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L M Lagrimini

Publications and source records attributed to L M Lagrimini.

At least 19 recordsLinked to original sources
PubMed2003

Expression and refolding of tobacco anionic peroxidase from E. coli inclusion bodies.

Coding DNA of the tobacco anionic peroxidase gene was cloned in pET40b vector. The problem of 11 arginine codons, rare in procaryotes, in the tobacco peroxidase gene was solved using E. coli BL21(DE3) Codon Plus strain. The expression level of the tobacco apo-peroxidase in the above strain was approximately 40% of the total E. coli protein. The tobacco peroxidase refolding was optimized based on the earlier developed protocol for horseradish peroxidase. The reactivation yield of recombinant tobacco enzyme was about 7% with the specific activity of 1100-1200 U/mg towards 2,2;-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was shown that the reaction of ABTS oxidation by hydrogen peroxide catalyzed by recombinant tobacco peroxidase proceeds via the ping-pong kinetic mechanism as for the native enzyme. In the presence of calcium ions, the recombinant peroxidase exhibits a 2.5-fold decrease in the second order rate constant for hydrogen peroxide and 1.5-fold decrease for ABTS. Thus, calcium ions have an inhibitory effect on the recombinant enzyme like that observed earlier for the native tobacco peroxidase. The data demonstrate that the oligosaccharide part of the enzyme has no effect on the kinetic properties and calcium inhibition of tobacco peroxidase.

Cloning, Molecular↗
PubMed2002

Effect of transgenic plants expressing high levels of a tobacco anionic peroxidase on the toxicity of Anagrapha falcifera nucleopolyhedrovirus to Helicoverpa zea (Lepidoptera: Noctuidae).

Wild type and corresponding transgenic tomato (Lycopersicon esculentum Miller) and two tobacco (Nicotiana spp.) plants that express high levels of a tobacco anionic peroxidase were used to determine what type of interactions occurred between peroxidase altered plant chemistry and the baculovirus Anagrapha falcifera nucleopolyhedrovirus (AfMNPV) for control of neonate corn earworms, Helicoverpa zea (Boddie). Transgenic plants expressed approximately five to 400 times higher peroxidase activity than corresponding tissues of wild type plants. The H. zea larvae typically fed 1.5 times less on transgenic compared with wild type leaf disks. There was only one experiment (of three with tomato leaves) where the larvae that fed on transgenic leaves were less susceptible to the virus based on nonoverlapping 95% confidence intervals for LC50 values. When the exposure dose was corrected for reduced feeding on the transgenic leaf disks, the insecticidal activity of the virus was not significantly different for larvae fed on transgenic versus wild type plants. Eight other experiments (with tomato and two species of tobacco) indicated either no significant effect or enhanced susceptibility (when corrected for feeding rates) to the virus of larvae fed on the transgenic leaves. These results indicate enhanced insect resistance in plants expressing high levels of a specific anionic peroxidase may be compatible with applications of AfMNPV. Potential reasons for this compatibility are discussed.

Animals↗
PubMed2001

Phenol-oxidizing peroxidases contribute to the protection of plants from ultraviolet radiation stress.

We have studied the mechanism of UV protection in two duckweed species (Lemnaceae) by exploiting the UV sensitivity of photosystem II as an in situ sensor for radiation stress. A UV-tolerant Spirodela punctata G.F.W. Meyer ecotype had significantly higher indole-3-acetic acid (IAA) levels than a UV-sensitive ecotype. Parallel work on Lemna gibba mutants suggested that UV tolerance is linked to IAA degradation rather than to levels of free or conjugated IAA. This linkage is consistent with a role for class III phenolic peroxidases, which have been implicated both in the degradation of IAA and the cross-linking of various UV-absorbing phenolics. Biochemical analysis revealed increased activity of a specific peroxidase isozyme in both UV-tolerant duckweed lines. The hypothesis that peroxidases play a role in UV protection was tested in a direct manner using genetically modified tobacco (Nicotiana sylvestris). It was found that increased activity of the anionic peroxidase correlated with increased tolerance to UV radiation as well as decreased levels of free auxin. We conclude that phenol-oxidizing peroxidases concurrently contribute to UV protection as well as the control of leaf and plant architecture.

Indoleacetic Acids↗
PubMed1999

High-sensitivity assay for pesticide using a peroxidase as chemiluminescent label.

