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Wireworm, the larval stages of click beetles, are a serious pest of tubers, brassicas and other important commercial crops throughout the northern hemisphere. No effective control agent has been developed specifically for them, and many of the pesticides marketed as having secondary application against them have been withdrawn from EU and Asian markets. Metarhizium brunneum, an effective entomopathogenic fungus, and its derived volatile metabolites are known to be effective plant biostimulants and plant protectants, although field efficacy has yet to be validated. Field validation of a combined M. brunneum and derived VOC treatments was conducted in Wales, UK, to assess the effects of each as a wireworm control agent and biostimulant. Plots were treated with Tri-Soil (Trichoderma atroviridae), M. brunneum, 1-octen-3-ol or 3-octanone, or combinations thereof. Treatments were applied subsurface during potato seeding (n = 52), and potatoes were harvested at the end of the growing season. Each potato was weighed individually and scored for levels of wireworm damage. Applications of both the VOCs and the M. brunneum individually were found to significantly decrease wireworm burden (p < 0.001). Combinations of M. brunneum and 3-octanone were also found to significantly decrease wireworm damage (p < 0.001), while no effect on yield was reported, resulting in an increased saleable mass over controls (p < 0.001). Herein, we present a novel 'stimulate and deter' wireworm control strategy that can be used to significantly enhance saleable potato yields and control wireworm populations, even under high pest pressure densities. [ABSTRACT FROM AUTHOR]

Metarhizium brunneum is a highly effective entomopathogenic fungus that also functions as a plant biostimulant. It can act as both an endophyte and rhizosphere colonizer; however, the mechanisms driving biostimulation are multifactorial. In this work, oilseed rape (Brassica napus) seeds were grown in composts treated with different concentrations of M. brunneum strains ARSEF 4556 or V275, or the M. brunneum-derived volatile organic compounds 1-octen-3-ol and 3-octanone. Biostimulation efficacy was found to be strongly dose dependent. Concentrations of 1 × 106 conidia g−1 compost were found to be most effective for the M. brunneum, whereas dosages of 1 µL 100 g−1 compost were found to be efficacious for the volatiles. These optimized doses were assessed individually and in combined formulations with a hydrogel against oilseed rape (Brassica napus), sitka spruce (Picea sitchensis), maize (Zea mays) and strawberry (Fragaria annanassa). Both volatile compounds were highly effective biostimulants and were found to increase in biostimulatory efficiency when combined with M. brunneum conidia. Hydrogels were not found to interact with the growth process and may offer avenues for novel formulation technologies. This study demonstrates that Metarhizium-derived volatile organic compounds are actively involved in plant growth promotion and have potential for use in novel formulations to increase the growth of a wide range of commercially relevant crops. [ABSTRACT FROM AUTHOR]

Plastics have become ubiquitous in both their adoption as materials and as environmental contaminants. Widespread pollution of these versatile, man‐made and largely petroleum‐derived polymers has resulted from their long‐term mass production, inappropriate disposal and inadequate end of life management. Polyethylene (PE) is at the forefront of this problem, accounting for one‐third of plastic demand in Europe in part due to its extensive use in packaging. Current recycling and incineration processes do not represent sustainable solutions to tackle plastic waste, especially once it becomes littered, and the development of new waste‐management and remediation technologies are needed. Mycoremediation (fungal‐based biodegradation) of PE has been the topic of several studies over the last two decades. The utility of these studies is limited by an inconclusive definition of biodegradation and a lack of knowledge regarding the biological systems responsible. This review highlights relevant features of fungi as potential bioremediation agents, before discussing the evidence for fungal biodegradation of both high‐ and low‐density PE. An up‐to‐date perspective on mycoremediation as a future solution to PE waste is provided. [ABSTRACT FROM AUTHOR]

The role of the salt bridges in the dimerization and thermal stability of dihydrofolate reductase from the hyperthermophile Thermotoga maritima (TmDHFR) is studied by generating a series of variants in which the residues are exchanged for residues whose side chains bear the opposite charge. The results have shown that although the rate of dihydrofolate reduction by TmDHFR is not affected by the loss of the K129-E136-E138 salt bridges, changes to the temperature dependence of the kinetic isotope effect on hydride transfer are observed.

