共查询到20条相似文献,搜索用时 15 毫秒
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Kopecný D Sebela M Briozzo P Spíchal L Houba-Hérin N Masek V Joly N Madzak C Anzenbacher P Laloue M 《Journal of molecular biology》2008,380(5):886-899
Cytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N6-(buta-2,3-dienyl)adenine (HA-8) and N6-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 Å resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent. 相似文献
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David Kopečný Pierre Briozzo Hana Popelková Marek Šebela Radka Končitíková Lukáš Spíchal Jaroslav Nisler Catherine Madzak Ivo Frébort Michel Laloue Nicole Houba-Hérin 《Biochimie》2010
Cytokinin oxidase/dehydrogenase (CKO) is a flavoenzyme, which irreversibly degrades the plant hormones cytokinins and thereby participates in their homeostasis. Several synthetic cytokinins including urea derivatives are known CKO inhibitors but structural data explaining enzyme–inhibitor interactions are lacking. Thus, an inhibitory study with numerous urea derivatives was undertaken using the maize enzyme (ZmCKO1) and the crystal structure of ZmCKO1 in a complex with N-(2-chloro-pyridin-4-yl)-N′-phenylurea (CPPU) was solved. CPPU binds in a planar conformation and competes for the same binding site with natural substrates like N6-(2-isopentenyl)adenine (iP) and zeatin (Z). Nitrogens at the urea backbone are hydrogen bonded to the putative active site base Asp169. Subsequently, site-directed mutagenesis of L492 and E381 residues involved in the inhibitor binding was performed. The crystal structures of L492A mutant in a complex with CPPU and N-(2-chloro-pyridin-4-yl)-N′-benzylurea (CPBU) were solved and confirm the importance of a stacking interaction between the 2-chloro-4-pyridinyl ring of the inhibitor and the isoalloxazine ring of the FAD cofactor. Amino derivatives like N-(2-amino-pyridin-4-yl)-N′-phenylurea (APPU) inhibited ZmCKO1 more efficiently than CPPU, as opposed to the inhibition of E381A/S mutants, emphasizing the importance of this residue for inhibitor binding. As highly specific CKO inhibitors without undesired side effects are of major interest for physiological studies, all studied compounds were further analyzed for cytokinin activity in the Amaranthus bioassay and for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4. By contrast to CPPU itself, APPU and several benzylureas bind only negligibly to the receptors and exhibit weak cytokinin activity. 相似文献
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The effects of three N(6)-substituted aminopurine derivatives containing either allenic or acetylenic side-chains on in vitro and in vivo cytokinin dehydrogenase (CKX; EC 1.5.99.12) activities were determined. At concentrations < or = 100 microM, the acetylenic derivative (HA-2) had no effect on in vitro CKX activity. In contrast, the two allenic derivatives (HA-1, HA-8) inhibited in vitro CKX activity in a dose-dependent manner with 50% inhibition occurring at HA-1 and HA-8 concentrations of 9.0 and 0.4 microM (respectively). HA-8 inhibited the degradation of both the free bases and ribosides of N6-(2-isopentenyl)adenine and zeatin. Pretreatment with HA-8 inhibited CKX activity in both a time- and concentration-dependent manner. In contrast to the reversible phenylurea inhibitor N-(chloro-4-pyridyl)-N'-phenylurea, inhibition of CKX activity by HA-8 was not relieved by 24 h of dialysis. Both HA-1 and HA-8 (but not HA-2) inhibited the metabolism of exogenous [3H]-N(6)-(2-isopentenyl)adenosine in excised aseptic potato (Solanum tuberosum) leaves. These results demonstrate that HA-8 is a mechanism-based irreversible (suicide) inhibitor of CKX and indicate that it may be useful in determining the role of CKX in cytokinin homeostasis in planta. 相似文献
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Cytokinin oxidase/cytokinin dehydrogenase assay: optimized procedures and applications 总被引:1,自引:0,他引:1
1H NMR spectroscopy has been used to assess long-term toxicological effects of a rare earth. Male Wistar rats were administrated orally with La(NO3)3 at doses of 0.1, 0.2, 2.0, 10, and 20 mg/kg body wt, resp., for 3-6 months. Urine was collected at 1, 2, and 3 months and serum samples were taken after 6 months. Numerous low-M(r) metabolites in rats serum and rats urine, including creatinine, citrate, glucose, ketone bodies, trimethylamine N-oxide (TMAO), and various amino acids, were identified on 400- and 500-MHz 1H NMR spectra. La3+-induced renal and liver damage is characterized by an increase in the amounts of the excreted ketone bodies, amino acids, lactate, ethanol, succinate, TMAO, dimethylamine, and taurine and a decrease in citrate, glucose, urea, and allantoin. Information on the molecular basis of the long-term toxicity of La(NO>3)3 was derived from the abnormal patterns of metabolite excretions. An assay of some biochemical indexes and analysis of some enzymes in plasma supported NMR results. 相似文献
