV-2 extracted genomic-RNA, LAMP outperformed both CPA and PSR assays. Moreover, the direct detection of SARS-CoV-2 in simulated patient samples (oropharyngeal and nasopharyngeal swabs) by colorimetric iNAATs was also successful. Further preparation of the lyophilized reagents for LAMP reactions revealed that the freeze-dried, ready-to-use kit maintained the sensitivity and LOD value of the liquid assays. These results strongly indicate that the colorimetric lyophilized LAMP test kit developed herein is highly suitable for detecting SARS-CoV-2 at POC.

X-ray absorption spectroscopy (XAS) was utilized to gain insights into the structure and electronic properties of the reduced copper active site in NCU08746, a polysaccharide monooxygenase (PMO) from Neurospora crassa that activates O2 to cleave glycosidic linkages in starch. The reaction of NCU08746 likely starts with binding of O2 to the copper(I) center. However, the solution structure of the reduced active site in NCU08746 has not been properly elucidated. In this study, we prepared Cu(I)-NCU08746 in solution, which was snap-frozen to preserve the solution structure of the copper(I) active site prior to XAS analysis. Results show that the copper(I) center in Cu(I)-NCU08746 exhibits a 4-coordinate geometry, which is different from the 3-coordinate geometry observed for some other PMOs. This difference likely arises from the coordination of the active site tyrosine residue and could contribute to the difference in activity between NCU08746 and other PMOs.

The increasing demand for the next generation of renewable fuels has driven extensive research in carbohydrate-active enzymes involved in converting plant biomass to fermentable sugars. Standing out of new enzymes discovered recently are polysaccharide monooxygenases (PMOs) that activate O2 with a Type 2 mono copper active site for the regioselective hydroxylation of the glycosidc linkages. This hydroxylation leads to the cleavage of glycosidic linkage on the substrate surfaces, creating new chain ends on the substrate surface that can be further processed by canonical glycoside hydrolases. PMOs can thus significantly boost the activity of industrial cellulases and have great potentials in the biofuel industry. Extensive research in the last 8 years have shed lights into the nature of the copper active site and how it activates O2. While other aspects of PMOs have been thoroughly discussed in recent outstanding reviews, the coordination chemistry aspects of the copper active site have not been reviewed in details. This article will provide in-depth analysis of the copper active site and surrounding residues, including structure, electronic and spectroscopic properties, copper-binding affinity, redox properties, modulation of geometry and activity by second-sphere residues, substrate binding affinity, O2 intermediates, and initial mechanistic insights, as well as some relevant model complexes. This review will also identify some key areas that need further work and predict the trend in PMOs research in the coming years.

The self-assembly of Amyloid beta (Aβ) peptides are widely accepted to associate with Alzheimer's disease (AD) via several proposed mechanisms. Because Aβ oligomers exist in a complicated environment consisting of various forms of Aβ, including oligomers, protofibrils, and fibrils, their structure has not been well understood. The negatively charged residue D23 is one of the critical residues of the Aβ peptide as it is located in the central hydrophobic domain of the Aβ N-terminal and forms a salt-bridge D23-K28, which helps stabilize the loop domain. In the familial Iowa (D23N) mutant, the total net charge of Aβ oligomers decreases, resulting in the decrease of electrostatic repulsion between D23N Aβ monomers and thus the increase in their self-aggregation rate. In this work, the impact of the D23N mutation on 3Aβ11–40 trimer was characterized utilizing temperature replica exchange molecular dynamics (REMD) simulations. Our simulation reveals that D23N mutation significantly enhances the affinity between the constituting chains in the trimer, increases the β-content (especially in the sequence 21–23), and shifts the β-strand hydrophobic core from crossing arrangement to parallel arrangement, which is consistent with the increase in self-aggregation rate. Molecular docking indicates that the Aβ fibril-binding ligands bind to the D23N and WT forms at different poses. These compounds prefer to bind to the N-terminal β-strand of the D23N mutant trimer, while they mostly bind to the N-terminal loop region of the WT. It is important to take into account the difference in the binding of ligands to mutant and wild type Aβ peptides in designing efficient inhibitors for various types of AD.