The article discusses the mechanisms, as well as the kinetics associated with the reaction catalyzed by dihydrofolate reductase obtained from Escherichia coli. The various effects of solvent composition on the temperature dependency of the various kinetic parameters are also explained.

The generation of a variant of dihydrofolate reductase (DHFR) from Thermotoga maritima, TmDHFR-V11D, in which a single amino acid replacement is sufficient to favor the monomeric form of the enzyme in the presence of the nondenaturing zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate is reported. The reduced catalytic activity of TmDHFR relative to the monomeric Escherichia coli enzyme is not caused by rigidity resulting from dimerization, but is a subtle consequence of the sequence and structure of its subunits, which appear to have evolved to permit thermostability at the expense of catalysis.

Solvent kinetic isotope effects are described for two dihydrofolate reductases, namely the monomers, mesophilic enzymes from Escherichia coli (EcDHFR) and the dimeric, thermophilic enzyme from Thermotoga maritime (TmDHFR). The reduced reaction rates of TmDHFR is due to its quaternary structure, which is needed for increased thermostability but which also prevents active modulation of the reactivity of the active site bound substrate observed in EcDHFR.

The role of protein motions in promoting the chemical step of enzyme catalysed reactions remains a subject of considerable debate. Here, a unified view of the role of protein dynamics in dihydrofolate reductase catalysis is described. Recently the role of such motions has been investigated by characterising the biophysical properties of isotopically substituted enzymes through a combination of experimental and computational analyses. Together with previous work, these results suggest that dynamic coupling to the chemical coordinate is detrimental to catalysis and may have been selected against during DHFR evolution. The full catalytic power of Nature's catalysts appears to depend on finely tuning protein motions in each step of the catalytic cycle. [ABSTRACT FROM AUTHOR]

Chemical ligation has been used to alter motions in specific regions of dihydrofolate reductase from E. coli and to investigate the effects of localized motional changes on enzyme catalysis. Two isotopic hybrids were prepared; one with the mobile N-terminal segment containing heavy isotopes (2H, 13C, 15N) and the remainder of the protein with natural isotopic abundance, and the other one with only the C-terminal segment isotopically labeled. Kinetic investigations indicated that isotopic substitution of the N-terminal segment affected only a physical step of catalysis, whereas the enzyme chemistry was affected by protein motions from the C-terminal segment. QM/MM studies support the idea that dynamic effects on catalysis mostly originate from the C-terminal segment. The use of isotope hybrids provides insights into the microscopic mechanism of dynamic coupling, which is difficult to obtain with other studies, and helps define the dynamic networks of intramolecular interactions central to enzyme catalysis. [ABSTRACT FROM AUTHOR]

Background Genetically customised Saccharomyces cerevisiae that can produce ethanol and additional bio-based chemicals from sustainable agro-industrial feedstocks (for example, residual plant biomass) are of major interest to the biofuel industry. We investigated the microbial biorefinery concept of ethanol and squalene co-production using S. cerevisiae (strain YUG37-ERG1) wherein ERG1 (squalene epoxidase) transcription is under the control of a doxycycline-repressible tet07-CYC1 promoter. The production of ethanol and squalene by YUG37-ERG1 grown using agriculturally sourced grass juice supplemented with doxycycline was assessed. Results Use of the tet07-CYC1 promoter permitted regulation of ERG1 expression and squalene accumulation in YUG37-ERG1, allowing us to circumvent the lethal growth phenotype seen when ERG1 is disrupted completely. In experiments using grass juice feedstock supplemented with 0 to 50 μg doxycycline mL-1, YUG37-ERG1 fermented ethanol (22.5 [±0.5] mg mL-1) and accumulated the highest squalene content (7.89 ± 0.25 mg g-1 dry biomass) and yield (18.0 ± 4.18 mg squalene L-1) with supplements of 5.0 and 0.025 μg doxycycline mL-1, respectively. Grass juice was found to be rich in water-soluble carbohydrates (61.1 [±3.6] mg sugars mL-1) and provided excellent feedstock for growth and fermentation studies using YUG37-ERG1. Conclusion Residual plant biomass components from crop production and rotation systems represent possible substrates for microbial fermentation of biofuels and bio-based compounds. This study is the first to utilise S. cerevisiae for the co-production of ethanol and squalene from grass juice. Our findings underscore the value of the biorefinery approach and demonstrate the potential to integrate microbial bioprocess engineering with existing agriculture. [ABSTRACT FROM AUTHOR]

