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The influence of viral infection caused by two different potyviruses, Potato virus Y (PVY) and Potato virus A (PVA) on plant metabolism and photosynthetic apparatus of Nicotiana tabacum L. cv. Samsun and cv. Petit Havana SR1 was studied. The main stress was focused on the activities of phospho enolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), and pyruvate phosphate dikinase (PPDK). The analysis of the presence of viral proteins, enzyme activities, and different photosynthetic parameters showed the time dependent progress of viral infection and NADP-ME and PEPC activities. PVY caused significant response, while PVA affected both tobacco cultivars only slightly. Viral infection, namely PVY, affected more negatively photosynthetic apparatus of cv. Petit Havana SR1 than cv. Samsun. [ABSTRACT FROM AUTHOR]

The non-photosynthetic NADP-malic enzyme EC 1.1.1.40 (NADP-ME), which catalyzes the oxidative decarboxylation of L-malate and NADP+ to produce pyruvate and NADPH, respectively, and which could be involved in plant defense responses, was isolated from Nicotiana tabacum L. leaves. The mechanism of the enzyme reaction was studied by the initial rate method and was found to be an ordered sequential one. Regulation possibilities of purified cytosolic NADP-ME by cell metabolites were tested. Intermediates of the citric acid cycle (á-ketoglutarate, succinate, fumarate), metabolites of glycolysis (pyruvate, phosphoenolpyruvate, glucose-6-phosphate), compounds connected with lipogenesis (coenzyme A, acetyl-CoA, palmitoyl-CoA) and some amino acids (glutamate, glutamine, aspartate) did not significantly affect the NADP-ME activity from tobacco leaves. In contrast, macroergic compounds (GTP, ATP and ADP) were strong inhibitors of NADP-ME; the type of inhibition and the inhibition constants were determined in the presence of the most effective cofactors (Mn2+ or Mg2+), required by NADP-ME. Predominantly non-competitive type of inhibitions of NADP-ME with respect to NADP+ and mixed type to L-malate were found.

The best-characterized functional motifs of the potyviral Helper-Component protease (HC-Pro) responding for aphid transmission, RNA silencing suppression, movement, symptom development, and replication are gathered in this review. The potential cellular protein targets of plant virus proteases remain largely unknown despite their multifunctionality. The HC-Pro catalytic domain, as a cysteine protease, autoproteolytically cleaves the potyviral polyproteins in the sequence motif YXVG/G and is not expected to act on host targets; however, 146 plant proteins in the Viridiplantae clade containing this motif were searched in the UniProtKB database and are discussed. On the other hand, more than 20 interactions within the entire HC-Pro structure are known. Most of these interactions with host targets (such as the 20S proteasome, methyltransferase, transcription factor eIF4E, and microtubule-associated protein HIP2) modulate the cellular environments for the benefit of virus accumulation or contribute to symptom severity (interactions with MinD, Rubisco, ferredoxin) or participate in the suppression of RNA silencing (host protein VARICOSE, calmodulin-like protein). On the contrary, the interaction of HC-Pro with triacylglycerol lipase, calreticulin, and violaxanthin deepoxidase seems to be beneficial for the host plant. The strength of these interactions between HC-Pro and the corresponding host protein vary with the plant species. Therefore, these interactions may explain the species-specific sensitivity to potyviruses.

