164 research outputs found

    The role of the C8 proton of ATP in the regulation of phosphoryl transfer within kinases and synthetases

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    <p>Abstract</p> <p>Background</p> <p>The kinome comprises functionally diverse enzymes, with the current classification indicating very little about the extent of conserved regulatory mechanisms associated with phosphoryl transfer. The apparent <it>K</it><sub>m </sub>of the kinases ranges from less than 0.4 μM to in excess of 1000 μM for ATP. It is not known how this diverse range of enzymes mechanistically achieves the regulation of catalysis via an affinity range for ATP varying by three-orders of magnitude.</p> <p>Results</p> <p>We have demonstrated a previously undiscovered mechanism in kinase and synthetase enzymes where the overall rate of reaction is regulated via the C8-H of ATP. Using ATP deuterated at the C8 position (C8D-ATP) as a molecular probe it was shown that the C8-H plays a direct role in the regulation of the overall rate of reaction in a range of kinase and synthetase enzymes. Using comparative studies on the effect of the concentration of ATP and C8D-ATP on the activity of the enzymes we demonstrated that not only did C8D-ATP give a kinetic isotope effect (KIE) but the KIE's obtained are clearly not secondary KIE effects as the magnitude of the KIE in all cases was at least 2 fold and in most cases in excess of 7 fold.</p> <p>Conclusions</p> <p>Kinase and synthetase enzymes utilise C8D-ATP in preference to non-deuterated ATP. The KIE obtained at low ATP concentrations is clearly a primary KIE demonstrating strong evidence that the bond to the isotopically substituted hydrogen is being broken. The effect of the ATP concentration profile on the KIE was used to develop a model whereby the C8H of ATP plays a role in the overall regulation of phosphoryl transfer. This role of the C8H of ATP in the regulation of substrate binding appears to have been conserved in all kinase and synthetase enzymes as one of the mechanisms associated with binding of ATP. The induction of the C8H to be labile by active site residues coordinated to the ATP purine ring may play a significant role in explaining the broad range of <it>K</it><sub>m </sub>associated with kinase enzymes.</p

    Differential metabolic reprogramming in paenibacillus alvei-primed sorghum bicolor seedlings in response to fusarium pseudograminearum infection

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    Metabolic changes in sorghum seedlings in response to Paenibacillus alvei (NAS-6G6)-induced systemic resistance against Fusarium pseudograminearum crown rot were investigated by means of untargeted ultra-high performance liquid chromatography-high definition mass spectrometry (UHPLC-HDMS). Treatment of seedlings with the plant growth-promoting rhizobacterium P. alvei at a concentration of 1 × 108 colony forming units mL- 1 prior to inoculation with F. pseudograminearum lowered crown rot disease severity significantly at the highest inoculum dose of 1 × 106 spores mL-1. Intracellular metabolites were subsequently methanol-extracted from treated and untreated sorghum roots, stems and leaves at 1, 4 and 7 days post inoculation (d.p.i.) with F. pseudograminearum. The extracts were analysed on an UHPLC-HDMS platform, and the data chemometrically processed to determine metabolic profiles and signatures related to priming and induced resistance. Significant treatment-related differences in primary and secondary metabolism post inoculation with F. pseudograminearum were observed between P. alvei-primed versus naïve S. bicolor seedlings. The differential metabolic reprogramming in primed plants comprised of a quicker and/or enhanced upregulation of amino acid-, phytohormone-, phenylpropanoid-, flavonoid- and lipid metabolites in response to inoculation with F. pseudograminearum.Supplementary Materials: Figure S1. (A) Microscopic identification of F. pseudograminearum at 400 × magnification. (B) Conidial morphology of F. pseudograminearum taken from Aoki et al. [65]. Figure S2. UHPLC-HDMS BPI chromatograms of ESI-positive data indicating the metabolomic profiles of untreated (black), naïve infected (blue) and primed infected (green) stems obtained at 1 d.p.i. with F. pseudograminearum. Figure S3. UHPLC-HDMS BPI chromatograms of ESI-positive data indicating the metabolomic profiles of untreated (black), naïve infected (blue) and primed infected (green) leaves obtained at 1 d.p.i. with F. pseudograminearum. Figure S4. PCA score/scatter plot of stem samples computed from ESI-positive data. Figure S5. PCA score/scatter plot of leaf samples computed from ESI-positive data. Figure S6. PCA score/scatter plot of root samples computed from ESI-negative data. Figure S7. PCA score/scatter plot of stems samples computed from ESI-negative data. Figure S8. PCA score/scatter plot of leaves samples computed from ESI-negative data. Figure S9. OPLS-DA modelling and variable/feature selection ESI-positive data (stem samples). Figure S10. OPLS-DA modelling and variable/feature selection ESI-positive data (leaf samples). Table S1. Summary of the description and validation of all the generated OPLS-DA models separating naïve versus primed S. bicolor plants. Figure S11. Summary of pathway analysis with MetPA. Figure S12. Venn diagram comparing the number of metabolites shown in Table 2 that were significantly upregulated at 1 d.p.i. (blue), 4 d.p.i. (yellow) and 7 d.p.i. (green) with F. pseudograminearum in primed versus naïve S. bicolor seedlings.https://www.mdpi.com/journal/metaboliteshj2020Plant Production and Soil Scienc