Dihydrofolate reductase (DHFR) from the hyperthermophile Thermotoga maritima (TmDHFR) forms a very stable homodimer, while DHFRs from other organisms are monomers. We investigated the effect of dimerization on DHFR catalysis by preparing a dimeric variant, Xet-3, of DHFR from Escherichia coli (EcDHFR). Introducing residues located at the TmDHFR dimer interface into EcDHFR increases the melting temperature to ~60 °C, approximately 9 °C higher than that measured for EcDHFR The steady-state and pre-steady-state rate constants measured for Xet-3 were similar to those of dimeric TmDHFR but significantly lower than those of the parent EcDHFR. This reduction in the degree of catalytic competence is likely a consequence of the loss of flexibility of catalytically important loop regions of EcDHFR on dimerization and a compromise of the electrostatic environment of the active site. In contrast, the reduced catalytic ability of TmDHFR relative to that of EcDHFR is not simply a consequence of reduced loop flexibility in the dimeric enzyme. Our studies demonstrate that EcDHFR is not a good model for understanding the properties of other DHFRs, including TmDHFR [ABSTRACT FROM AUTHOR]

The question of whether protein motions play a role in the chemical step of enzymatic catalysis has generated much controversy in recent years. Debate has recently reignited over possible dynamic contributions to catalysis in dihydrofolate reductase, following conflicting conclusions from studies of the N23PP/S148A variant of the Escherichia coli enzyme. By investigating the temperature dependence of kinetic isotope effects, we present evidence that the reduction in the hydride transfer rate constants in this variant is not a direct result of impairment of conformational fluctuations. Instead, the conformational state of the enzyme immediately before hydride transfer, which determines the electrostatic environment of the active site, affects the rate constant for the reaction. Although protein motions are clearly important for binding and release of substrates and products, there appears to be no detectable dynamic coupling of protein motions to the hydride transfer step itself. [ABSTRACT FROM AUTHOR]

Dihydrofolate reductase has long been used as a model system to study the coupling of protein motions to enzymatic hydride transfer. By studying environmental effects on hydride transfer in dihydrofolate reductase (DHFR) from the cold-adapted bacterium Moritella profunda (MpDHFR) and comparing the flexibility of this enzyme to that of DHFR from Escherichia coli (EcDHFR), we demonstrate that factors that affect large-scale (i.e., long-range, but not necessarily large amplitude) protein motions have no effect on the kinetic isotope effect on hydride transfer or its temperature dependence, although the rates of the catalyzed reaction are affected. Hydrogen/deuterium exchange studies by NMR-spectroscopy show that MpDHFR is a more flexible enzyme than EcDHFR. NMR experiments with EcDHFR in the presence of cosolvents suggest differences in the conformational ensemble of the enzyme. The fact that enzymes from different environmental niches and with different flexibilities display the same behavior of the kinetic isotope effect on hydride transfer strongly suggests that, while protein motions are important to generate the reaction ready conformation, an optimal conformation with the correct electrostatics and geometry for the reaction to occur, they do not influence the nature of the chemical step itself; large-scale motions do not couple directly to hydride transfer proper in DHFR. [ABSTRACT FROM AUTHOR]

Post-translational glycosylation is one of the most abundant forms of covalent protein modification in eukaryotic cells. It plays an important role in determining the properties of proteins, and stabilizes many proteins against thermal denaturation. Protein glycosylation may establish a surface microenvironment that resembles that of unglycosylated proteins in concentrated solutions of sugars and other polyols. We have used site-directed mutagenesis to introduce a series of unique cysteine residues into a cysteine-free double mutant (DM, C85A/C152S) of dihydrofolate reductase from Escherichia coli (EcDHFR). The resulting triple mutants, DM-N18C, DM-R52C, DM-D87C and DM-D132C EcDHFR, were alkylated with glucose, N-acetylglucosamine, lactose and maltotriose iodoacetamides. We found little effect on catalysis or stability in three cases. However, when DM-D87C EcDHFR is glycosylated, stability is increased by between 1.5 and 2.6 kcal·mol−1 in a sugar-dependent manner. D87 is found in a hinge region of EcDHFR that loses structure early in the thermal denaturation process, whereas the other glycosylation sites are found in regions involved in the later stages of temperature-induced unfolding. Glycosylation at this site may improve the stability of EcDHFR by protecting a region of the enzyme that is particularly prone to denaturation. [ABSTRACT FROM AUTHOR]