Several 1,2,4-triazoles are widely used as systemic fungicides in agriculture because they inhibit fungal 14ɑ-demethylase. However, they can also act on many non-target plant enzymes, thereby affecting phytohormonal balance, free amino acid content, and adaptation to stress. In this study, tomato plants (Solanum lycopersicum L. var. 'Cherrola') were exposed to penconazole, tebuconazole, or their combination, either by foliar spraying or soil drenching, every week, as an ecotoxicological model. All triazole-exposed plants showed a higher content (1.7-8.8 ×) of total free amino acids than the control, especially free glutamine and asparagine were increased most likely in relation to the increase in active cytokinin metabolites 15 days after the first application. Conversely, the Trp content decreased in comparison with control (0.2-0.7 ×), suggesting depletion by auxin biosynthesis. Both triazole application methods slightly affected the antioxidant system (antioxidant enzyme activity, antioxidant capacity, and phenolic content) in tomato leaves. These results indicated that the tomato plants adapted to triazoles over time. Therefore, increasing the abscisic and chlorogenic acid content in triazole-exposed plants may promote resistance to abiotic stress., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Triazoles inhibit lanosterol 14α-demethylase and block ergosterol biosynthesis in fungal pathogens. However, they also interact with other cytochrome P450 enzymes and influence non-target metabolic pathways. Disturbingly, triazoles may interact with essential elements. The interaction of penconazole (Pen), cyproconazole (Cyp) and tebuconazole (Teb) with Zn 2+ results in the formation of deprotonated ligands in their complexes or in the creation of complexes with Cl - as a counterion or doubly charged complexes. Triazoles, as well as their equimolar cocktails with Zn 2+ (10 -6  mol/L), decreased the activities of the non-target enzymes CYP19A1 and CYP3A4. Pen most decreased CYP19A1 activity and was best bound to its active centre to block the catalytic cycle in computational analysis. For CYP3A4, Teb was found to be the most effective inhibitor by both, activity assay and interaction with the active centre. Teb/Cyp/Zn 2+ and Teb/Pen/Cyp/Zn 2+ cocktails also decreased the CYP19A1 activity, which was in correlation with the formation of numerous triazole-Zn 2+ complexes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Triazole fungicides can threaten plants as abiotic stressors but can also positively affect plant defense by inducing priming. Thus, plant yield is also both protected and endangered by triazoles that may influence several metabolic pathways during maturation processes, such as the biosynthesis of saccharides or secondary metabolites. Here, Solanum lycopersicum L. plants were exposed to foliar and soil applications of penconazole, tebuconazole, or their combination, and their resulting effect on tomato fruits was followed. The exposure to the equimolar mixture of both triazoles influenced the representation of free proteinogenic amino acids, especially Gln, Glu, Gly, Ile, Lys, Ser and Pro, saccharide content, and led to a significant increase in the contents of total phenolics and flavonoids as well as positive stimulation of the non-enzymatic antioxidant system. Among the identified secondary metabolites, the most abundant was naringenin, followed by chlorogenic acid in tomato peel. In turn, all triazole-treated groups showed a significantly lower content of rosmarinic acid in comparison with the control. Foliar application of penconazole affected the fruit more than other single triazole applications, showing a significant decrease in antioxidant capacity, the total content of secondary metabolites, and the activities of total membrane-bound peroxidases and ascorbate peroxidase.

In contrast to inorganic nitrogen (N) assimilation, the role of organic N forms, such as proteins and peptides, as sources of N and their impact on plant metabolism remains unclear. Simultaneously, organic biostimulants are used as priming agents to improve plant defense response. Here, we analysed the metabolic response of tobacco plants grown in vitro with casein hydrolysate or protein. As the sole source of N, casein hydrolysate enabled tobacco growth, while protein casein was used only to a limited extent. Free amino acids were detected in the roots of tobacco plants grown with protein casein but not in the plants grown with no source of N. Combining hydrolysate with inorganic N had beneficial effects on growth, root N uptake and protein content. The metabolism of casein-supplemented plants shifted to aromatic (Trp), branched-chain (Ile, Leu, Val) and basic (Arg, His, Lys) amino acids, suggesting their preferential uptake and/or alterations in their metabolic pathways. Complementarily, proteomic analysis of tobacco roots identified peptidase C1A and peptidase S10 families as potential key players in casein degradation and response to N starvation. Moreover, amidases were significantly upregulated, most likely for their role