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Arbuscular mycorrhizal fungi (AMF) and potassium humate (KH) are separately known as significant biostimulants, but their combined effect on plants remains elusive. This study investigated the single and combined roles of AMF and KH on the antioxidant defense system in Russian olive (Elaeagnus angustifoliaL.) leaves. Soil below the seeds was inoculated with indigenous AMF spores (Funneliformis,Claroideoglomus; 500 spores per seed). The KH (1.5 g/ per 1 kg of seed) was applied during sowing. Growth, leaf-water ratio, chlorophyll fluorescence, lipid peroxidation, H(2)O(2)content, antioxidant enzymes, and antioxidant capacity were analyzed in treated and untreated plants. Combined AMF and KH applications had a greater recovery effect on vegetative organ growth than separate treatments. With combined treatment, plants maintained leaf water status and chlorophyll fluorescence, while peroxidation of lipid membranes and H(2)O(2)content was reduced. Moreover, increases in superoxide dismutase and glutathione reductase activity prevented cellular damage from reactive oxygen species. Total phenolic content and antioxidant capacity values were remarkably higher in plants grown under the combined treatment. As a result, compared with their separate applications, a combination of AMF and KH enhanced the antioxidant defense system by increasing antioxidant enzymes and antioxidant capacity and, thus, could be used to enhance plant growth. WOS:000556718200001 2-s2.0-85089259000

Objectives: Earthworms have a large impact on the soil ecosystem. They are quite sensitive to pollutants. Purification and biochemical characterization of glucose-6-phosphate dehydrogenases (G6PD) from the earthworm species Eisenia fetida were aimed. The determination of the toxicity potentials of some soil pollutants on G6PD activity was intended. Methods: G6PD was isolated using 2',5'-ADP-Sepharose 4B affinity column. Enzyme purity and molecular mass were determined by SDS-PAGE. Its biochemical properties investigated. The effects of some soil pollutants on the enzyme were studied in vitro. Results: Enzyme was purified with 28% yields and 232 fold. Optimum pH and buffer concentration, optimal and stable temperature was determined as pH: 8.5, 60 mM, 25 degrees C and 20 degrees C. Its molecular weight estimated as 36 kDa. The Ni2+, Hg2+, Pb2+, Cr2+, and Fe2+ ions with IC50 values in the range of 56 +/- 06-120 +/- 20 mu M and the diniconazole, metalaxyl, methomyl, carboxyl, and oxamyl with IC50 values in the range of 7.6 +/- 1.2-77 +/- 12 mu M exhibited an inhibitory effect on G6PD. Conclusions: G6PD was isolated and characterized from E. fetida. Its catalytic activity decreased with very low concentration by pesticides and metal ions. The results indicated that the inhibition of G6PD may be important in the toxicity mechanism of pollutants on this earthworm., Cankiri Karatekin University [FF200217B42], This research was supported by Cankiri Karatekin University (Project No: FF200217B42).

This study describes in vitro the effect of three plant growth regulators (PGRs) such as mepiquat chloride, beta-naphthoxyacetic acid and gibberellic acid on the human blood antioxidant enzyme activites of glucose-6-phosphate dehydrogenase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, catalase and superoxide dismutase. Enzyme activities have determined spectrophotometrically. PGRs have different effects on antioxidant enzyme activities. I-50 values of PGRs on antioxidant enzymes were determined by means of PGR concentration-activity % graph. The activities of antioxidant enzymes were changed by different concentrations of PGRs. PGRs behaved as an inhibitor on the antioxidant enzymes. The inhibition power of PGRs was different from enzyme to enzyme. The most power inhibitor for all enzymes studied was gibberellic acid, followed by beta-naphthoxyacetic acid and mepiquat chloride. When I-50 values compared with the results of metal ions in literature, PGRs had more inhibition powder than the metal ions. PGR toxicity caused a significant decrease in the antioxidant enzyme activity.

Posttranslational modifications (PTM) are one of the first responses of plants to environmental stress and involve changing the location and activity of proteins in the cell. Addition of poly(ADP-ribose) (PAR) to proteins, poly(ADP-ribose)ylation (PARylation), is a posttranslational modification resulting from the binding of ADP-ribose from NAD+ to target proteins by PAR polymerases (PARP). PARylation is involved in many physiological events in plants including abiotic and biotic stress response. The aim of this work was to understand involvement of PARylation in inducing enzymatic antioxidant defence and alternative electron sinks in response to salinity. For this purpose A. thaliana plants were treated with 3-aminobenzamide (3-AB), which is a PARP inhibitor, in the presence of 100 mM NaCl. The 3-AB treatment induced plant fresh weight under control and salinity conditions. Moreover, 100 mM NaCl + 3-AB treated plants had lower lipid peroxidation when compared to 100 mM NaCl group indicating mitigation of oxidative stress. This oxidative stress mitigation was achieved by significantly induced superoxide dismutase (SOD) activity and transcriptional activation of genes related to ROS scavenging such as MSD1, CAT1, APX1, GR1. On the other hand, transcriptional regulation of mitochondrial alternative oxidase (AOX) pathway was also induced with 3-AB treatment (AOX1a and AOX1d) under salt stress indicating that ROS avoidance mechanisms are also activated along with ROS scavenging. However, in contrast to AOX, chloroplastic plastid terminal oxidase pathway was not induced with 3-AB., Ege University Scientific Research Projects Coordination Grant [18/B?, L/003], Funding This work was supported by Ege University Scientific Research Projects Coordination Grant (18/B?L/003) .