    Unravelling the metabolic reconfiguration of the post-challenge primed state in Sorghum bicolor responding to Colletotrichum sublineolum infection

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    Priming is a natural phenomenon that pre-conditions plants for enhanced defence against a wide range of pathogens. It represents a complementary strategy, or sustainable alternative that can provide protection against disease. However, a comprehensive functional and mechanistic understanding of the various layers of priming events is still limited. A non-targeted metabolomics approach was used to investigate metabolic changes in plant growth-promoting rhizobacteria (PGPR)-primed Sorghum bicolor seedlings infected with the anthracnose-causing fungal pathogen, Colletotrichum sublineolum, with a focus on the post-challenge primed state phase. At the 4-leaf growth stage, the plants were treated with a strain of Paenibacillus alvei at 108 cfu mL1. Following a 24 h PGPR application, the plants were inoculated with a C. sublineolum spore suspension (106 spores mL1), and the infection monitored over time: 1, 3, 5, 7 and 9 days post-inoculation. Non-infected plants served as negative controls. Intracellular metabolites from both inoculated and non-inoculated plants were extracted with 80% methanol-water. The extracts were chromatographically and spectrometrically analysed on an ultra-high performance liquid chromatography (UHPLC) system coupled to high-definition mass spectrometry. The acquired multidimensional data were processed to create data matrices for chemometric modelling. The computed models indicated time-related metabolic perturbations that reflect primed responses to the fungal infection. Evaluation of orthogonal projection to latent structure-discriminant analysis (OPLS-DA) loading shared and unique structures (SUS)-plots uncovered the di erential stronger defence responses against the fungal infection observed in primed plants. These involved enhanced levels of amino acids (tyrosine, tryptophan), phytohormones (jasmonic acid and salicylic acid conjugates, and zeatin), and defence-related components of the lipidome. Furthermore, other defence responses in both naïve and primed plants were characterised by a complex mobilisation of phenolic compounds and de novo biosynthesis of the flavones, apigenin and luteolin and the 3-deoxyanthocyanidin phytoalexins, apigeninidin and luteolinidin, as well as some related conjugates.Supplementary Material: Figure S1: Evaluation of disease symptoms in Colletotrichum sublineolum infected sorghum plants; Figure S2: Representative BPI MS chromatograms of ESI(+) data (3 d.p.i.); Figure S3: Unsupervised chemometric modelling of ESI(-) data; Figure S4: OPLS-DA modelling and variable/feature selection. Table S1: Annotated (MSI-level 2) metabolites reported in Table 1, with fragmentation information.The South African National Research Foundation (NRF)http://www.mdpi.com/journal/metabolitesam2020Plant Production and Soil Scienc

    Metabolomic Analysis of Defense-Related Reprogramming in Sorghum bicolor in Response to Colletotrichum sublineolum Infection Reveals a Functional Metabolic Web of Phenylpropanoid and Flavonoid Pathways