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Mietha M. Van der Walt Gisella Terre’Blanche Anél Petzer Jacobus P. Petzer 《Bioorganic & medicinal chemistry letters》2012,22(21):6632-6635
Monoamine oxidase (MAO) plays an essential role in the catabolism of neurotransmitter amines. The two isoforms of this enzyme, MAO-A and -B, are considered to be drug targets for the therapy of depression and neurodegenerative diseases, respectively. Based on a recent report that the phthalimide moiety may be a useful scaffold for the design of potent MAO-B inhibitors, the present study examines a series of 5-sulfanylphthalimide analogues as potential inhibitors of both human MAO isoforms. The results document that 5-sulfanylphthalimides are highly potent and selective MAO-B inhibitors with all of the examined compounds possessing IC50 values in the nanomolar range. The most potent inhibitor, 5-(benzylsulfanyl)phthalimide, exhibits an IC50 value of 0.0045 μM for the inhibition of MAO-B with a 427-fold selectivity for MAO-B compared to MAO-A. We conclude that 5-sulfanylphthalimides represent an interesting class of MAO-B inhibitors and may serve as lead compounds for the design of antiparkinsonian therapy. 相似文献
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P Galuszka I Frébort M Sebela P Sauer S Jacobsen P Pec 《European journal of biochemistry》2001,268(2):450-461
An enzyme degrading cytokinins with isoprenoid side chain, previously named cytokinin oxidase, was purified to near homogeneity from wheat and barley grains. New techniques were developed for the enzyme activity assay and staining on native electrophoretic gels to identify the protein. The purified wheat enzyme is a monomer 60 kDa, its N-terminal amino-acid sequence shows similarity to hypothetical cytokinin oxidase genes from Arabidopsis thaliana, but not to the enzyme from maize. N6-isopentenyl-2-(2-hydroxyethylamino)-9-methyladenine is the best substrate from all the cytokinins tested. Interestingly, oxygen was not required and hydrogen peroxide not produced during the catalytic reaction, so the enzyme behaves as a dehydrogenase rather than an oxidase. This was confirmed by the ability of the enzyme to transfer electrons to artificial electron acceptors, such as phenazine methosulfate and 2,6-dichlorophenol-indophenol. 2,3-Dimethoxy-5-methyl-1,4-benzoquinone, a precursor of the naturally occurring electron acceptor ubiquinone, readily interacts with the enzyme in micromolar concentrations. Typical flavoenzyme inhibitors such as acriflavine and diphenyleneiodonium inhibited this enzyme activity. Presence of the flavin cofactor in the enzyme was confirmed by differential pulse polarography and by measuring the fluorescence emission spectrum. Possible existence of a second redox centre is discussed. 相似文献
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Irina Vaseva Dessislava Todorova Jiří Malbeck Alena Trávníčková Ivana Macháčková 《Acta Physiologiae Plantarum》2008,30(2):151-155
Changes in cytokinin pool and cytokinin oxidase/dehydrogenase activity (CKX EC: 1.5.99.12) in response to increasing abscisic
acid (ABA) concentrations (0.5–10 μM) were assessed in the last fully expanded leaves and secondary roots of two pea (Pisum sativum) varieties with different vegetation periods. Certain organ diversity in CKX response to exogenous ABA was observed. Treatment
provoked altered cytokinin pool in the aboveground parts of both studied cultivars. Specific CKX activity was influenced significantly
basically in roots of the treated plants. Results suggest that ABA-mediated cytokinin pool changes are leaf-specific and involve
certain root signals in which CKX activity presents an important link. This enzymatic activity most probably regulates vascular
transport of active cytokinins from roots to shoots. 相似文献
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Maturing maize kernels are a rich source of cytokinins and cytokinin oxidase/dehydrogenase activity, but the relationship between kernel development, cytokinin levels, the induction of cytokinin oxidase/dehydrogenase and the control of cell division is not known. Using polyclonal antibodies raised against recombinant maize cytokinin oxidase/dehydrogenase, we investigated the appearance of cytokinin oxidase/dehydrogenase (ZmCKX1) in both hybrid and inbred maize kernels as a function of time after pollination. Cytokinin oxidase/dehydrogenase was detected by five days after pollination (5 DAP) in a hybrid line, but significantly later in inbred lines. The bulk of the cytokinin oxidase/dehydrogenase detected was associated with the embryo and placental/chalazal region of the kernels rather than with the endosperm. We identified additional maize sequences in the database that appear to encode cytokinin oxidase/dehydrogenase gene family members and correspond closely with a subset of the ten cytokinin oxidase/dehydrogenase genes identified in the rice genome. Gene expression of Zmckx1 was examined by RT-PCR in immature kernels and compared with that of three putative maize cytokinin oxidase/dehydrogenase homologs. We conclude that the manipulation of kernel cytokinin levels to increase endosperm cell division will require a more detailed understanding of specific expression patterns and localization of multiple cytokinin oxidase/dehydrogenases within kernels. 相似文献