in ammonia release and impact on auxin synthesis. In phytohormonal analysis, both forms of casein influenced phenylacetic acid and cytokinin contents, suggesting a root system response to scarce N availability. In turn, metabolomics highlighted the stimulation of some plant defense mechanisms under such growth conditions, that is, the high concentrations of secondary metabolites (e.g., ferulic acid) and heat shock proteins., (© 2023 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Pseudomonas aeruginosa is one of the most antibiotic multi-resistant bacteria, causing chronic pulmonary disease and leading to respiratory failure and even mortality. Thus, there has been an ever-increasing search for novel and preferably natural antimicrobial compounds. Agrimonia eupatoria L. and Origanum vulgare L. shoots are commonly used as teas or alcoholic tinctures for their human health-promoting and antibacterial properties. Here, we explored the antimicrobial effects of all plant parts, i.e., leaf, flower, stem, and root extracts, prepared in water or in 60% ethanol, against P. aeruginosa . The impact of these extracts on bacterial survival was determined using a luminescent strain of P. aeruginosa , which emits light when alive. In addition, the antimicrobial effects were compared with the antioxidant properties and content of phenolic compounds of plant extracts. Ethanolic extracts of O. vulgare roots and flowers showed the highest antimicrobial activity, followed by A. eupatoria roots. In particular, chlorogenic acid, the ethanolic extract of O. vulgare roots contained high levels of protocatechuic acid, hesperidin, shikimic acid, rutin, quercetin, and morin. The synergistic effects of these phenolic compounds and flavonoids may play a key role in the antibacterial activity of teas and tinctures.

Pythium oligandrum , strain M1, is a soil oomycete successfully used as a biological control agent (BCA), protecting plants against fungal, yeast, and oomycete pathogens through mycoparasitism and elicitor-dependent plant priming. The not yet described Pythium strains, X42 and 00X48, have shown potential as BCAs given the high activity of their secreted proteases, endoglycosidases, and tryptamine. Here, Solanum lycopersicum L. cv. Micro-Tom seeds were coated with Pythium strains, and seedlings were exposed to fungal pathogens, either Alternaria brassicicola or Verticillium albo-atrum . The effects of both infection and seed-coating on plant metabolism were assessed by determining the activity and isoforms of antioxidant enzymes and endoglycosidases and the content of tryptamine, amino acids, and heat shock proteins. Dual culture competition testing and microscopy analysis confirmed mycoparasitism in all three Pythium strains. In turn, seed treatment significantly increased the total free amino acid content, changing their abundance in both non-infected and infected plants. In response to pathogens, plant Hsp70 and Hsp90 isoform levels also varied among Pythium strains, most likely as a strategy for priming the plant against infection. Overall, our results show in vitro mycoparasitism between Pythium strains and fungal pathogens and in planta involvement of heat shock proteins in priming.

Pythium is a genus of parasitic oomycetes which target plants and both nonvertebrate and vertebrate animals, including fish and mammalian species. However, several Pythium spp., such as P. oligandrum, function as mycoparasites of pathogenic fungi, bacteria, and oomycetes in soil and thus as advantageous biocontrol agents. This review primarily focuses on biochemical processes underlying their positive effects. For example, P. oligandrum degrades host cell wall polysaccharides using chitinases, cellulases, endo-β-1,3-glucanases, and various exoglycosidases. Proteases from various classes also participate in the cell wall hydrolysis. All these processes can modify cell surface structures and help Pythium spp. compete for space and nutrition. Accordingly, enzyme secretion most likely plays a key role in plant root colonisation. Plant-P. oligandrum interactions, nevertheless, do not involve tissue injury but instead activate plant defence mechanisms, thereby strengthening future plant responses to pathogen attacks. Priming induces the phenylpropanoid and terpenoid pathways and thus synthesis of secondary metabolites, including lignin, for cell wall fortification and other metabolic adjustments. Such metabolic changes are mediated by elicitins, cell wall glycoproteins and oligandrins produced by P. oligandrum. As homologous proteins of β-cinnamomin from Phytophthora cinnamomi with similar essential amino acids for sterol binding, oligandrins stand out for their structure, which they share with cell wall glycoproteins, albeit without the Ser-Thr-rich O-glycosylated domain for cell wall attachment. P. oligandrum also provides plant with tryptamine used for auxin synthesis, promoting plant growth. Overall, in addition to discussing plant metabolic and phytohormonal changes after P. oligandrum inoculation, we review data on P. oligandrum applications as