Type 2 copper active sites, one of the several important copper active sites in biology, were recently found in the novel superfamily of polysaccharide monooxygenases (PMOs) that cleave recalcitrant polysaccharides via an unprecedented oxidative mechanism. The copper center in PMOs is ligated by the bidentate N-terminal histidine residue and another conserved histidine residue, forming a unique T-shaped core termed as

Degradation of polysaccharides is central to numerous biological and industrial processes. Starch-active polysaccharide monooxygenases (AA13 PMOs) oxidatively degrade starch and can potentially be used with industrial amylases to convert starch into a fermentable carbohydrate. The oxidative activities of the starch-active PMOs from the fungi Neurospora crassa and Myceliophthora thermophila, NcAA13 and MtAA13, respectively, on three different starch substrates are reported here. Using high-performance anion-exchange chromatography coupled with pulsed amperometry detection, we observed that both enzymes have significantly higher oxidative activity on amylose than on amylopectin and cornstarch. Analysis of the product distribution revealed that NcAA13 and MtAA13 more frequently oxidize glycosidic linkages separated by multiples of a helical turn consisting of six glucose units on the same amylose helix. Docking studies identified important residues that are involved in amylose binding and suggest that the shallow groove that spans the active-site surface of AA13 PMOs favors the binding of helical amylose substrates over nonhelical substrates. Truncations of NcAA13 that removed its native carbohydrate-binding module resulted in diminished binding to amylose, but truncated NcAA13 still favored amylose oxidation over other starch substrates. These findings establish that AA13 PMOs preferentially bind and oxidize the helical starch substrate amylose. Moreover, the product distributions of these two enzymes suggest a unique interaction with starch substrates.

Alzheimer's disease (AD) is associated with the oligomerization and/or fibrillation of amyloid beta (Aβ) peptides, which cause damage to brain cells. Aβ oligomers and fibrils contain hydrophobic cores formed with parallel beta sheets. Mutations of F19, a residue in the hydrophobic core of Aβ peptides, slow down their aggregation process but do not alter the overall structure of the resulting fibrils. However, the effects of F19 mutations on the toxic Aβ oligomers have not been elucidated. We studied the F19W mutant of the 11–40 truncated Aβ trimer (F19W 3Aβ11–40) using replica exchange molecular dynamics (REMD) simulations. While most structural terms do not change significantly, critical polar contacts decrease by 20%, and notably, RMSD almost doubles upon F19W mutation. Six minima were found in the free energy surface of F19W 3Aβ11–40, which have lower energy barriers (by ∼1 kJ mol−1) and significantly lower total population (∼20%) compared to those of the three minima found for 3Aβ11–40 (∼60%). The binding free energy between constituting chains of the mutant trimer increases by ∼28 kcal mol−1 but fluctuates significantly (±27.1 kcal mol−1). Our results indicate that while the hydrophobic core of amyloid beta peptide is capable of adapting to structural changes, F19W mutation results in a significantly more flexible trimer. The more flexible F19W mutant oligomers would require more time to self-assemble into fibrils. Our results contribute to a better understanding of the behavior of Aβ peptides and their oligomerization/aggregation process, which is necessary to understand AD pathogenesis.

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.

Oligomerization of amyloid beta (Aβ) peptides has been considered as the crucially causative agent in the development of Alzheimer's disease. Etersalate, a nonsteroidal anti-inflammatory oral drug (United State Food and Drug Administration-Unique Ingredient Identifier: 653GN04T2G) was previously suggested to bind well to proto-fibrils of Aβ peptides in silico. Here, the effect of etersalate on the oligomerization of soluble Aβ16-22 hexamer (6Aβ16-22) were extensively investigated using temperature replica exchange molecular dynamics (REMD) simulations over ~16.8 μs in total for 48 replicas (350 ns per replica). The results reveal that etersalate can enter the inner space or bind on the surface of 6Aβ16-22 conformations, which destabilizes the hexamer. Etersalate was predicted to able to cross the blood brain barrier using prediction of absorption, distribution, metabolism, and excretion-toxicity (preADMET) tools. Overall, although the investigation was performed with the low concentration of trial inhibitor, the obtained results indicate that etersalate is a potential drug candidate for AD through inhibiting formation of Aβ oligomers with the average binding free energy of -11.7 kcal/mol.