The present work aims to provide insight on the role of phytohormone application in developing efficient practical defense strategies to improve plants tolerance under heavy metal contamination. For this purpose, pea (Pisum sativum L.) seeds were germinated in an aqueous solution of 200 mu M CuCl2 up to the 3rd day and then continued to germinate in the presence of distilled water (stress cessation) or were subjected to following combinations: Cu + 1 mu M IAA and Cu + 1 mu M GA(3) for 3 additional days. The results showed that copper excess induced oxidative stress in germinating seeds, which resulted in changes of the redox state of glutathione and cysteine, and proteomics revealed Cu-induced modifications of thiols (SH) and carbonyls (CO) (indicators of protein oxidation). However, application of IAA or GA(3) in the germination medium after 3 days of Cu exposure alleviated toxicity on seedlings, despite the persistence of Cu up to 6th day. This improving effect seems to be mediated by a cell Cu accumulation decrease and a protein reduced status recovery, since phytohormones modulate thioredoxin/ferredoxin systems in favor of protecting proteins against oxidation. In addition, an IAA and GA(3) protective effect was evidenced by a cellular homeostasis amelioration resulting from the balance conservation between the regeneration and consumption processes of glutathione and cysteine reduced forms. The exogenous effectors also induced modifications of profiles of SH and CO, suggesting changes in the regulation and expression of proteins that could be involved in defense mechanism against Cu stress.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; The aim of this work was to investigate the effects of cadmium (Cd) on thiol and especially glutathione (GSH)-dependent reactions (glutathione content, glutaredoxin (Grx) content and activity, "glutathione" peroxidase (Gpx) activity, and glutathione reductase (GR) activity) in germinating pea seeds. Under Cd stress conditions, the overall activity as well as more specifically the expression of Grx C4 and Grx S12 increased. On the contrary, when incubated with Cd ions in vitro, the disulfide reductase activity of both isoforms was drastically inhibited. In the case of Grx C4, this correlated with the formation of protein dimers of 28 kDa as evidenced by electrophoresis analysis. Oxidative stress also affected the GSH status, since Cd treatment provoked (1) a pronounced stimulation in Gpx (a thioredoxin-dependent enzyme in plants) expression and (2) a drastic decrease in GR activity. These results are discussed in relation with the known contribution of Grx system to the thiol status during the germination of Cd-poisoned pea seeds.

Forster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET-FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactive.

Tyrosine nitration is a hallmark for nitrosative stress caused by the release of reactive oxygen and nitrogen species by activated macrophages and neutrophilic granulocytes at sites of inflammation and infection. In the first part of the study, we used an informative host¿parasite animal model to describe the differential contribution of macrophages and neutrophilic granulocytes to in vivo tissue nitration. To this purpose common carp (Cyprinus carpio) were infected with the extracellular blood parasite Trypanoplasma borreli (Kinetoplastida). After infection, serum nitrite levels significantly increased concurrently to the upregulation of inducible nitric oxide synthase (iNOS) gene expression. Tyrosine nitration, as measured by immunohistochemistry using an anti-nitrotyrosine antibody, dramatically increased in tissues from parasite-infected fish, demonstrating that elevated NO production during T. borreli infection coincides with nitrosative stress in immunologically active tissues. The combined use of an anti-nitrotyrosine antibody with a panel of monoclonal antibodies specific for several carp leukocytes, revealed that fish neutrophilic granulocytes strongly contribute to in vivo tissue nitration most likely through both, a peroxynitrite- and an MPO-mediated mechanism. Conversely, fish macrophages, by restricting the presence of radicals and enzymes to their intraphagosomal compartment, contribute to a much lesser extent to in vivo tissue nitration. In the second part of the study, we examined the effects of nitrosative stress on the parasite itself. Peroxynitrite, but not NO donor substances, exerted strong cytotoxicity on the parasite in vitro. In vivo, however, nitration of T. borreli was limited if not absent despite the presence of parasites in highly nitrated tissue areas. Further, we investigated parasite susceptibility to the human anti-trypanosome drug Melarsoprol (Arsobal), which directly interferes with the parasite-specific trypanothione anti-oxidant system. Arsobal treatment strongly decreased T. borreli viability both, in vitro and in vivo. All together, our data suggest an evolutionary conservation in modern bony fish of the function of neutrophilic granulocytes and macrophages in the nitration process and support the common carp as a suitable animal model for investigations on nitrosative stress in host¿parasite interactions. The potential of T. borreli to serve as an alternative tool for pharmacological studies on human anti-trypanosome drugs is discussed.

Aim: to study selenium, glutathione, glutathione-peroxidase and glutathione-reductase levels in blood in patients with anemia cardiomyopathy previously and after the treatment of Sorbifer and Neoselenium. Selenium levels were investigated by mass concentration method in 48 patients with anemia cardiomyopathy and 15 healthy people. The decreased of studying indices in patients with anemia cardiomyopathy were shown. These indices were normalized under the influence of treatment.