researchers increasingly search for effective and environmentally friendly ways to protect crops. In this context, P. oligandrum emerges as a highly suitable biotechnological solution., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Inhibitors of the shikimate pathway are widely used as herbicides, antibiotics, and anti-infectious drugs. However, the regulation of the shikimic pathway is complex, and little is known about the feedback regulation of the shikimate dehydrogenase (SDH, EC 1.1.1.25) in plants. Thus, the aim of this study was to elucidate the kinetic mechanism of SDH purified from the root of Petroselinum crispum (parsley), to determine all possible reaction products and to identify phenylpropanoid compounds that affect its activity. Our results showed that the bisubstrate reaction catalyzed by P. crispum SDH follows a sequential ordered mechanism, except for three dead-end complexes. The main and lateral reactions of SDH were monitored by mass spectrometry, thereby detecting protocatechuic acid as a byproduct. Gallic acid was formed non-enzymatically, whereas quinate was not detected. Several polyphenolic compounds inhibited SDH activity, especially tannic, caffeic and chlorogenic acids, with IC 50 0.014 mM, 0.15 mM, and 0.19 mM, respectively. The number of hydroxyl groups influenced their inhibition effect on SDH, and p-coumaric, t-ferulic, sinapic, syringic and salicylic acids were less effective SDH inhibitors. Nevertheless, one branch of the phenylpropanoid pathway may affect SDH activity through feedback regulation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Triazoles are used as antifungal agents, they mostly inhibit two enzymes: 14α-demethylase and aromatase. These enzymes are utilised also in other species and therefore the affection in non-target species in the environment is expected as well. Besides, triazoles are often being applied in a mixture and they can also interact with other substances present. This study clarifies how three selected representative triazoles (tebuconazole, penconazole and cyproconazole) interact with each other (group effect) and in mixtures (cocktail effect) with copper, essential/toxic for all organisms. Within the experiments on electrospray and collision-induced dissociations (both ESI-MS), it has been found that the fragments correspond to typical triazole metabolites. For their formation, the presence of copper ions is crucial. The inhibitory effect of Cu cocktails on aromatase enzymatic activity has been studied. The presence of Cu ions together with triazole(s) significantly increases the inhibitory effect on aromatase activity. The highest inhibitory effect (more than 60%) on aromatase activity is produced by cocktails containing penconazole and Cu ions, namely by penconazole/Cu and penconazole/tebuconazole/Cu. The reactivity of triazoles in groups is not significantly affected by the interactions among them. Additionally, the role of triazoles in copper Fenton reaction regulation has been observed and described. These changes may be attributed to the formation and stabilization of the complexes with the central Cu ion, with usually one, two or three triazolic ligands, depending on the mixture. The study demonstrates that the interaction of triazoles and Cu ions is a complex process; their impact on metabolism seems to be rather extensive and must be evaluated in the context of biochemical reactions., Competing Interests: Declaration of competing interest The authors declare that there are no competing interests associated with the manuscript., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Pythium oligandrum is a unique biological control agent. This soil oomycete not only acts as a mycoparasite, but also interacts with plant roots and stimulates plant defense response via specific elicitors. In addition, P. oligandrum can synthetize auxin precursors and stimulate plant growth. We analyzed the secretomes and biochemical properties of eleven Pythium isolates to find a novel and effective strain with advantageous features for plants. Our results showed that even closely related P. oligandrum isolates significantly differ in the content of compounds secreted into the medium, and that all strains secrete proteins, amino acids, tryptamine, phenolics, and hydrolytic enzymes capable of degrading cell walls (endo-β-1,3-glucanase, chitinase, and cellulase), exoglycosidases (especially β-glucosidase), proteases, and phosphatases. The most different strain was identified as a not yet described Pythium species. The changes in metabolism of Brassica napus plants grown from seeds coated with the tested Pythium spp. were characterized. Enhanced levels of jasmonates, ethylene precursor, and salicylic acid may indicate better resistance to a wide variety of pathogens. Glucosinolates, as defense compounds against insects and herbivores, were enhanced in young plants. Altogether, P. oligandrum strains varied in their life strategies, and either they could perform equally as plant growth promoters and mycoparasites or they had developed one of these strategies better.

