Conformational Plasticity of the Essential Membrane-associated Mannosyltransferase PimA from Mycobacteria

Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of peripheral membrane-associated GTs for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here we used single molecule force spectroscopy techniques to study the mechanical and conformational properties of PimA. In our studies, we engineered a polyprotein containing PimA flanked by four copies of the well characterized I27 protein, which provides an unambiguous mechanical fingerprint. We found that PimA exhibits weak mechanical stability albeit displaying β-sheet topology expected to unfold at much higher forces. Notably, PimA unfolds following heterogeneous multiple step mechanical unfolding pathways at low force akin to molten globule states. Interestingly, the ab initio low resolution envelopes obtained from small angle x-ray scattering of the unliganded PimA and the PimA·GDP complexed forms clearly demonstrate that not only the “open” and “closed” conformations of the GT-B enzyme are largely present in solution, but in addition, PimA experiences remarkable flexibility that undoubtedly corresponds to the N-terminal “Rossmann fold” domain, which has been proved to participate in protein-membrane interactions. Based on these results and on our previous experimental data, we propose a model wherein the conformational transitions are important for the mannosyltransferase to interact with the donor and acceptor substrates/membrane.     

Dispersal of aphids, whiteflies and their natural enemies under photoselective nets

Integrated Pest Management of insects includes several control tactics, such as the use of photoselective nets, which may reduce the flight activity of insects. Limiting the dispersal of pests such as aphids and whiteflies is important because of their major role as vectors of plant viruses, while a minor impact on natural enemies is desired. In this study, we examined for the first time the dispersal ability of three vector species, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), Macrosiphum euphorbiae (Thomas) (Hemiptera: Aphididae) and Myzus persicae (Sulzer) (Hemiptera: Aphididae), in cages covered with photoselective nets. Contrary to the results obtained with aphids, the ability of the whitefly B. tabaci, to reach the target plant was reduced by photoselective nets. In a second set of experiments, the impact of UV-absorbing nets on the visual cues of two important predator species, Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) and Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae), was evaluated. The anthocorid was caught in higher numbers in traps placed under regular nets, whereas the mites preferably chose environments in which the UV radiation was attenuated. We have observed a wide range of effects that impedes generalization, although photoselective nets have a positive effect on pest management of whiteflies and aphids under protected environments.     

Mechanistic insights into the retaining glucosyl-3-phosphoglycerate synthase from mycobacteria

Considerable progress has been made in recent years in our understanding of the structural basis of glycosyl transfer. Yet the nature and relevance of the conformational changes associated with substrate recognition and catalysis remain poorly understood. We have focused on the glucosyl-3-phosphoglycerate synthase (GpgS), a "retaining" enzyme, that initiates the biosynthetic pathway of methylglucose lipopolysaccharides in mycobacteria. Evidence is provided that GpgS displays an unusually broad metal ion specificity for a GT-A enzyme, with Mg(2+), Mn(2+), Ca(2+), Co(2+), and Fe(2+) assisting catalysis. In the crystal structure of the apo-form of GpgS, we have observed that a flexible loop adopts a double conformation L(A) and L(I) in the active site of both monomers of the protein dimer. Notably, the L(A) loop geometry corresponds to an active conformation and is conserved in two other relevant states of the enzyme, namely the GpgS·metal·nucleotide sugar donor and the GpgS·metal·nucleotide·acceptor-bound complexes, indicating that GpgS is intrinsically in a catalytically active conformation. The crystal structure of GpgS in the presence of Mn(2+)·UDP·phosphoglyceric acid revealed an alternate conformation for the nucleotide sugar β-phosphate, which likely occurs upon sugar transfer. Structural, biochemical, and biophysical data point to a crucial role of the β-phosphate in donor and acceptor substrate binding and catalysis. Altogether, our experimental data suggest a model wherein the catalytic site is essentially preformed, with a few conformational changes of lateral chain residues as the protein proceeds along the catalytic cycle. This model of action may be applicable to a broad range of GT-A glycosyltransferases.     

SENSE tool: easy-to-use web-based tool to calculate food product environmental impact

Purpose

The purpose of the European SENSE project was to define an integral system to assess and communicate the environmental impacts of food products and to develop a web-based tool for Small and Medium size Enterprises (SMEs). The tool has been tested in salmon, beef-and-dairy, and fruit juice production sectors.

Methods

The SENSE project has evaluated several existing methodologies for environmental impact assessment over the life cycle including also social aspects, in order to deliver a new integral system for the environmental and social assessment of agricultural and aquaculture food products.