The application of a newly isolated transgenic tobacco peroxidase (TOP) as a chemiluminescent label for immunoassay purposes is described for the first time. The enzyme has been oxidized with m-periodate and subsequently coupled to the model compound 2,4-dichlorophenoxyacetic acid (2,4-D) using a carbodiimide method. As compared to the native horseradish peroxidase used in control experiments, the TOP enzyme showed significantly higher efficiency of coupling to the antigen and no loss of the specific activity was observed. The obtained 2,4-D-TOP conjugate demonstrated unique properties in chemiluminescent detection. The latter allowed the minimization of the conjugate concentration due to the superior chemiluminescent activity of the enzyme. A highly sensitive capillary chemiluminescent immunoassay using the 2,4-D-TOP conjugate as labeled competitor is reported. Direct competitive ELISA has been performed using a specific monoclonal antibody immobilized onto the sol-gel treated glass capillary surface. A modified photomultiplier tube with a special holder for a capillary was used for the resulting chemiluminescent signal detection. The typical standard calibration curve for the 2,4-D pesticide detection is linear between 30 pg and 500 ng/mL.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1999

Oxidation of indole-3-acetic acid by dioxygen catalysed by plant peroxidases: specificity for the enzyme structure.

Indole-3-acetic acid (IAA) can be oxidized via two mechanisms: a conventional hydrogen-peroxide-dependent pathway, and one that is hydrogen-peroxide-independent and requires oxygen. It has been shown here for the first time that only plant peroxidases are able to catalyse the reaction of IAA oxidation with molecular oxygen. Cytochrome c peroxidase (CcP), fungal peroxidases (manganese-dependent peroxidase, lignin peroxidase and Arthromyces ramosus peroxidase) and microperoxidase were essentially inactive towards IAA in the absence of added H2O2. An analysis of amino acid sequences allowed five structurally similar fragments to be identified in auxin-binding proteins and plant peroxidases. The corresponding fragments in CcP and fungal peroxidases showed no similarity with auxin-binding proteins. Five structurally similar fragments form a subdomain including the catalytic centre and two residues highly conserved among 'classical' plant peroxidases only, namely His-40 and Trp-117. The subdomain identified above with the two residues might be responsible for the oxidation of the physiological substrate of classical plant peroxidases, IAA.

Amino Acid Sequence↗
PubMed1998

Bioelectrochemical monitoring of phenols and aromatic amines in flow injection using novel plant peroxidases.

An amperometric flow system combined with a glucose oxidase-mutarotase reactor was optimized and used to determine aromatic amines and phenols using peroxidase-modified graphite electrodes. An increase in currents upon injection of the analyzed substrate was shown to be approximated by a Michaelis-Menten type dependence. The detection limit was calculated as 3 times the noise, and the sensitivity was calculated as Imax/K(m)app. Commercially available horseradish peroxidase was compared with tobacco anionic and peanut cationic peroxidases for determination of aromatic amines and phenols. Detection limits of 10 nM for determination of o-aminophenol and o- and p-phenylenediamine achieved with a tobacco peroxidase-modified electrode give a promise for further improvements in sensitivities and detection limits of biosensors.

Aniline Compounds↗
PubMed1998

Identification of skatolyl hydroperoxide and its role in the peroxidase-catalysed oxidation of indol-3-yl acetic acid.

Indol-3-yl acetic acid (IAA, auxin) is a plant hormone whose degradation is a key determinant of plant growth and development. The first evidence for skatolyl hydroperoxide formation during the plant peroxidase-catalysed degradation of IAA has been obtained by electrospray MS. Skatolyl hydroperoxide degrades predominantly non-enzymically to oxindol-3-yl carbinol but in part enzymically into indol-3-yl methanol via a peroxidase cycle in which IAA acts as an electron donor. Skatolyl hydroperoxide is degradable by catalase. Horseradish peroxidase isoenzyme C (HRP-C) and anionic tobacco peroxidase (TOP) exhibit differences in their mechanisms of reaction. The insensitivity of the HRP-C-catalysed reaction to catalase is ascribed to the formation of HRP-C Compound III at the initiation step and its subsequent role in radical propagation. This is in contrast with the TOP-catalysed process in which skatolyl hydroperoxide has a key role. Indol-3-yl aldehyde is produced not via the peroxidase cycle but by catalysis involving ferrous peroxidase. Because indol-3-yl aldehyde is one of the main IAA-derived products identified in planta, we conclude that ferrous peroxidases participate in IAA catalytic transformations in vivo. A general scheme for peroxidase-catalysed IAA oxidation is presented.