Resveratrol is antioxidant naturally occurring in wine grapes. It is thought to have a preventive biological activity against number of diseases. However, it has been recently shown that in the presence of metal ions, such as Cu 2+ , resveratrol forms oxidative radicals. Cu 2+ is usually present in wine due to former usage of bluestone in vineyards. Fungicide tebuconazole has substituted bluestone and is presently one of the most widely used agrochemicals in wine industry; wine thus may contain traces of tebuconazole. Here, we study the ternary system of resveratrol, Cu 2+ , and tebuconazole experimentally and theoretically (using mass spectrometry, antioxidant capacity assay and quantum-chemical calculations) to model the redox behaviour of resveratrol in wine. We show that tebuconazole prevents formation of oxidative resveratrol radicals (induced by Cu 2+ reaction with resveratrol) via preferential Cu 2+ capture and protection of the binding sites of resveratrol. This positive effect of tebuconazole has not been observed before., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Pancreatic α-amylase plays an important role in dietary starch hydrolysis in the small intestine and participates in enhanced glucose concentration after meals. It seems to be a problem for diabetic patients, who suffer from longer postprandial hyperglycemia after meal consumption than healthy people. There are commercially available drugs that inhibit α-amylase and thus reduce the postprandial hyperglycemia effect. However, these drugs may cause severe side effects. Conversely, some naturally occurring flavonoids were suggested to have an α-amylase-inhibiting effect without any side effects. There had been no rapid, undemanding method in terms of sample and reagent preparation that would enable screening of many potential inhibitors. Therefore, we developed an online capillary electrophoresis method to monitor α-amylase activity in the presence of an inhibitor. Each reaction constituent was introduced separately, directly into a capillary where the reagents were mixed by diffusion, which resulted in a 5-min analysis including conditioning of the capillary. We applied the method to test the inhibitory effect of flavonoid standards and their mixture and we investigated the inhibitory effect of ethanolic extract from Betula pendula bark. The developed method presents a faster and less expensive alternative to previously described offline methods. Graphical abstract Online CE screening of α-amylase inhibitors.

Salt stress is one of the most damaging plant stressors, whereas hypoosmotic stress is not considered to be a dangerous type of stress in plants and has been less extensively studied. This study was performed to compare the metabolism of cucumber plants grown in soil with plants transferred to distilled water and to a 100 mM NaCl solution. Even though hypoosmotic stress caused by distilled water did not cause such significant changes in the relative water content, Na+/K+ ratio and Rubisco content as those caused by salt stress, it was accompanied by more pronounced changes in the specific activities of NADP-dependent enzymes. After 3 days, the specific activities of NADP-isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, NADP-malic enzyme and non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in leaves were highest under hypoosmotic stress, and lowest in plants grown in soil. In roots, salt stress caused a decrease in the specific activities of major NADP-enzymes. However, at the beginning of salt stress, NADP-galactose-1-dehydrogenase and ribose-1-dehydrogenase were involved in a plant defense response in both roots and leaves. Therefore, the enhanced demands of NADPH in stress can be replenished by a wide range of NADP-dependent enzymes.