Results and discussion

The system includes a standardization of a data gathering system, a selection of relevant key environmental performance indicators for food supply chains and a common methodology to perform simplified life cycle impact assessment. The results are based on collected information on the use of resources and emissions generated along the supply chain of food or drink products. The main result is a web-based software tool that is based on a summation of the partial impacts of the different steps in food supply chains. In this software, different actors in the supply chain can enter their own data and link them to the data of other companies. The results obtained in the tool could be used for at least six different approaches: (i) environmental impact assessment of the product, (ii) food chain hot spot identification, (iii) comparison of hypothetical or real improvement scenarios, (iv) assessment of the environmental impact development over the years, (v) benchmarking opportunity for the companies, and (vi) a business to business communication strategy. The scientific robustness of the tool has been tested comparing the obtained results with the same analysis with commercial software.

Conclusions

The SENSE tool is a simplified tool designed for food and drink SMEs to assess their sustainability on their own. This cannot be fully compared to a complete LCA study. The testing with SMEs showed that they need additional support for filling in the questionnaires correctly and interpret the results. The simplified evaluation of environmental impacts based on a life cycle approach could lead to benefits to SMEs within the food industry. The future application and development of the tool will be focused on adapting the tool to the Product Environmental Footprint initiative requirements and self-assessment opportunities.

     

The Redox State Regulates the Conformation of Rv2466c to Activate the Antitubercular Prodrug TP053

Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin-fold with a Cys¹⁹-Pro²⁰-Trp²¹-Cys²² motif, and an insertion consisting of a four α-helical bundle and a short α-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in α-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle x-ray scattering showed that the fully reduced form of Rv2466c adopts a “closed” compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an “open” conformation, where tertiary structural changes in the α-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical, and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily.     

Impaired LXRα Phosphorylation Attenuates Progression of Fatty Liver Disease

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High irradiance increases NH(4)(+) tolerance in Pisum sativum: Higher carbon and energy availability improve ion balance but not N assimilation

The widespread use of NO₃⁻ fertilization has had a major ecological impact. NH₄⁺ nutrition may help to reduce this impact, although high NH₄⁺ concentrations are toxic for most plants. The underlying tolerance mechanisms are not yet fully understood, although they are thought to include the limitation of C, the disruption of ion homeostasis, and a wasteful NH₄⁺ influx/efflux cycle that carries an extra energetic cost for root cells.In this study, high irradiance (HI) was found to induce a notable tolerance to NH₄⁺ in the range 2.5-10 mM in pea plants by inducing higher C availability, as shown by carbohydrate content. This capacity was accompanied by a general lower relative N content, indicating that tolerance is not achieved through higher net N assimilation on C-skeletons, and it was also not attributable to increased GS content or activity in roots or leaves. Moreover, HI plants showed higher ATP content and respiration rates. This extra energy availability is related to the internal NH₄⁺ content regulation (probably NH₄⁺ influx/efflux) and to an improvement of the cell ionic balance.The limited C availability at lower irradiance (LI) and high NH₄⁺ resulted in a series of metabolic imbalances, as reflected in a much higher organic acid content, thereby suggesting that the origin of the toxicity in plants cultured at high NH₄⁺ and LI is related to their inability to avoid large-scale accumulation of the NH₄⁺ ion.     

Evidence for the Introduction of Lethal Chytridiomycosis Affecting Wild Betic Midwife Toads (Alytes dickhilleni)

Batrachochytrium dendrobatidis is an unpredictable pathogen for European amphibian species, and existing field surveillance studies likely underestimate the scope of its distribution and effects. Mass mortality episodes recorded in Europe indicate that investigations of unstudied species should focus on members of the frog family Alytidae. Here, we report the combined results of a field survey and laboratory observations of field collected Alytes dickhilleni. Our data support the hypothesis that B. dendrobatidis has recently emerged in at least two disjunct locations in the species range and populations across much of the species range lack evidence of infection pathogen. Tadpoles taken into the laboratory from sites with infection experienced 70% mortality, unlike those taken into the laboratory from uninfected sites, and both infection and strength of infection was associated with mortality in animals collected from infected locations. Several conservation interventions are underway in response to our study, including the establishment of a captive assurance colony, a public awareness campaign, and experimental tests of disease mitigation schemes.     

New 1,4-di-N-oxide-quinoxaline-2-ylmethylene isonicotinic acid hydrazide derivatives as anti-Mycobacterium tuberculosis agents

The increase in the prevalence of drug-resistant tuberculosis cases demonstrates the need of discovering new and promising compounds with antimycobacterial activity. As a continuation of our research and with the aim of identifying new antitubercular drugs candidates, a new series of quinoxaline 1,4-di-N-oxide derivatives containing isoniazid was synthesized and evaluated for in vitro anti-tuberculosis activity against Mycobacterium tuberculosis H37Rv strain. Moreover, various drug-like properties of new compounds were predicted. Taking into account the biological results and the promising drug-likeness profile of these compounds, make them valid leads for further experimental research.