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    The metabolome of a biological system provides a functional readout of the cellular state, thus serving as direct signatures of biochemical events that define the dynamic equilibrium of metabolism and the correlated phenotype. Hence, to elucidate biochemical processes involved in sorghum responses to fungal infection, a liquid chromatography-mass spectrometry-based untargeted metabolomic study was designed. Metabolic alterations of three sorghum cultivars responding to Colletotrichum sublineolum, were investigated. At the 4-leaf growth stage, the plants were inoculated with fungal spore suspensions and the infection monitored over time: 0, 3, 5, 7, and 9 days post inoculation. Non-infected plants were used as negative controls. The metabolite composition of aqueous-methanol extracts were analyzed on an ultra-high performance liquid chromatography system coupled to high-definition mass spectrometry. The acquired multidimensional data were processed to create data matrices for multivariate statistical analysis and chemometric modeling. The computed chemometric models indicated time- and cultivar-related metabolic changes that reflect sorghum responses to the fungal infection. Metabolic pathway and correlation-based network analyses revealed that this multi-component defense response is characterized by a functional metabolic web, containing defense-related molecular cues to counterattack the pathogen invasion. Components of this network are metabolites from a range of interconnected metabolic pathways with the phenylpropanoid and flavonoid pathways being the central hub of the web. One of the key features of this altered metabolism was the accumulation of an array of phenolic compounds, particularly de novo biosynthesis of the antifungal 3-deoxyanthocynidin phytoalexins, apigeninidin, luteolinidin, and related conjugates. The metabolic results were complemented by qRT-PCR gene expression analyses that showed upregulation of defense-related marker genes. Unraveling key characteristics of the biochemical mechanism underlying sorghum—C. sublineolum interactions, provided valuable insights with potential applications in breeding crop plants with enhanced disease resistance. Furthermore, the study contributes to ongoing efforts toward a comprehensive understanding of the regulation and reprogramming of plant metabolism under biotic stress

    Rhizobacteria-induced systemic resilience in Sorghum bicolor (L.) moench against Fusarium pseudograminearum crown rot under drought stress conditions

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    The potential of 77 rhizobacterial isolates to elicit induced systemic resilience (ISResilience) against combined biotic (Fusarium pseudograminearum crown rot) and abiotic (drought) stress in Sorghum bicolor was investigated. ISResilience was determined by assessing disease incidence and severity, plant height and biomass (root and shoots) in rhizobacteria-primed and untreated (naïve) plants inoculated with F. pseudograminearum and subjected to drought stress. Three rhizobacterial isolates (Paenibacillus alvei NAS-6G6, Pseudomonas taiwanensis N66 and Bacillus velezensis N54) showed significant protection of S. bicolor seedlings against biotic, abiotic and combined biotic and abiotic stress. Isolate N54, identified in this study as B. velezensis by 16S rRNA sequencing, was considered as the best performing rhizobacterial isolate to elicit ISResilience. Untargeted ultra-high performance liquid chromatography-high definition mass spectrometry (UHPLC-HDMS) based metabolomics was used to investigate the mechanism by which ISResilience was elicited in S. bicolor by strain N54 (B. velezensis). Comparisons were made with isolates that were previously selected for induced systemic tolerance (ISTolerance) against drought stress (strain N66, Ps. taiwanensis) and induced systemic resistance (ISResistance) against F. pseudograminearum crown rot (strain NAS-6G6, Pa. alvei). The stress alleviation that resulted from treatment with the respective rhizobacterial isolates, was visually confirmed by the use of infrared (IR) thermography. For the metabolomics study, intracellular metabolites were methanol-extracted from rhizobacteria-primed and untreated (naïve) S. bicolor shoots. Extracts were analyzed on an UHPLC-HDMS platform, and the data were chemometrically analyzed to determine metabolite bio-markers related to ISResistance, ISTolerance and ISResilience. The results demonstrated significant treatment-related differences, reflecting differential metabolic reprogramming in S. bicolor in response to the biotic, abiotic and combined stresses. Synergistic effects involved in the lowered susceptibility to crown rot of rhizobacteria-primed S. bicolor seedlings, compared to those left naïve (untreated control) under drought stress conditions and the upregulation of the signatory molecules myo-inositol and riboflavin, provided evidence for the role of crosstalk in the ISResilience observed.The National Research Foundation (NRF) of South Africa and the University of Pretoria.http://www.elsevier.com/locate/ybcon2021-08-01hj2021Plant Production and Soil Scienc