Effects of salt stress on the time course of stomatal behaviors and the activity of antioxidative enzymes such as catalase (CAT) (EC 1.11.1.6), ascorbate peroxidase (APX) (EC 1.11.1.11), and glutathione reductase (GR) (EC. 1.6.4.2) were studied in three strawberry cultivars. The responses of the cultivars 'Camarosa', 'Tioga,' and 'Chandler' were compared when they were irrigated with nutrient solution containing 0, 8.5, 17.0, and 34.0 mM sodium chloride (NaCl) for 30 days. A significant reduction in stomatal conductance (g(s)) was seen particularly on the 30th day of the salt treatments only in Camarosa, which is parallel to transpiration rate (E). CAT activities decreased in all of the salt treatments only in Tioga, while it remained almost unchanged or slightly increased depending on the period in Camarosa and Chandler. APX activity sharply increased in 17.0 and 8.5-mM NaCl treatments for 30 days in Camarosa and Tioga, respectively, whereas it linearly increased based on the NaCl treatments in Chandler. On the other hand, only Camarosa demonstrated a sharp increase in GR activity induced by salinity applied for 30 days. All the data indicated that control of the stomatal behavior, the higher salt-stress tolerance (LT50) and higher constitutive activity of antioxidant enzymes made Camarosa and Tioga relatively salt-tolerant cultivars.

Conformational heterogeneity of the FAD cofactor in p-hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations; the 'in' conformation with the isoalloxazine ring located in the active site, and the 'out' conformation with the isoalloxazine ring disposed towards the protein surface. Fluorescence-lifetime analysis of these complexes revealed similar lifetime distributions for the 'in' and 'out' conformations. The reason for this is twofold. First, the active site of PHBH contains various potential fluorescence-quenching sites close to the flavin. Fluorescence analysis of uncomplexed PHBH Y222V and Y222A showed that Tyr222 is responsible for picosecond fluorescence quenching free enzyme. In addition, other potential quenching sites, including a tryptophan and two tyrosines involved in substrate binding, are located nearby. Since the shortest distance between these quenching sites and the isoalloxazine ring differs only little on average, these aromatic residues are likely to contribute to fluorescence quenching. Second, the effect of flavin conformation on the fluorescence lifetime distribution is blurred by binding of the aromatic substrates: saturation with aromatic substrates induces highly efficient fluorescence quenching. The flavin conformation is therefore only reflected in the small relative contributions of the longer lifetimes. © Indian Academy of Sciences.

International audience; Ergovaline (EV) produced by symbiotic association of Epichloe coenophiala with tall fescue (Lolium arundinaceum) causes toxicoses in livestock. In this study, 16 lactating ewes (BW 76.0 +/- 0.6 kg) were used to determine the effects of feeding endophyte-infected (FE+) or endophyte free (FE-) tall fescue hay on animal health and performances and to investigate the putative mechanisms of action of EV. The mean EV concentrations in FE+ and FE- diets were 497 +/- 52 and

Alcohol oxidase (AO) is a homo-octameric flavoenzyme which catalyzes methanol oxidation in methylotrophic yeasts. AO protein is synthesized in the cytosol and subsequently sorted to peroxisomes where the active enzyme is formed. To gain further insight in the molecular mechanisms involved in AO activation, we studied spectroscopically native AO from Hansenula polymorpha and Pichia pastoris and three putative assembly intermediates. Fluorescence studies revealed that both Trp and FAD are suitable intramolecular markers of the conformation and oligomeric state of AO. A direct relationship between dissociation of AO octamers and increase in Trp fluorescence quantum yield and average fluorescence lifetime was found. The time-resolved fluorescence of the FAD cofactor showed a rapid decay component which reflects dynamic quenching due to the presence of aromatic amino acids in the FAD-binding pocket. The analysis of FAD fluorescence lifetime profiles showed a remarkable resemblance of pattern for purified AO and AO present in intact yeast cells. Native AO contains a high content of ordered secondary structure which was reduced upon FAD-removal. Dissociation of octamers into monomers resulted in a conversion of beta-sheets into alpha-helices. Our results are explained in relation to a 3D model of AO, which was built based on the crystallographic data of the homologous enzyme glucose oxidase from Aspergillus niger. The implications of our results for the current model of the in vivo AO assembly pathway are discussed.

[EN] Because of the biological importance of thiols, the development of probes for thiols has been an active research area in recent years. In this review, we summarize the results of recent exciting reports regarding thiol-addition reactions and their applications in thiol recognition. The examples reported can be classified into four reaction types including 1,1, 1,2, 1,3, 1,4 addition reactions, according to their addition mechanisms, based on different Michael acceptors. In all cases, the reactions are coupled to color and/or emission changes, although some examples dealing with electrochemical recognition have also been included. The use of thiol-addition reactions is a very simple and straightforward procedure for the preparation of thiol-sensing probes., The work was supported by the National Natural Science Foundation of China (no. 21072119, 21102086), the Shanxi Province Science Foundation for Youths (no. 2012021009-4), the Shanxi Province Foundation for Returnee (no. 2012-007), the Taiyuan Technology star special (No. 12024703), the Shanxi Province Foundation for Selected Returnees (no. 2010), and CAS Key Laboratory of Analytical Chemistry for Living Biosystems Open Foundation (ACL201304). Financial support from the Spanish Government (project MAT2012-38429-C04-01) and the Generalitat Valencia (project PROMETEO/2009/016) is gratefully acknowledged.