Carbon Monoxide↗
PubMed1998

Anaerobic stopped-flow studies of indole-3-acetic acid oxidation by dioxygen catalysed by horseradish C and anionic tobacco peroxidase at neutral pH: catalase effect.

The effect of order of reagent mixing in the absence and in the presence of catalase on the transient kinetics of indole-3-acetic acid (IAA) oxidation by dioxygen catalysed by horseradish peroxidase C and anionic tobacco peroxidase at neutral pH has been studied. The data suggest that haem-containing plant peroxidases are able to catalyse the reaction in the absence of exogenous hydroperoxide. The initiation proceeds via the formation of the ternary complex enzyme-->IAA-->oxygen responsible for IAA primary radical generation. The horseradish peroxidase-catalysed reaction is independent of catalase indicating a significant contribution of free radical processes into the overall mechanism. This is in contrast to the tobacco peroxidase-catalysed reaction where the peroxidase cycle plays an important role. The transient kinetics of IAA oxidation catalysed by tobacco peroxidase exhibits a biphasic character with the first phase affected by catalase. The first phase is therefore associated with the common peroxidase cycle while the second is ascribed to native enzyme interaction with skatole peroxy radicals yielding directly Compound II.

Journal Article↗
PubMed1998

Differential leaf resistance to insects of transgenic sweetgum (Liquidambar styraciflua) expressing tobacco anionic peroxidase.

Leaves of transgenic sweetgum (Liquidambar styraciflua) trees that expressed tobacco anionic peroxidase were compared with leaves of L. styraciflua trees that did not express the tobacco enzyme. Leaves of the transgenic trees were generally more resistant to feeding by caterpillars and beetles than wild-type leaves. However, as for past studies with transgenic tobacco and tomato expressing the tobacco anionic peroxidase, the degree of relative resistance depended on the size of insect used and the maturity of the leaf. Decreased growth of gypsy moth larvae appeared mainly due to decreased consumption, and not changes in the nutritional quality of the foliage. Transgenic leaves were more susceptible to feeding by the corn earworm, Helicoverpa zea. Thus, it appears the tobacco anionic peroxidase can contribute to insect resistance, but its effects are more predictable when it is expressed in plant species more closely related to the original gene source.

Animals↗
PubMed1998

Expression of the tobacco anionic peroxidase gene is tissue-specific and developmentally regulated.

Transcriptionally regulated expression of tobacco anionic peroxidase was investigated with regard to tissue specificity and developmental regulation. Two tobacco species, Nicotiana sylvestris and Nicotiana tabacum cv. Xanthi, were stably transformed with a gene chimera composed of 3 kb of the tobacco anionic peroxidase promoter, the Escherichia coli beta-glucuronidase (GUS) coding region and the nopaline synthase terminator. Gene expression was regulated spatially and developmentally in all organs, and generally increased with age and maturity of the plant, tissue or organ. In the aerial portions of the plant, GUS activity was strongly expressed in trichomes and epidermis at nearly all developmental stages. In later stages of development, activity was also detected in ground tissue and parenchyma cells associated with vascular tissues. Activity in roots was limited to cortical cells and vascular-associated parenchyma cells. In reproductive tissue, expression was observed in sepals and petals before anthesis, and in all floral organs after anthesis. Expression was never detected in vascular tissue and was poorly correlated with lignification except in the cells surrounding primary xylem and pericyclic fibers in N. sylvestris. These studies suggest that this peroxidase isoenzyme is only limitedly involved in lignification but may be important in plant defense, growth and development.

Cell Wall↗
PubMed1998

Relative resistance of transgenic tomato tissues expressing high levels of tobacco anionic peroxidase to different insect species.

Different parts of genetically transformed tomato (Lycopersicon esculentum L.) plants that express the tobacco anionic peroxidase were compared for insect resistance with corresponding wild type plants. Leaf feeding by first instar Helicoverpa zea and Manduca sexta was often significantly reduced on intact transgenic plants and/or leaf disks compared to wild type plants, but the effect could depend on leaf age. Leaves of transgenic plants were generally as susceptible to feeding damage by third instar Helicoverpa zea (Boddie) and Manduca sexta (L.) as wild type plants. Green fruit was equally susceptible to third instar larvae of H. zea in both type plants, but fruit of transgenic plants were more resistant to first instar larvae as indicated by significantly greater mortality. Basal stem sections were more resistant to neonate larvae of H. zea and adults of Carpophilus lugubris Murray compared to wild type plants as indicated by significantly greater mortality and/or reduced feeding damage. Thus, tobacco anionic peroxidase activity can increase plant resistance to insects in tomato, a plant species closely related to the original source plant species, when expressed at sufficiently high levels. However, the degree of resistance is dependent on the size of insect and plant tissue involved.