    A metabolomics-guided exploration of the phytochemical constituents of Vernonia fastigiata with the aid of pressurized hot water extraction and liquid chromatography-mass spectrometry

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    CITATION: Masike, K., et al. 2017. A metabolomics-guided exploration of the phytochemical constituents of Vernonia fastigiata with the aid of pressurized hot water extraction and liquid chromatography-mass. Molecules, 22(8):1200, doi:10.3390/molecules22081200.The original publication is available at http://www.mdpi.comVernonia fastigiata is a multi-purpose nutraceutical plant with interesting biological properties. However, very little is known about its phytochemical composition and, thus the need for its phytochemical characterization. In the current study, an environmentally friendly method, pressurized hot water extraction (PHWE), was used to extract metabolites from the leaves of V. fastigiata at various temperatures (50 °C, 100 °C, 150 °C and 200 °C). Ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-qTOF-MS) analysis in combination with chemometric methods, particularly principal component analysis (PCA) and liquid/gas chromatography mass spectrometry (XCMS) cloud plots, were used to descriptively visualize the data and identify significant metabolites extracted at various temperatures. A total of 25 different metabolites, including hydroxycinnamic acid derivatives, clovamide, deoxy-clovamide and flavonoids, were noted for the first time in this plant. Overall, an increase in extraction temperature resulted in an increase in metabolite extraction during PHWE. This study is the first scientific report on the phytochemical composition of V. fastigiata, providing insight into the components of the chemo-diversity of this important plant.http://www.mdpi.com/1420-3049/22/8/1200Publisher's versio

    Metabolomic evaluation of PGPR defence priming in wheat (Triticum aestivum L.) cultivars infected with Puccinia striiformis f. sp. tritici (stripe rust)

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    Plant-microbe interactions are a phenomenal display of symbiotic/parasitic relationships between living organisms. Plant growth-promoting rhizobacteria (PGPR) are some of the most widely investigated plant-beneficial microbes due to their capabilities in stimulating plant growth and development and conferring protection to plants against biotic and abiotic stresses. As such, PGPR-mediated plant priming/induced systemic resistance (ISR) has become a hot topic among researchers, particularly with prospects of applications in sustainable agriculture. The current study applies untargeted ultra-high performance liquid chromatography-high-definition mass spectrometry (UHPLC-HDMS) to investigate PGPR-based metabolic reconfigurations in the metabolome of primed wheat plants against Puccinia striiformis f. sp. tricti (Pst). A seed bio-priming approach was adopted, where seeds were coated with two PGPR strains namely Bacillus subtilis and Paenibacillus alvei (T22) and grown under controlled conditions in a glasshouse. The plants were infected with Pst one-week post-germination, followed by weekly harvesting of leaf material. Subsequent metabolite extraction was carried out for analysis on a UHPLC-HDMS system for data acquisition. The data was chemometrically processed to reveal the underlying trends and data structures as well as potential signatory biomarkers for priming against Pst. Results showed notable metabolic reprogramming in primary and secondary metabolism, where the amino acid and organic acid content of primed-control, primed-challenged and non-primed-challenged plants were differentially reprogrammed. Similar trends were observed from the secondary metabolism, in which primed plants (particularly primed-challenged) showed an up-regulation of phenolic compounds (flavonoids, hydroxycinnamic acids-HCAs- and HCA amides) compared to the non-primed plants. The metabolomics-based semi-quantitative and qualitative assessment of the plant metabolomes revealed a time-dependent metabolic reprogramming in primed-challenged and primed-unchallenged plants, indicating the metabolic adaptations of the plants to stripe rust infection over time

    The Effect of Geometrical Isomerism of 3,5-Dicaffeoylquinic Acid on Its Binding Affinity to HIV-Integrase Enzyme: A Molecular Docking Study