International audience; Lipid peroxidation is a major biochemical consequence of the oxidative deterioration of polyunsaturated lipids in cell membranes and causes damage to membrane integrity and loss of protein function. 4-hydroxy-2-nonenal (HNE), one of the most reactive products of n-6 polyunsaturated fatty acid peroxidation of membrane phospholipids, has been shown to be capable of affecting both nicotinamide adenine dinucleotide (phosphate) reduced [NAD(P)H] as well as NADH production. However, the understanding of its effects in living cardiac cells is still lacking. Our goal was to therefore investigate HNE effects on NAD(P)H noninvasively in living cardiomyocytes. Spectrally resolved lifetime detection of endogenous fluorescence, an innovative noninvasive technique, was employed. Individual fluorescence components were resolved by spectral linear unmixing approach. Gathered results revealed that HNE reduced the amplitude of both resolved NAD(P)H components in a concentration-dependent manner. In addition, HNE increased flavoprotein fluorescence and responsiveness of the NAD(P)H component ratio to glutathione reductase (GR) inhibitor. HNE also increased the percentage of oxidized nucleotides and decreased maximal NADH production. Presented data indicate that HNE provoked an important cell oxidation by acting on NAD(P) H regulating systems in cardiomyocytes. Understanding the precise role of oxidative processes and their products in living cells is crucial for finding new noninvasive tools for biomedical diagnostics of pathophysiological states. (C) 2013 Society of Photo-Optical Instrumentation Engineers (SPIE)

Plant acclimation to environmental stress is governed by several mechanisms including alterations in antioxidant defense systems. The purpose of this study was to investigate the effects of Gisela 5 and Mazzard rootstocks on the cold-hardiness of the sweet cherry cultivars (cvs.) 0900 Ziraat and Lambert, as well as to analyze the changes in activities of antioxidative enzymes including catalase (CAT; EC 1.11.1.6), soluble peroxidase (S-PRX; EC 1.11.1.7), cell wall-bound peroxidase (CWB-PRX; EC 1.11.1.7) and glutathione reductase (GR; EC 1.6.4.2) in cold-acclimated (CA) and non-acclimated (NA) stages. One-year shoots of 4 years-old sweet cherry tree cvs. 0900 Ziraat and Lambert grafted either on Gisela 5 or Mazzard rootstock were collected in CA (in January) or NA (in July) stage and subjected to artificial low temperature tests (4 degrees C, -5 degrees C, -15 degrees C and -25 degrees C) in order to determine the degree of cell membrane injury in bark tissues by ion leakage method. Antioxidant enzyme activities were assayed in another set of samples that were not subjected to low temperature treatments. Our data showed that ion leakage in bark tissues was greater in NA stage compared to CA stage. Ion leakage was below 50% in barks exposed to 4 degrees C and -5 degrees C in either period and above 50% in those exposed to -15 degrees C and -25 degrees C in NA stage. Ion leakage was lower if cultivars were grafted on Mazzard compared with grafting on Gisela 5. In addition, activities in both cultivars of CAT, as well as S-PRX and S-PRX, but not GR, were significantly higher during CA stage compared with those in NA stage. Elevations in CAT and PRX (both cell wall and soluble) activities were more prominent if the cultivars were grafted on Mazzard rootstock. We conclude that, cold hardiness in the sweet cherry involves changes in certain antioxidant enzyme activities depending usually on whether the plant is in CA or NA stage. Our finding that activities of CAT, as well as S-PRX and CWB-PRX in cultivars grafted on Mazzard were higher than those in cultivars grafted on Gisela 5 suggests that cold hardiness may be affected by the rootstock.

Relative growth rate of winter wheat at low temperature (day/night, 4/2 degrees C) was about 0.02 g g(-1) fresh weight day(-1). There was a substantial increase in dry matter content of shoots and roots, especially during the first week of low temperature exposure, which for the greater part could be attributed to an accumulation of soluble sugars and starch. The freezing tolerance (LTS,) of the leaf tissue gradually increased with 6 degrees C during the 6 weeks exposure, whereas freezing tolerance of roots was hardly affected. Upon low temperature exposure there was an increase in the content of water-soluble non-protein thiols (mainly glutathione), soluble sugars, starch, amino acids and total nitrogen in both shoots and roots. The most substantial increases in the content of thiols, sugars and amino acids already occurred within hours or a few days after the onset of the exposure. The nitrate content in shoots showed a rapid and strong decrease during low temperature exposure, however, in roots its content remained unaffected. It is proposed that the observed rapid increases in the content of various metabolites at low temperature are primarily due to an altered balance in their synthesis and their consumption in growth. There was no direct relation between increases in the content of the various metabolites and the degree of freezing tolerance of the tissue.

Assembly of glutenin polymers was examined for two contrasted durum wheat cultivars in connection with changes in the redox status of the endosperm cells that accompanied grain development. The evolutions of the redox state of ascorbate and glutathione, as well as the activities of antioxidant enzymes were measured. Changes in the size distribution profile and redox state of storage proteins were evaluated, with particular emphasis on protein-bound glutathione (PSSG). At the beginning of grain filling phase, the size distribution profile of proteins included an extra peak shoulder at about 40,000 g mol(-1). The shoulder was assimilated to free glutenin subunits as it disappeared concomitantly with the upturn in glutenin polymers accumulation. Irrespective of cultivars, small SDS-soluble polymers accumulated first, followed by larger and insoluble ones, attesting for a progressive polymerization. During the grain filling phase, catalase (EC 1.11.1.6) activity dropped, reaching a very low level at physiological maturity. During the same period, superoxide dismutase (EC 1.15.1.1) and glutathione reductase (EC 1.6.4.2) activities increased steadily while the equilibrium constant between GSSG and PSSG shifted from 10(-2) to unity. These results demonstrated that grain filling was accompanied by a continuous decrease in cellular redox potential. In this context, formation of protein-bound glutathione would represent a protective mechanism against irreversible thiol oxidation. Storage protein S-gluta-thionylation instead of limiting glutenin polymer assembly as it has been proposed might be a required intermediate step for glutenin subunits pairing. (C) 2012 Elsevier Masson SAS. All rights reserved.