In this study, we optimized a method for the determination of free amino acids in Nicotiana tabacum leaves. Capillary electrophoresis with contactless conductivity detector was used for the separation of 20 proteinogenic amino acids in acidic background electrolyte. Subsequently, the conditions of extraction with HCl were optimized for the highest extraction yield of the amino acids because sample treatment of plant materials brings some specific challenges. Central composite face-centered design with fractional factorial design was used in order to evaluate the significance of selected factors (HCl volume, HCl concentration, sonication, shaking) on the extraction process. In addition, the composite design helped us to find the optimal values for each factor using the response surface method. The limits of detection and limits of quantification for the 20 proteinogenic amino acids were found to be in the order of 10 -5 and 10 -4  mol l -1 , respectively. Addition of acetonitrile to the sample was tested as a method commonly used to decrease limits of detection. Ambiguous results of this experiment pointed out some features of plant extract samples, which often required specific approaches. Suitability of the method for metabolomic studies was tested by analysis of a real sample, in which all amino acids, except for L-methionine and L-cysteine, were successfully detected. The optimized extraction process together with the capillary electrophoresis method can be used for the determination of proteinogenic amino acids in plant materials. The resulting inexpensive, simple, and robust method is well suited for various metabolomic studies in plants. As such, the method represents a valuable tool for research and practical application in the fields of biology, biochemistry, and agriculture.

The anticonvulsant drug carbamazepine is considered as an indicator of sewage water pollution: however, its uptake by plants and effect on metabolism have not been sufficiently documented, let alone its metabolite (10,11-epoxycarbamazepine). In a model system of sterile, hydroponically cultivated Zea mays (as C4 plant) and Helianthus annuus (as C3 plant), the uptake and effect of carbamazepine and 10,11-epoxycarbamazepine were studied in comparison with those of acetaminophen and ibuprofen. Ibuprofen and acetaminophen were effectively extracted from drug-supplemented media by both plants, while the uptake of more hydrophobic carbamazepine was much lower. On the other hand, the carbamazepine metabolite, 10,11-epoxycarbamazepine, was, unlike sunflower, willingly taken up by maize plants (after 96 h 88 % of the initial concentration) and effectively stored in maize tissues. In addition, the effect of the studied pharmaceuticals on the plant metabolism (enzymes of Hatch-Slack cycle, peroxidases) was followed. The activity of bound peroxidases, which could cause xylem vessel lignification and reduction of xenobiotic uptake, was at the level of control plants in maize leaves contrary to sunflower. Therefore, our results indicate that maize has the potential to remove 10,11-epoxycarbamazepine from contaminated soils.

Faster or stronger response to pathogen occurs if plants undergo prior priming. Cytokinins seem to be also involved in plant priming and in response to pathogens. Susceptibility to Potato virus Y(NTN) (PVY(NTN)) was studied in transgenic cytokinin overproducing (Pssu-ipt) tobacco and compared with nontransgenic plants. Since cytokinin overproduction inhibits development of plant roots and grafting overcomes this limitation, both types were grown as rooted and/or grafted plants to check also the effect of grafting. Control rooted tobacco (C), the most susceptible to PVY(NTN), showed always symptoms during the infection together with the rising virus content and a systemic response, such as accumulation of H2O2, salicylic acid (SA) and other phenolic acids, and stress-induced enzyme activities. In transgenic and grafted plants, the response to PVY(NTN) was dependent on protective mechanisms activated prior to the inoculation. In Pssu-ipt tobacco, cytokinin active forms and SA contents exceeded manifold their content in C. Grafting promoted the accumulation of phenolics, but SA, and stimulated peroxidase activities. Thus, the pre-infection barrier established in both transgenic and grafted plants helped to suppress partly the virus multiplication and resulted in milder symptom development. However, only the synergic effect of both grafting and the high cytokinins led to PVY(NTN) tolerance in transgenic grafts. Possible mechanisms were discussed., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The kinetic properties of β-N-acetylhexosaminidase purified from tobacco (Nicotiana tabacum L.) leaves have been investigated. In addition to chromogenic pNP derivates, N,N'-diacetylchitobiose and N,N',N″-triacetylchitotriose were also used as substrates of β-N-acetylhexosaminidase. The highest reaction rate and the affinity for the substrate were observed for pNP-GlcNAc; however, an excess of this substrate inhibits the reaction. The reaction rate with pNP-GalNAc as the substrate was found to be about 85% of that obtained with pNP-GlcNAc. The hydrolysis of acetylated chitooligomers by β-N-acetylhexosaminidase followed by separation and quantification using capillary electrophoresis was slower compared to pNP-GlcNAc. The pH optimum of β-N-acetylhexosaminidase for individual substrates was found at 4.3-5.0 and the temperature optimum was 50-55 °C. Gel permeation chromatography and red native electrophoresis determined the relative molecular weight as 280 000 and the isoelectric point as 5.3. The inhibition of β-N-acetylhexosaminidase by monosaccharides GlcN, GalN, GlcNAc, GalNAc in combination with substrates pNP-GlcNAc and pNP-GalNAc was studied and the type of inhibition and the inhibition constants were determined., (Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.)