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    A potent plant-derived HIV-1 inhibitor, 3,5-dicaffeoylquinic acid (diCQA), has been shown to undergo isomerisation upon UV exposure where the naturally occurring 3trans,5trans-diCQA isomer gives rise to the 3cis,5trans-diCQA, 3trans,5cis-diCQA, and 3cis,5cis-diCQA isomers. In this study, inhibition of HIV-1 INT by UV-induced isomers was investigated using molecular docking methods. Here, density functional theory (DFT) models were used for geometry optimization of the 3,5-diCQA isomers. The YASARA and Autodock VINA software packages were then used to determine the binding interactions between the HIV-1 INT catalytic domain and the 3,5-diCQA isomers and the Discovery Studio suite was used to visualise the interactions between the isomers and the protein. The geometrical isomers of 3,5-diCQA were all found to bind to the catalytic core domain of the INT enzyme. Moreover, the cis geometrical isomers were found to interact with the metal cofactor of HIV-1INT, a phenomenon which has been linked to antiviral potency. Furthermore, the 3trans,5cis-diCQA isomer was also found to interact with both LYS156 and LYS159 which are important residues for viral DNA integration. The differences in binding modes of these naturally coexisting isomers may allow wider synergistic activity which may be beneficial in comparison to the activities of each individual isomer

    Roots and nodules response differently to P starvation in the Mediterranean-type legume Virgilia divaricata

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    Virgilia divaricata is a tree legume that grows in the Cape Floristic Region (CFA) in poor nutrient soils. A comparison between high and low phosphate growth conditions between roots and nodules was conducted and evaluated for the plants ability to cope under low phosphate stress conditions in V. divaricata. We proved that the plant copes with low phosphate stress through an increased allocation of resources, reliance on BNF and enhanced enzyme activity, especially PEPC. Nodules had a lower percentage decline in P compared to roots to uphold its metabolic functions. These strategies partly explain how V. divaricata can sustain growth despite LP conditions. Although the number of nodules declined with LP, their biomass remained unchanged in spite of a plant decline in dry weight. This is achieved via the high efficiency of BNF under P stress. During LP, nodules had a lower % decline at 34% compared to the roots at 88%. We attribute this behavior to P conservation strategies in LP nodules that imply an increase in a metabolic bypass that operates at the PEP branch point in glycolysis. The enhanced activities of nodule PEPC, MDH, and ME, whilst PK declines, suggests that under LP conditions an adenylate bypass was in operation either to synthesize more organic acids or to mediate pyruvate via a non-adenylate requiring metabolic route. Both possibilities represent a P-stress adaptation route and this is the first report of its kind for legume trees that are indigenous to low P, acid soils. Although BNF declined by a small percentage during LP, this P conservation was evident in the unchanged BNF efficiency per weight, and the increase in BNF efficiency per mol of P. It appears that legumes that are indigenous to acid soils, may be able to continue their reliance on BNF via increased allocation to nodules and also due to increase their efficiency for BNF on a P basis, owing to P-saving mechanisms such as the organic acid routes.Figure S1 : A section of the 13C spectra for (a) roots after 1 h, (b) roots after 2 h grown under high phosphate (500 mM P) conditions of V. divaricata.Figure S2 : A section of the 13C spectra of (a) roots after 1 h, (b) roots after 2 h grown under low phosphate (5 mM P) conditions of V. divaricata.Figure S3 : A section of the 13C spectra of (a) nodules after 1 h, (b) nodules after 2 h grown under high phosphate (500 mM P) conditions of V. divaricata.Figure S4 :| A section of the 13C spectra of (a) nodules after 1 h, (b) nodules after 2 h grown under low phosphate (5 mM P) conditions of V. divaricata.Figure S5 : A sample of the full 13C spectra of roots after 1 h, from plants grown under high phosphate (500 mM P) conditions of V. divaricata.Figure S6 : A sample of the full 13C spectra of nodules after 1 h, from plants grown under high phosphate (500 mM P) conditions of V. divaricata.This work is based on the Ph.D. thesis (University of Stellenbosch) of one of the authors, Gary Grant Stevens (GGS).The DST-NRF Centre of Excellence in Tree Health Biotechnology (CTHB)http://www.frontiersin.org/Plant_Scienceam2020Forestry and Agricultural Biotechnology Institute (FABI)Microbiology and Plant Patholog
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