The aim of this study was to compare the salinity tolerances of 2 oriental tobacco varieties (İzmir Özbaş and Akhisar 97). Salinity stress experiments were performed under both in vitro and in vivo conditions. Seedlings of each variety were subjected to 0, 50, 100, 150, 200, 250, 300, and 350 mM NaCl. Photosynthetic pigment levels, lipid peroxidation rate, total protein content, antioxidant enzyme activities, and proline concentrations were determined for seedlings treated with salt for 14 days. The Akhisar 97 variety was found to be more sensitive to salinity stress than the İzmir Özbaş variety. Although proline is thought to accumulate in salt-tolerant plants, we found a negative correlation between salinity tolerance and proline accumulation in the plants. According to biochemical analyses, there were no differences in SOD, APX, GPX, or CAT activity levels between the 2 varieties, either in vivo or in vitro. However, differences in glutathione reductase (GR) activity between control plants and plants under NaCl stress were statistically significant in both varieties, both in vitro and in vivo. Our results support the hypothesis that GR is a key element in the evaluation of salinity tolerance of tobacco varieties.

The catalytic domain of dihydrolipoyl transacetylase (E2pCD) forms the core of the pyruvate dehydrogenase multienzyme complex and catalyzes the acetyltransferase reaction using acetylCoA as acetyl donor and dihydrolipoamide (Lip(SH)2) as acceptor. The crystal structures of six complexes and derivatives of Azotobacter vinelandii E2pCD were solved. The binary complexes of the enzyme with CoA and Lip-(SH)2 were determined at 2.6- and 3.0-angstrom resolutions, respectively. The two substrates are found in an extended conformation at the two opposite entrances of the 30 angstrom long channel which runs at the interface between two 3-fold-related subunits and forms the catalytic center. The reactive thiol groups of both substrates are within hydrogen-bond distance from the side chain of His 610. This fact supports the indication, derived from the similarity with chloramphenicol acetyl transferase, that the histidine side chain acts as general-base catalyst in the deprotonation of the reactive thiol of CoA. The conformation of Asn 614 appears to be dependent on the protonation state of the active site histidine, whose function as base catalyst is modulated in this way. Studies on E2pCD soaked in a high concentration of dithionite lead to the structure of the binary complex between E2pCD and hydrogen sulfite solved at 2.3-angstrom resolution. It appears that the anion is bound in the middle of the catalytic center and is therefore capable of hosting and stabilizing a negative charge, which is of special interest since the reaction catalyzed by E2pCD is thought to proceed via a negatively charged tetrahedral intermediate. The structure of the binary complex between E2pCD and hydrogen sulfite suggests that transition-state stabilization can be provided by a direct hydrogen bond between the side chain of Ser 558 and the oxy anion of the putative intermediate. In the binary complex with CoA, the hydroxyl group of Ser 558 is hydrogen bonded to the nitrogen atom of one of the two peptide-like units of the substrate. Thus, CoA itself is involved in keeping the Ser hydroxyl group in the proper position for transition-state stabilization. Quite unexpectedly, the structure at 2.6-angstrom resolution of a ternary complex in which CoA and Lip(SH)2 are simultaneously bound to E2pCD reveals that CoA has an alternative, nonproductive binding mode. In this abortive ternary complex, CoA adopts a helical conformation with two intramolecular hydrogen bonds and the reactive sulfur of the pantetheine arm positioned 12 angstrom away from the active site residues involved in the transferase reaction. The same CoA conformation is observed in the ternary complex with CoA and dithiothreitol. The latter behaves like a substrate analogue which binds in the Lip(SH)2 binding pocket. The strict conservation of the active site residues among the transacylases forming the core of the branched-chain oxo acid dehydrogenase and the oxoglutarate dehydrogenase multienzyme complexes indicates that these enzymes all share the same catalytic mechanism as dihydrolipoyl transacetylase. The investigation of the three-dimensional structure of E2pCD combined with the analysis of the amino acid sequences suggests that the nature and size of only a few side chains might be the major determinants for tuning substrate specificity among the various transacylases.

BACKGROUND: Inappropriate utilisation of biosolids may adversely impact agrosystem productivity. Here, we address the response of wheat (Triticum durum) to different doses (0, 40, 100, 200 and 300 t ha(-1)) of either municipal solid waste (MSW) compost or sewage sludge in a greenhouse pot experiment. Plant growth, heavy metal uptake, and antioxidant activity were considered. RESULTS: Biomass production of treated plants was significantly enhanced at 40 t ha(-1) and 100 t ha(-1) of MSW compost (+48% and +78% relative to the control, respectively). At the same doses of sewage sludge, the increase was only 18%. Higher doses of both biosolids restricted significantly the plant growth, in concomitance with the significant accumulation of heavy metals (Ni2+, Pb2+, Cu2+ and Zn2+), especially in leaves. Leaf activities of antioxidant enzymes (ascorbate peroxidase, glutathione reductase, catalase and superoxide dismutase) were unchanged at 40 t ha-1 MSW compost or sewage sludge, but were significantly stimulated at higher doses (200-300 t ha(-1)), together with higher leaf concentration of reduced glutathione. CONCLUSION: This preliminary study suggests that a MSW supply at moderate doses (100 t ha-1) could be highly beneficial for wheat productivity. C) 2010 Society of Chemical Industry