Drought stress is one of the most frequent forms of abiotic stresses, which occurs under condition of limited water availability. In this work, the possible participation of phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC), NADP-malic enzyme (EC 1.1.1.40; NADP-ME), and pyruvate, phosphate dikinase (EC 2.7.9.1; PPDK) in response to drought of tobacco plants (Nicotiana tabacum L., cv. W38) was investigated. Enzyme specific activities in tobacco leaves of drought stressed plants were significantly increased after 11 days of stress, PEPC 2.3-fold, NADP-ME 3.9-fold, and PPDK 2.7-fold compared to control plants. The regulation of PEPC and NADP-ME activities were studied on transcriptional level by the quantitative RT PCR and on translational level - immunochemically. The amount of NADP-ME protein and transcription of mRNA for chloroplastic NADP-ME isoform were increased indicating their enhanced synthesis de novo. On the other hand, mRNA for cytosolic isoform of NADP-ME was decreased. The changes in PEPC protein and PEPC mRNA were not substantial. Therefore regulation of PEPC activity by phosphorylation was evaluated and found to be involved in the stress response. During recovery, activities of the tested enzymes returned close to their basal levels., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The detailed examination of enzyme molecules by mass spectrometry and other techniques continues to identify hundreds of distinct PTMs. Recently, global analyses of enzymes using methods of contemporary proteomics revealed widespread distribution of PTMs on many key enzymes distributed in all cellular compartments. Critically, patterns of multiple enzymatic and nonenzymatic PTMs within a single enzyme are now functionally evaluated providing a holistic picture of a macromolecule interacting with low molecular mass compounds, some of them being substrates, enzyme regulators, or activated precursors for enzymatic and nonenzymatic PTMs. Multiple PTMs within a single enzyme molecule and their mutual interplays are critical for the regulation of catalytic activity. Full understanding of this regulation will require detailed structural investigation of enzymes, their structural analogs, and their complexes. Further, proteomics is now integrated with molecular genetics, transcriptomics, and other areas leading to systems biology strategies. These allow the functional interrogation of complex enzymatic networks in their natural environment. In the future, one might envisage the use of robust high throughput analytical techniques that will be able to detect multiple PTMs on a global scale of individual proteomes from a number of carefully selected cells and cellular compartments. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Enzyme assays of β-N-acetylhexosaminidase from Aspergillus oryzae using capillary electrophoresis in the offline and online setup have been developed. The pH value and concentration of the borate-based background electrolyte were optimized in order to achieve baseline separation of N,N',N″-triacetylchitotriose, N,N'-diacetylchitobiose, and N-acetyl-D-glucosamine. The optimized method using 25 mM tetraborate buffer, pH 10.0, was evaluated in terms of repeatability, limits of detection, quantification, and linearity. The method was successfully applied to the offline enzyme assay of β-N-acetylhexosaminidase, which was demonstrated by monitoring the hydrolysis of N,N',N″-triacetylchitotriose. The presented method was also utilized to study the pH dependence of enzyme activity. An online assay with N,N'-diacetylchitobiose as a substrate was developed using the Transverse Diffusion of Laminar Flow Profiles model to optimize the injection sequence and in-capillary mixing of substrate and enzyme plugs. The experimental results were in good agreement with predictions of the model. The online assay was successfully used to observe the inhibition effect of N,N'-dimethylformamide on the activity of β-N-acetylhexosaminidase with nanoliter volumes of reagents used per run and a high degree of automation. After adjustment of background electrolyte pH, an online assay with N,N',N″-triacetylchitotriose as a substrate was also performed.