Animals↗
PubMed1998

Effect of pH on tobacco anionic peroxidase stability and its interaction with hydrogen peroxide.

The effect of extremely acidic pH on the stability of tobacco peroxidase and lignin peroxidase holoenzymes has been studied. Stabilization of tobacco peroxidase holoenzyme in the presence of calcium cations at pH < 2 and stabilization of lignin peroxidase at pH > 2 in the presence of veratryl alcohol have been shown. The dependence of the reaction rate constant for hydrogen peroxide interaction with tobacco peroxidase on pH suggests that the reaction rate is under control of a group with pK of 2.5. A tobacco peroxidase model structure has been created by means of homology modeling on the basis of the tobacco peroxidase sequence and the coordinates of peanut peroxidase crystal structure. The model structure demonstrates the presence of the negatively charged Glu-141 at the entrance to the active site and its electrostatic repulsion from heme propionates and triad of Asp-76, -79, and -80 residues. The results on tobacco holoperoxidase stabilization at pH 1.8 in the presence of calcium cations and drop in reaction rate constant for the enzyme interaction with hydrogen peroxide are explained by a hypothetical formation of ionic bonds between Glu-141 and the triad of aspartic acid residues via calcium cation lowering the accessibility of the active site and stabilizing the holoenzyme.

Amino Acid Sequence↗
PubMed1997

The consequence of peroxidase overexpression in transgenic plants on root growth and development.

Transgenic tobacco plants that overproduce the tobacco anionic peroxidase wilt upon reaching maturity, although having functional stomata and normal vascular anatomy and physiology. These plants were examined further to determine the cause for wilting, and thus better understand how the anionic peroxidase functions in plant growth and development. Shoots from young peroxidase overproducing plants were grafted onto wild-type tobacco root stock to determine if the roots could absorb and transmit sufficient water to maintain leaf turgidity. These grafted plants never wilted when grown in the greenhouse though shoot peroxidase activity remained ten-fold greater than in control plants, thus indicating that wilting is a consequence of peroxidase expression in the roots. Close examination of root systems revealed considerably less root mass in the transformed plant, primarily exhibited through a decrease in branching. At flowering, root growth rate and total root mass in transformed plants were less than 50% of control plants although shoot mass and growth rate were unchanged. This is in contrast to root growth in young seedlings where transformed plants performed equivalently to controls. Root hydraulic conductivity was measured to evaluate the effect of elevated peroxidase expression on water absorption and transport; however, no significant change in hydraulic conductivity was found in transformed plants. The consequence of anionic peroxidase overexpression on indoleacetic acid (IAA) metabolism was also examined. No significant difference in IAA levels was observed; however, root elongation in plants overexpressing peroxidase was insensitive to exogenous IAA. It can be concluded that the overexpression of the tobacco anionic peroxidase in transformed plants results in diminished root mass from fewer root branches, which contributes to the wilting phenomenon seen in these plants. Further, this developmental change in transformed plants may be a consequence of the metabolism of IAA by the anionic peroxidase.

Indoleacetic Acids↗
PubMed1996

Anionic tobacco peroxidase is active at extremely low pH: veratryl alcohol oxidation with a pH optimum of 1.8.

Tobacco peroxidase (36 kDa, pI 3.5) exhibits unique catalytic and spectral properties that are modulated by pH, calcium and magnesium ions. It catalyses the oxidation of veratryl alcohol by hydrogen peroxide over a wide pH range (1.5-5.0) in the presence of these metal ions with a pH optimum of 1.8. This is the only example of a holoperoxidase described so far that is active and comparatively stable at such a low pH. The enhancement of tobacco peroxidase activity by magnesium ions is to our knowledge the first example of a magnesium-induced peroxidase activation. UV/visible spectra of tobacco peroxidase showed that the Soret band shifted and its absorption coefficient increased upon the addition of calcium or magnesium ions and on lowering the pH. The tobacco peroxidase spectrum at pH 1.85, in the presence of calcium chloride (> 50 mM), is similar to that of lignin peroxidase at pH 6.0, with the Soret band shifting from 403 to 409 nm and the molar absorption coefficient increasing from 108,000 to 148,000 +/- 2000 M-1.cm-1 (results given +/- S.E.M.; n = 3). The data provide evidence for a low-affinity site for bivalent metal ion binding in addition to the two constitutive calcium sites that are present in all plant peroxidases. The presence of a glutamic acid residue (Glu-141) at the entrance to the haem-binding pocket, analogous to Glu-146 in lignin peroxidase and not present in other plant peroxidases, may account for these novel properties.