The three-dimensional structure of one of the three lipoamide dehydrogenases occurring in Pseudomonas putida, LipDH Val, has been determined at 2.45 A resolution. The orthorhombic crystals, grown in the presence of 20 mM NAD+, contain 458 residues per asymmetric unit. A crystallographic 2-fold axis generates the dimer which is observed in solution. The final crystallographic R-factor is 21.8% for 18,216 unique reflections and a model consisting of 3,452 protein atoms, 189 solvent molecules and 44 NAD+ atoms, while the overall B-factor is unusually high: 47 angstrom2. The structure of LipDH Val reveals the conformation of the C-terminal residues which fold "back" into the putative lipoamide binding region. The C-terminus has been proven to be important for activity by site-directed mutagenesis. However, the distance of the C-terminus to the catalytically essential residues is surprisingly large, over 6 angstrom, and the precise role of the C-terminus still needs to be elucidated. In this crystal form LipDH Val contains one NAD+ molecule per subunit. Its adenine-ribose moiety occupies an analogous position as in the structure of glutathione reductase. However, the nicotinamide-ribose moiety is far removed from its expected position near the isoalloxazine ring and points into solution. Comparison of LipDH Val with Azotobacter vinelandii lipoamide dehydrogenase yields an rms difference of 1.6 angstrom for 440 well defined C(alpha) atoms per subunit. Comparing LipDH Val with glutathione reductase shows large differences in the tertiary and quaternary structure of the two enzymes. For instance, the two subunits in the dimer are shifted by 6 angstrom with respect to each other. So, LipDH Val confirms the surprising differences in molecular architecture between glutathione reductase and lipoamide dehydrogenase, which were already observed in Azotobacter vinelandii LipDH. This is the more remarkable since the active sites are located at the subunit interface and are virtually identical in all three enzymes.

Fumigation of winter wheat with H2S during low temperature acclimation substantially reduced the development of freezing tolerance of the leaves. After 6 weeks of low temperature exposure (3-degrees-C), the freezing tolerance was increased by 6 and 2-degrees-C at 0 and 0.25-mu-l l-1 H2S, respectively. Changes in levels of cellular constituents (c. g. amino acids, sugars), which are characteristic for an increased freezing tolerance, were much less pronounced upon H2S fumigation. Besides, H2S fumigation diminished shoot growth at low temperature. At 20-degrees-C, H2S fumigation did not affect both freezing tolerance of the leaves and shoot growth. The role of glutathione in the development of freezing tolerance was evaluated. Low temperature exposure resulted in a rapid increase in the water-soluble non-protein-sulfhydryl content (mainly glutathione) of the leaves: it was maximal after 3 days of exposure (two-fold). Fumigation of wheat with 0.25-mu-l l-1 H2S during low temperature exposure resulted in a further increase (up to six-fold) in the sulfhydryl content.

The conformational stability of holo-lipoamide and apo-lipoamide dehydrogenase from Azotobacter vinelandii was studied by thermoinactivation, unfolding and limited proteolysis. The oxidized holoenzyme is thermostable, showing a melting temperature, t(m) = 80-degrees-C. The thermal stability of the holoenzyme drastically decreases upon reduction. Unlike the oxidized and lipoamide two-electron reduced enzyme species, the NADH four-electron reduced enzyme is highly sensitive to unfolding by urea. Loss of energy transfer from Trp199 to flavin reflects the unfolding of the oxidized holoenzyme by guanidine hydrochloride. Unfolding of the monomeric apoenzyme is a rapid fully reversible process, following a simple two-state mechanism. The oxidized and two-electron reduced holoenzyme are resistant to limited proteolysis by trypsin and endoproteinase Glu-C. Upon cleavage of the apoenzyme or four-electron reduced holoenzyme by both proteases, large peptide fragments (molecular mass > 40 kDa) are transiently produced. Sequence studies show that limited trypsinolysis of the NADH-reduced enzyme starts mainly at the C-terminus of Arg391. In the apoenzyme, limited proteolysis by endoproteinase Glu-C starts from the C-terminus at the carboxyl ends of Glu459 and/or Glu435. From crystallographic data it is deduced that the susceptible amino acid peptide bonds are situated near the subunit interface. Thus, these bonds are inaccessible to the proteases in the dimeric enzyme and become accessible after monomerization. It is concluded that reduction of lipoamide dehydrogenase to the four-electron reduced state(s) is accompanied by conformational changes promoting subunit dissociation.

Photoinhibition of photosynthesis in vivo is shown to be considerably promoted by O2 under circumstances where energy turnover by photorespiration and photosynthetic carbon metabolism are low. Intact protoplasts of Valerianella locusta L. were photoinhibited by 30 min irradiation with 3000-mu-mol photons.m-2.s-1 at 4-degrees-C in saturating [CO2] at different oxygen concentrations, corresponding to 2-40% O2 in air. The photoinhibition of light-limited CO2-dependent photosynthetic O2 evolution increased with increasing oxygen concentration. The uncoupled photochemical activity of photosystem II, measured in the presence of the electron acceptor 1,4-benzoquinone, and maximum variable fluorescence, F(V), were strongly affected and this inhibition was closely correlated to the O2 concentration. The effect of O2 did not saturate at the highest concentrations applied. An increase in photoinhibitory fluorescence quenching with [O2], although less pronounced than in protoplasts, was also observed with intact leaves irradiated at 4-degrees-C in air. Initial fluorescence, F(O), was slightly (about 10%) increased by the inhibitory treatments but not influenced by [O2]. A long-term cold acclimation of the plants did not substantially alter the O2-sensitivity of the protoplasts under the high-light treatment. From these experiments we conclude that oxygen is involved in the photoinactivation of photosystem II by excess light in vivo.