Lytic transglycosylases such as Slt35 from E. coli are enzymes involved in bacterial cell wall remodelling and recycling, which represent potential targets for novel antibacterial agents. Here, we investigated a series of known glycosidase inhibitors for their ability to inhibit Slt35. While glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, thiamet G and miglitol had no effect, the phenothiazinium dye thionine acetate was found to be a weak inhibitor. IC50 values and binding constants for thionine acetate were similar for Slt35 and the hen egg white lysozyme. Molecular docking simulations suggest that thionine binds to the active site of both Slt35 and lysozyme, although it does not make direct interactions with the side-chain of the catalytic Asp and Glu residues as might be expected based on other inhibitors. Thionine acetate also increased the potency of the beta-lactam antibiotic ampicillin against a laboratory strain of E. coli. [ABSTRACT FROM AUTHOR]

Pseudomonas mesoacidophila ATCC 31433 is a Gram-negative bacterium, first isolated from Japanese soil samples, that produces the monobactam isosulfazecin and the ß-lactam-potentiating bulgecins. To characterize the biosynthetic potential of P. mesoacidophila ATCC 31433, its complete genome was determined using single-molecule real-time DNA sequence analysis. The 7.8-Mb genome comprised four replicons, three chromosomal (each encoding rRNA) and one plasmid. Phylogenetic analysis demonstrated that P. mesoacidophila ATCC 31433 was misclassified at the time of its deposition and is a member of the Burkholderia cepacia complex, most closely related to Burkholderia ubonensis. The sequenced genome shows considerable additional biosynthetic potential; known gene clusters for malleilactone, ornibactin, isosulfazecin, alkylhydroxyquinoline, and pyrrolnitrin biosynthesis and several uncharacterized biosynthetic gene clusters for polyketides, nonribosomal peptides, and other metabolites were identified. Furthermore, P. mesoacidophila ATCC 31433 harbors many genes associated with environmental resilience and antibiotic resistance and was resistant to a range of antibiotics and metal ions. In summary, this bioactive strain should be designated B. cepacia complex strain ATCC 31433, pending further detailed taxonomic characterization. [ABSTRACT FROM AUTHOR]

The Bcl-2 family of proteins includes the major regulators and effectors of the intrinsic apoptosis pathway. Cancers are frequently formed when activation of the apoptosis mechanism is compromised either by misregulated expression of prosurvival family members or, more frequently, by damage to the regulatory pathways that trigger intrinsic apoptosis. Short peptides derived from the pro-apoptotic members of the Bcl-2 family can activate mechanisms that ultimately lead to cell death. The recent development of photocontrolled peptides that are able to change their conformation and activity upon irradiation with an external light source has provided new tools to target cells for apoptosis induction with temporal and spatial control. Here, we report the first NMR solution structure of a photoswitchable peptide derived from the proapoptotic protein Bak in complex with the antiapoptotic protein Bcl-xL. This structure provides insight into the molecular mechanism, by which the increased affinity of such photopeptides compared to their native forms is achieved, and offers a rationale for the large differences in the binding affinities between the helical and nonhelical states. [ABSTRACT FROM AUTHOR]