Benzyl Alcohols↗
PubMed1996

Mechanism of indole-3-acetic acid oxidation by plant peroxidases: anaerobic stopped-flow spectrophotometric studies on horseradish and tobacco peroxidases.

Indole-3-acetic acid (IAA) is a powerful plant growth regulator. The oxidative decarboxylation of IAA by plant peroxidases is thought to be a major degradation reaction involved in controlling the in vivo level of IAA. Horseradish peroxidase isoenzyme C and an anionic tobacco peroxidase isolated from transgenic Nicotiana sylvestris have been used in experiments in vitro designed to determine the mechanism of IAA oxidation. In particular, the initial reduction of ferric to ferrous enzyme, a key step in previously proposed mechanisms, has been investigated by rapid-scan stopped-flow spectrophotometry under strictly anaerobic conditions and at defined oxygen concentrations. The data provide the first evidence for a ternary complex comprising peroxidase, IAA and oxygen that is kinetically competent both at the initiation stage and during the catalytic cycle of IAA oxidation. A general scheme describing the oxidative cycles of both anionic and cationic peroxidases is proposed that includes native ferric enzyme and compound II as kinetically competent intermediates. For anionic peroxidases, addition of hydrogen peroxide switches on the oxidative cycle thereby promoting IAA oxidation. 2-Methyl-IAA is not a substrate of the oxidase reaction, suggesting a specific interaction between plant peroxidases and IAA.

Anaerobiosis↗
PubMed1996

Phytohormone control of the tobacco anionic peroxidase promoter.

The tobacco anionic peroxidase gene encodes the predominant peroxidase isoenzyme in the aerial portions of tobacco. Three kb of the peroxidase promoter was joined to the coding region of the Escherichia coli beta-glucuronidase gene (GUS), and transiently expressed in tobacco mesophyll protoplasts in the presence or absence of plant growth regulators. Benzyladenine, ethylene, and gibberellic acid did not affect peroxidase gene expression. Abscisic acid slightly inhibited expression at high concentrations. The auxins indole-3-acetic acid (IAA) and naphthaleneacetic acid strongly suppressed peroxidase expression. We observed half maximal suppression at 30 microM IAA. An anti-auxin, p-chlorophenoxyisobutyric acid (PCIB), enhanced expression from the peroxidase promoter above that of untreated controls or restored activity when used in combination with IAA. Sequencing 3 kb of the peroxidase promoter revealed many potential regulatory elements based on sequence homology to previously characterized genes. This includes several consensus transcription factor binding sites found in auxin-regulated promoters. 5' deletions of the peroxidase promoter/GUS fusion revealed several positive and negative regulatory elements. An upstream enhancer element was found between -3146 and -638 from the start of transcription. A strong silencer element was observed between -638 and -220. Removal of this silencer resulted in a truncated promoter (-220) with 100% activity of the full-length promoter (-3146). Inhibition by auxin was observed with all 5' deletions.

Base Sequence↗
PubMed1996

Purification and unusual kinetic properties of a tobacco anionic peroxidase.

The tobacco anionic peroxidase has been isolated from the leaves of transgenic Nicotiana sylvestris plants overproducing this enzyme. The plant expression system and the purification protocol developed allow the preparation of greater than 60 mg of homogeneous enzyme (M(r) 36 kDa, pI 3.5) from 1 kg of fresh leaves, which is an order of magnitude higher than for wild-type tobacco plants. The tobacco anionic peroxidase exhibits rather unusual catalytic properties in comparison with horseradish peroxidase (HRP C). Compound I is less active than Compound II in the tobacco enzyme. The enzyme is nearly inactive towards iodide, reflecting the peculiarities of its molecular structure. In particular, the presence of the negatively charged glutamate residue 141 at the entrance of the haeme-binding pocket seems to affect the stabilities of Compounds I, II and III, leading to a different enzyme substrate specificity than that of HRP C. Investigation of thermal stability towards a number of electron donors reveals the following 'order of stabilities': ferrocyanide > guaiacol > 2,2'-azino-bis(3-ethyl-6- benzothiazoline sulphonate) > iodide > o-dianisidine, which may indicate different binding sites and rate-limiting steps in the mechanism of the substrate oxidation.

Amino Acid Sequence↗