Familial Alzheimer’s disease (FAD) is passed down in family, which account for 2–3% of about 40 million AD cases worldwide. The Flemish (A21G) mutant of amyloid β (Aβ) exhibits unique properties among all hereditary mutants of FAD, including the lowest aggregation rate. Recent studies showed that Aβ oligomers play a key role in Alzheimer’s disease (AD) pathogenesis. They could insert themselves in brain cell membrane, disrupting the membrane integrity and ion homeostasis. However, experimental studies of transmembrane Aβ oligomers have been limited due to their intrinsic heterogeneity. In this work, we extensively studied the A21G mutant of the transmembrane 3Aβ11–40 (A21G 3Aβ11–40) using temperature replica exchange molecular dynamics (REMD) simulations. Results provide detailed information on the conformational distribution and dynamics of transmembrane A21G 3Aβ11–40. Minimal local change from A to G leads to significant conformational changes and wider free energy holes on the free energy surface as we...

Human deoxyhypusine hydroxylase (hDOHH) is an enzyme that is involved in the critical post-translational modification of the eukaryotic translation initiation factor 5A (eIF5A). Following the conversion of a lysine residue on eIF5A to deoxyhypusine (Dhp) by deoxyhypusine synthase, hDOHH hydroxylates Dhp to yield the unusual amino acid residue hypusine (Hpu), a modification that is essential for eIF5A to promote peptide synthesis at the ribosome, among other functions. Purification of hDOHH overexpressed in E. coli affords enzyme that is blue in color, a feature that has been associated with the presence of a peroxo-bridged diiron(III) active site. To gain further insight into the nature of the diiron site and how it may change as hDOHH goes through the catalytic cycle, we have conducted X-ray absorption spectroscopic studies of hDOHH on five samples that represent different species along its reaction pathway. Structural analysis of each species has been carried out, starting with the reduced diferrous state, proceeding through its O2 adduct, and ending with a diferric decay product. Our results show that the Fe•••Fe distances found for the five samples fall within a narrow range of 3.4–3.5 Å, suggesting that hDOHH has a fairly constrained active site. This pattern differs significantly from what has been associated with canonical dioxygen activating nonheme diiron enzymes such as soluble methane monooxygenase and Class 1A ribonucleotide reductases, for which the Fe•••Fe distance can change by as much as 1 Å during the redox cycle. These results suggest that the O2 activation mechanism for hDOHH deviates somewhat from that associated with the canonical nonheme diiron enzymes, opening the door to new mechanistic possibilities for this intriguing family of enzymes.

Deoxyhypusine hydroxylase is the key enzyme in the biosynthesis of hypusine containing eukaryotic translation initiation factor 5A (eIF5A), which plays an essential role in the regulation of cell proliferation. Recombinant human deoxyhypusine hydroxylase (hDOHH) has been reported to have oxygen- and iron-dependent activity, an estimated iron/holoprotein stoichiometry of 2, and a visible band at 630 nm responsible for the blue color of the as-isolated protein. EPR, Mössbauer, and XAS spectroscopic results presented herein provide direct spectroscopic evidence that hDOHH has an antiferromagnetically coupled diiron center with histidines and carboxylates as likely ligands, as suggested by mutagenesis experiments. Resonance Raman experiments show that its blue chromophore arises from a (μ-1,2-peroxo)diiron(III) center that forms in the reaction of the reduced enzyme with O 2 , so the peroxo form of hDOHH is unusually stable. Nevertheless we demonstrate that it can carry out the hydroxylation of the deoxyhypusine residue present in the elF5A substrate. Despite a lack of sequence similarity, hDOHH has a nonheme diiron active site that resembles both in structure and function those found in methane and toluene monooxygenases, bacterial and mammalian ribonucleotide reductases, and stearoyl acyl carrier protein Δ 9 -desaturase from plants, suggesting that the oxygen-activating diiron motif is a solution arrived at by convergent evolution. Notably, hDOHH is the only example thus far of a human hydroxylase with such a diiron active site.