Olfactory systems are indispensable for insects as they, including Western Flower Thrips ( Frankliniella occidentalis ), use olfactory cues for ovipositing and feeding. F. occidentalis use odorant binding proteins (OBPs) to transport semiochemicals to odorant receptors to induce a behavioural response from the sensillum lymph of the insect's antennae. This study identifies four OBPs of F. occidentalis and analyses their expression at three stages of growth: larvae, adult males and adult females. Further, it investigates the presence of conserved motifs and their phylogenetic relationship to other insect species. Moreover, FoccOBP3 was in silico characterized to analyse its structure along with molecular docking and molecular dynamics simulations to understand its binding with semiochemicals of F. occidentalis . Molecular docking revealed the interactions of methyl isonicotinate, p-anisaldehyde and (S)-(-)-verbenone with FoccOBP3 . Moreover, molecular dynamics simulations showed bonding stability of these ligands with FoccOBP3, and field trials validated that Lurem TR (commercial product) and p-anisaldehyde had greater attraction as compared to (S)-(-)-verbenone, given the compound's binding with FoccOBP3 . The current study helps in understanding the tertiary structure and interaction of FoccOBP3 with lures using computational and field data and will help in the identification of novel lures of insects in the future, given the importance of binding with OBPs.Communicated by Ramaswamy H. Sarma.

A new iboga-vobasine-type isomeric bisindole alkaloid named voacamine A ( 1 ), along with eight known compounds-voacangine ( 2 ), voacristine ( 3 ), coronaridine ( 4 ), tabernanthine ( 5 ), iboxygaine ( 6 ), voacamine ( 7 ), voacorine ( 8 ) and conoduramine ( 9 )-were isolated from the stem bark of Voacangaafricana . The structures of the compounds were determined by comprehensive spectroscopic analyses. Compounds 1 , 2 , 3 , 4 , 6 , 7 and 8 were found to inhibit the motility of both the microfilariae (Mf) and adult male worms of Onchocerca ochengi , in a dose-dependent manner, but were only moderately active on the adult female worms upon biochemical assessment at 30 μM drug concentrations. The IC 50 values of the isolates are 2.49-5.49 µM for microfilariae and 3.45-17.87 µM for adult males. Homology modeling was used to generate a 3D model of the O. ochengi thioredoxin reductase target and docking simulation, followed by molecular dynamics and binding free energy calculations attempted to offer an explanation of the anti-onchocercal structure-activity relationship (SAR) of the isolated compounds. These alkaloids are new potential leads for the development of antifilarial drugs. The results of this study validate the traditional use of V. africana in the treatment of human onchocerciasis.

The HLA-A*02:01-restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T-cells-1 (MART-1) protein, represents one of the best-studied tumor associated T-cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA-A*02:01 and TCRs from clinically relevant T-cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5-HLA-A*02:01-AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide-MHC anchoring. This "flexing" at the TCR-peptide-MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well-studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells., (© 2019 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pterin-containing natural products have diverse functions in life, but an efficient and easy scheme for their in vitro synthesis is not available. Here we report a chemoenzymatic 14-step, one-pot synthesis that can be used to generate 13 C- and 15 N-labeled dihydrofolates (H 2 F) from glucose, guanine, and p-aminobenzoyl-l-glutamic acid. This synthesis stands out from previous approaches to produce H 2 F in that the average yield of each step is >91% and it requires only a single purification step. The use of a one-pot reaction allowed us to overcome potential problems with individual steps during the synthesis. The availability of labeled dihydrofolates allowed the measurement of heavy-atom isotope effects for the reaction catalyzed by the drug target dihydrofolate reductase and established that protonation at N5 of H 2 F and hydride transfer to C6 occur in a stepwise mechanism. This chemoenzymatic pterin synthesis can be applied to the efficient production of other folates and a range of other natural compounds with applications in nutritional, medical, and cell-biological research.

Chemical ligation has been used to alter motions in specific regions of dihydrofolate reductase from E. coli and to investigate the effects of localized motional changes on enzyme catalysis. Two isotopic hybrids were prepared; one with the mobile N-terminal segment containing heavy isotopes ((2) H, (13) C, (15) N) and the remainder of the protein with natural isotopic abundance, and the other one with only the C-terminal segment isotopically labeled. Kinetic investigations indicated that isotopic substitution of the N-terminal segment affected only a physical step of catalysis, whereas the enzyme chemistry was affected by protein motions from the C-terminal segment. QM/MM studies support the idea that dynamic effects on catalysis mostly originate from the C-terminal segment. The use of isotope hybrids provides insights into the microscopic mechanism of dynamic coupling, which is difficult to obtain with other studies, and helps define the dynamic networks of intramolecular interactions central to enzyme catalysis., (© 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.)