Molecular dynamics (MD) simulations and polarized subnanosecond time-resolved flavin fluorescence spectroscopy have been used to study the conformational dynamics of the flavin adenine dinucleotide (FAD) cofactor in aqueous solution. FAD displays a highly heterogeneous fluorescence intensity decay, resulting in lifetime spectra with two major components: a dominant 7-ps contribution that is characteristic of ultrafast fluorescence quenching and a 2.7-ns contribution resulting from moderate quenching. MD simulations were performed in both the ground state and first excited state. The simulations showed transitions from "open" conformations to "closed" conformations in which the flavin and adenine ring systems stack coplanarly. Stacking generally occurred within the lifetime of the flavin excited state (4.7 ns in water), and yielded a simulated fluorescence lifetime on the order of the nanosecond lifetime that was observed experimentally. Hydrogen bonds in the ribityl-pyrophosphate-ribofuranosyl chain connecting both ring systems form highly stable cooperative networks and dominate the conformational transitions of the molecule. Fluorescence quenching in FAD is mainly determined by the coplanar stacking of the flavin and adenine ring systems, most likely through a mechanism of photoinduced electron transfer. Whereas in stacked conformations fluorescence is quenched nearly instantaneously, open fluorescent conformations can stack during the lifetime of the flavin excited state, resulting in immediate fluorescence quenching upon stacking.

Using spectroscopic techniques we studied the effect of the nucleophilic reagents cyanide, cyanate and thiocyanate on three flavo-oxidases namely alcohol oxidase (AO), glucose oxidase (GOX) and D-amino acid oxidase (DAOX). All three ions, added at concentrations in the mM range, caused release of the flavin adenine dinucleotide (FAD) co-factors from the enzyme molecules. In the case of AO this was accompanied by significant conformational perturbations, which was not observed for GOX and DAOX. As suggested from fluorescence, absorption and circular dichroism spectral changes at least one phenolic hydroxyl group became ionized upon FAD release from AO and a new class of Trp residues, fluorescent only in apo-AO protein, was demasked.

This article is a report on a symposium held at Experimental Biology '98 in San Francisco, California. Recent developments in site-directed mutagenesis, computer-modeling, and mechanistic analysis of cytochromes P450 and flavin-containing monooxygenases are described. A unifying theme is the elaboration of general approaches for understanding and predicting the function of individual forms of these enzymes. A related goal is the production of soluble forms of mammalian cytochromes P450 for X-ray crystallography.

The catalytic domain of dihydrolipoyl transacetylase (E2pCD) forms the core of the pyruvate dehydrogenase multienzyme complex and catalyzes the acetyltransferase reaction using acetylCoA as acetyl donor and dihydrolipoamide (Lip(SH)2) as acceptor. The crystal structures of six complexes and derivatives of Azotobacter vinelandii E2pCD were solved. The binary complexes of the enzyme with CoA and Lip-(SH)2 were determined at 2.6- and 3.0-angstrom resolutions, respectively. The two substrates are found in an extended conformation at the two opposite entrances of the 30 angstrom long channel which runs at the interface between two 3-fold-related subunits and forms the catalytic center. The reactive thiol groups of both substrates are within hydrogen-bond distance from the side chain of His 610. This fact supports the indication, derived from the similarity with chloramphenicol acetyl transferase, that the histidine side chain acts as general-base catalyst in the deprotonation of the reactive thiol of CoA. The conformation of Asn 614 appears to be dependent on the protonation state of the active site histidine, whose function as base catalyst is modulated in this way. Studies on E2pCD soaked in a high concentration of dithionite lead to the structure of the binary complex between E2pCD and hydrogen sulfite solved at 2.3-angstrom resolution. It appears that the anion is bound in the middle of the catalytic center and is therefore capable of hosting and stabilizing a negative charge, which is of special interest since the reaction catalyzed by E2pCD is thought to proceed via a negatively charged tetrahedral intermediate. The structure of the binary complex between E2pCD and hydrogen sulfite suggests that transition-state stabilization can be provided by a direct hydrogen bond between the side chain of Ser 558 and the oxy anion of the putative intermediate. In the binary complex with CoA, the hydroxyl group of Ser 558 is hydrogen bonded to the nitrogen atom of one of the two peptide-like units of the substrate. Thus, CoA itself is involved in keeping the Ser hydroxyl group in the proper position for transition-state stabilization. Quite unexpectedly, the structure at 2.6-angstrom resolution of a ternary complex in which CoA and Lip(SH)2 are simultaneously bound to E2pCD reveals that CoA has an alternative, nonproductive binding mode. In this abortive ternary complex, CoA adopts a helical conformation with two intramolecular hydrogen bonds and the reactive sulfur of the pantetheine arm positioned 12 angstrom away from the active site residues involved in the transferase reaction. The same CoA conformation is observed in the ternary complex with CoA and dithiothreitol. The latter behaves like a substrate analogue which binds in the Lip(SH)2 binding pocket. The strict conservation of the active site residues among the transacylases forming the core of the branched-chain oxo acid dehydrogenase and the oxoglutarate dehydrogenase multienzyme complexes indicates that these enzymes all share the same catalytic mechanism as dihydrolipoyl transacetylase. The investigation of the three-dimensional structure of E2pCD combined with the analysis of the amino acid sequences suggests that the nature and size of only a few side chains might be the major determinants for tuning substrate specificity among the various transacylases.