Streptomyces venezuelae CmlI catalyzes the six-electron oxygenation of the arylamine precursor of chloramphenicol in a nonribosomal peptide synthetase (NRPS)-based pathway to yield the nitroaryl group of the antibiotic. Optical, EPR, and Mössbauer studies show that the enzyme contains a nonheme dinuclear iron cluster. Addition of O(2) to the diferrous state of the cluster results in an exceptionally long-lived intermediate (t(1/2) = 3 h at 4 °C) that is assigned as a peroxodiferric species (CmlI-peroxo) based upon the observation of an (18)O(2)-sensitive resonance Raman (rR) vibration. CmlI-peroxo is spectroscopically distinct from the well characterized and commonly observed cis-μ-1,2-peroxo (μ-η(1):η(1)) intermediates of nonheme diiron enzymes. Specifically, it exhibits a blue-shifted broad absorption band around 500 nm and a rR spectrum with a ν(O-O) that is at least 60 cm(-1) lower in energy. Mössbauer studies of the peroxo state reveal a diferric cluster having iron sites with small quadrupole splittings and distinct isomer shifts (0.54 and 0.62 mm/s). Taken together, the spectroscopic comparisons clearly indicate that CmlI-peroxo does not have a μ-η(1):η(1)-peroxo ligand; we propose that a μ-η(1):η(2)-peroxo ligand accounts for its distinct spectroscopic properties. CmlI-peroxo reacts with a range of arylamine substrates by an apparent second-order process, indicating that CmlI-peroxo is the reactive species of the catalytic cycle. Efficient production of chloramphenicol from the free arylamine precursor suggests that CmlI catalyzes the ultimate step in the biosynthetic pathway and that the precursor is not bound to the NRPS during this step.

The recently discovered fungal and bacterial polysaccharide monooxygenases (PMOs) are capable of oxidatively cleaving chitin, cellulose, and hemicelluloses that contain β(1→4) linkages between glucose or substituted glucose units. They are also known collectively as lytic PMOs, or LPMOs, and individually as AA9 (formerly GH61), AA10 (formerly CBM33), and AA11 enzymes. PMOs share several conserved features, including a monocopper center coordinated by a bidentate N-terminal histidine residue and another histidine ligand. A bioinformatic analysis using these conserved features suggested several potential new PMO families in the fungus Neurospora crassa that are likely to be active on novel substrates. Herein, we report on NCU08746 that contains a C-terminal starch-binding domain and an N-terminal domain of previously unknown function. Biochemical studies showed that NCU08746 requires copper, oxygen, and a source of electrons to oxidize the C1 position of glycosidic bonds in starch substrates, but not in cellulose or chitin. Starch contains α(1→4) and α(1→6) linkages and exhibits higher order structures compared with chitin and cellulose. Cellobiose dehydrogenase, the biological redox partner of cellulose-active PMOs, can serve as the electron donor for NCU08746. NCU08746 contains one copper atom per protein molecule, which is likely coordinated by two histidine ligands as shown by X-ray absorption spectroscopy and sequence analysis. Results indicate that NCU08746 and homologs are starch-active PMOs, supporting the existence of a PMO superfamily with a much broader range of substrates. Starch-active PMOs provide an expanded perspective on studies of starch metabolism and may have potential in the food and starch-based biofuel industries.

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.

X-ray absorption and resonance Raman spectroscopies show that CmlA, the β-hydroxylase of the chloramphenicol biosynthetic pathway, contains a (μ-oxo)-(μ-1,3-carboxylato)diiron(III) cluster with 6-coordinate iron centers and 3 – 4 His ligands. This active site is found within a unique β-lactamase fold and is distinct from those of soluble methane monooxygenase and related enzymes that utilize a highly conserved diiron cluster with a 2-His-4-carboxylate ligand set within a 4-helix bundle motif. These structural differences may have an impact on the nature of the activated oxygen species of the reaction cycle.