A new type of native electrophoresis was developed to separate and characterize proteins. In this modification of the native blue electrophoresis, the dye Ponceau Red S is used instead of Coomassie Brilliant Blue to impose uniform negative charge on proteins to enable their electrophoretic separation according to their relative molecular masses. As Ponceau Red S binds less tightly to proteins, in comparison with Coomassie Blue, it can be easily removed after the electrophoretic separation and a further investigation of protein properties is made possible (e.g. an enzyme detection or electroblotting). The tested proteins also kept their native properties (enzyme activity or aggregation state)., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Fungal β-N-acetylhexosaminidases are inducible extracellular enzymes with many biotechnological applications. The enzyme from Penicillium oxalicum has unique enzymatic properties despite its close evolutionary relationship with other fungal hexosaminidases. It has high GalNAcase activity, tolerates substrates with the modified N-acyl group better and has some other unusual catalytic properties. In order to understand these features, we performed isolation, biochemical and enzymological characterization, molecular cloning and molecular modelling. The native enzyme is composed of two catalytic units (65 kDa each) and two propeptides (15 kDa each), yielding a molecular weight of 160 kDa. Enzyme deglycosylated by endoglycosidase H had comparable activity, but reduced stability. We have cloned and sequenced the gene coding for the entire hexosaminidase from P. oxalicum. Sufficient sequence identity of this hexosaminidase with the structurally solved enzymes from bacteria and humans with complete conservation of all catalytic residues allowed us to construct a molecular model of the enzyme. Results from molecular dynamics simulations and substrate docking supported the experimental kinetic and substrate specificity data and provided a molecular explanation for why the hexosaminidase from P. oxalicum is unique among the family of fungal hexosaminidases., (© 2011 The Authors Journal compilation © 2011 FEBS.)

Phosphoenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), and pyruvate, phosphate dikinase (PPDK) participate in the process of concentrating CO₂ in C₄ photosynthesis. Non-photosynthetic counterparts of these enzymes, which are present in all plants, play important roles in the maintenance of pH and replenishment of Krebs cycle intermediates, thereby contributing to the biosynthesis of amino acids and other compounds and providing NADPH for biosynthesis and the antioxidant system. Enhanced activities of PEPC and/or NADP-ME and/or PPDK were found in plants under various types of abiotic stress, such as drought, high salt concentration, ozone, the absence of phosphate and iron or the presence of heavy metals in the soil. Moreover, the activities of all of these enzymes were enhanced in plants under biotic stress caused by viral infection. The functions of PEPC, NADP-ME and PPDK appear to be more important for plants under stress than under optimal growth conditions., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) from mature maize seeds (Zea mays L.) was purified to homogeneity and a final specific activity of 13.3 μmol min⁻¹ mg⁻¹. Purified PEPC was treated with phosphatase from bovine intestinal mucosa or protein kinase A to study its apparent phosphorylation level. Kinetic parameters of the enzyme reaction catalyzed by phosphorylated and dephosphorylated forms under different conditions were compared, as well as an effect of modulators. The enzyme dephosphorylation resulted in the change of hyperbolic kinetics to the sigmoidal one (with respect to PEP), following with the decrease of maximal reaction rate and the increase of sensitivity to L-malate inhibition. The hyperbolic kinetics of native PEPC present in dry maize seeds was not changed after the protein kinase A treatment, while it was converted to the sigmoidal one after dephosphorylation. Level of PEPC phosphorylation was not affected during seed imbibition., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)