The three-dimensional structure of one of the three lipoamide dehydrogenases occurring in Pseudomonas putida, LipDH Val, has been determined at 2.45 A resolution. The orthorhombic crystals, grown in the presence of 20 mM NAD+, contain 458 residues per asymmetric unit. A crystallographic 2-fold axis generates the dimer which is observed in solution. The final crystallographic R-factor is 21.8% for 18,216 unique reflections and a model consisting of 3,452 protein atoms, 189 solvent molecules and 44 NAD+ atoms, while the overall B-factor is unusually high: 47 angstrom2. The structure of LipDH Val reveals the conformation of the C-terminal residues which fold "back" into the putative lipoamide binding region. The C-terminus has been proven to be important for activity by site-directed mutagenesis. However, the distance of the C-terminus to the catalytically essential residues is surprisingly large, over 6 angstrom, and the precise role of the C-terminus still needs to be elucidated. In this crystal form LipDH Val contains one NAD+ molecule per subunit. Its adenine-ribose moiety occupies an analogous position as in the structure of glutathione reductase. However, the nicotinamide-ribose moiety is far removed from its expected position near the isoalloxazine ring and points into solution. Comparison of LipDH Val with Azotobacter vinelandii lipoamide dehydrogenase yields an rms difference of 1.6 angstrom for 440 well defined C(alpha) atoms per subunit. Comparing LipDH Val with glutathione reductase shows large differences in the tertiary and quaternary structure of the two enzymes. For instance, the two subunits in the dimer are shifted by 6 angstrom with respect to each other. So, LipDH Val confirms the surprising differences in molecular architecture between glutathione reductase and lipoamide dehydrogenase, which were already observed in Azotobacter vinelandii LipDH. This is the more remarkable since the active sites are located at the subunit interface and are virtually identical in all three enzymes.

Photoinhibition of photosynthesis in vivo is shown to be considerably promoted by O2 under circumstances where energy turnover by photorespiration and photosynthetic carbon metabolism are low. Intact protoplasts of Valerianella locusta L. were photoinhibited by 30 min irradiation with 3000-mu-mol photons.m-2.s-1 at 4-degrees-C in saturating [CO2] at different oxygen concentrations, corresponding to 2-40% O2 in air. The photoinhibition of light-limited CO2-dependent photosynthetic O2 evolution increased with increasing oxygen concentration. The uncoupled photochemical activity of photosystem II, measured in the presence of the electron acceptor 1,4-benzoquinone, and maximum variable fluorescence, F(V), were strongly affected and this inhibition was closely correlated to the O2 concentration. The effect of O2 did not saturate at the highest concentrations applied. An increase in photoinhibitory fluorescence quenching with [O2], although less pronounced than in protoplasts, was also observed with intact leaves irradiated at 4-degrees-C in air. Initial fluorescence, F(O), was slightly (about 10%) increased by the inhibitory treatments but not influenced by [O2]. A long-term cold acclimation of the plants did not substantially alter the O2-sensitivity of the protoplasts under the high-light treatment. From these experiments we conclude that oxygen is involved in the photoinactivation of photosystem II by excess light in vivo.

Submolecular details of Azotobacter vinelandii apoflavodoxin (apoFD) (un)folding are revealed by time-resolved fluorescence anisotropy using wild-type protein and variants lacking one or two of apoFD's three tryptophans. ApoFD equilibrium (un)folding by guanidine hydrochloride follows a three-state model: native ↔ unfolded ↔ intermediate. In native protein, W128 is a sink for Förster resonance energy transfer (FRET). Consequently, unidirectional FRET with a 50-ps transfer correlation time occurs from W167 to W128. FRET from W74 to W167 is much slower (6.9ns). In the intermediate, W128 and W167 have native-like geometry because the 50-ps transfer time is observed. However, non-native structure exists between W74 and W167 because instead of 6.9ns the transfer correlation time is 2.0ns. In unfolded apoFD this 2.0-ns transfer correlation time is also detected. This decrease in transfer correlation time is a result of W74 and W167 becoming solvent accessible and randomly oriented toward one another. Apparently W74 and W167 are near-natively separated in the folding intermediate and in unfolded apoFD. Both tryptophans may actually be slightly closer in space than in the native state, even though apoFD's radius increases substantially upon unfolding. In unfolded apoFD the 50-ps transfer time observed for native and intermediate folding states becomes 200ps as W128 and W167 are marginally further separated than in the native state. Apparently, apoFD's unfolded state is not a featureless statistical coil but contains well-defined substructures. The approach presented is a powerful tool to study protein folding.

Fumigation of winter wheat with H2S during low temperature acclimation substantially reduced the development of freezing tolerance of the leaves. After 6 weeks of low temperature exposure (3-degrees-C), the freezing tolerance was increased by 6 and 2-degrees-C at 0 and 0.25-mu-l l-1 H2S, respectively. Changes in levels of cellular constituents (c. g. amino acids, sugars), which are characteristic for an increased freezing tolerance, were much less pronounced upon H2S fumigation. Besides, H2S fumigation diminished shoot growth at low temperature. At 20-degrees-C, H2S fumigation did not affect both freezing tolerance of the leaves and shoot growth. The role of glutathione in the development of freezing tolerance was evaluated. Low temperature exposure resulted in a rapid increase in the water-soluble non-protein-sulfhydryl content (mainly glutathione) of the leaves: it was maximal after 3 days of exposure (two-fold). Fumigation of wheat with 0.25-mu-l l-1 H2S during low temperature exposure resulted in a further increase (up to six-fold) in the sulfhydryl content.