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D. Todorova I. Vaseva J. Malbeck A. Trávníčková I. Macháčková E. Karanov 《Biologia Plantarum》2007,51(3):579-583
Changes in endogenous cytokinin (CK) content and cytokinin oxidase/dehydrogenase activity (CKX) in response to gibberellic
acid (GA3) in two pea cultivars with different life span were assessed. The control leaves of cv. Scinado, which developed faster,
had higher initial cytokinin content and lower CKX activity, while opposite trend was observed in cv. Manuela with longer
life span. Increased CKX and decreased CK content were detected in leaves of cv. Scinado after treatments with 0.5, 1 and
5 μM GA3. Changes in CK content and CKX activity in GA3-treated cv. Manuela leaves were reciprocal to those in cv. Scinado. CK content and CKX activity in roots were not significantly
influenced by the application of GA3. The slight repression of CKX activity in some of the root samples was accompanied by increased isopentenyladenine and isopentenyladenine
riboside content. Obtained results suggest that CKX was responsible for the changes in endogenous cytokinin pool in GA3-treated plants and most probably this enzyme represents an important link in GA/cytokinin cross talk. 相似文献
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Kopecný D Pethe C Sebela M Houba-Hérin N Madzak C Majira A Laloue M 《Biochimie》2005,87(11):1011-1022
Cytokinin oxidase/dehydrogenase (CKO/CKX) is a flavoenzyme, which irreversibly inactivates cytokinins by severing the isoprenoid side chain from the adenine/adenosine moiety. There are several genes coding for the enzyme in maize (Zea mays). A Z. mays CKO1 cDNA was cloned in the yeast Yarrowia lipolytica to achieve heterologous protein expression. The recombinant ZmCKO1 was recovered from cultures of transformed yeasts and purified using several chromatographic steps. The enzyme was obtained as a homogeneous protein in a remarkably high-yield and its molecular and kinetic properties were characterized. The enzyme showed a molecular mass of 69 kDa, pI was 6.3. Neutral sugar content of the molecule was 22%. Absorption and fluorescence spectra were in accordance with the presence of FAD as a cofactor. Peptide mass fingerprinting using MALDI-MS correctly assigned the enzyme in MSDB protein database. The enzyme showed a relatively high degree of thermostability (T50=55 degrees C for 30 min incubation). The following pH optimum and K(m) values were determined for natural substrates (measured in the oxidase mode): pH 8.0 for isopentenyl adenine (K(m)=0.5 microM), pH 7.6 for isopentenyl adenosine (K(m)=1.9 microM), pH 7.9 for zeatin (K(m)=1.5 microM) and pH 7.3 for zeatin riboside (K(m)=2.0 microM). ZmCKO1, functioning in the oxidase mode, catalyzes the production of one molecule of H2O2 per one molecule of cytokinin substrate. This finding represents clear evidence for the existence of dual enzyme functionality (oxygen serves as a cosubstrate in the absence of better electron acceptors). 相似文献
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James G. Laskey Paige Patterson Kristin Bilyeu Roy O. Morris 《Plant Growth Regulation》2003,40(3):189-196
Cytokinin oxidase/dehydrogenase degrades cytokinins by dehydrogenating the N6-C1 bond of cytokinins. The resulting imine is then hydrolyzed. For example, isopentenyl-adenine is cleaved into 3-methyl-2-butenal (isopentenyl-aldehyde) and adenine . The reducing equivalents from dehydrogenation are transferred to an unknown sink, in vivo. It has been hypothesized that the enzyme requires oxygen , possibly resulting in the formation of hydrogen peroxide. 2,6-dichloroindophenol (DCPIP) can function as an acceptor of reducing equivalents for in vitro cytokinin oxidase/dehydrogenase reactions. For the predominant cytokinin oxidase/dehydrogenase in maize, ZmCKX1, the addition of DCPIP to in vitro reactions increases the reaction rate to nearly 4000-fold faster than the oxygen-dependent rate. Further, the change in absorbance of DCPIP at 600 nm, as it is reduced, forms the basis for an assay suitable for following biochemical purification of cytokinin oxidase/dehydrogenases , detailed kinetic studies , and rapid measurement of cytokinin oxidase/dehydrogenase activity in large numbers of samples. 相似文献
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Milana Trifunović Aleksandar Cingel Ana Simonović Slađana Jevremović Marija Petrić Ivana Č. Dragićević Václav Motyka Petre I. Dobrev Lenka Zahajská Angelina Subotić 《Plant Cell, Tissue and Organ Culture》2013,115(2):139-150
Cytokinin oxidase/dehydrogenase (CKX) is the only known enzyme involved in cytokinin catabolism. Genes coding for two Arabidopsis CKX isoforms, AtCKX1 and AtCKX2, were introduced separately into a binary cloning vector, immobilized into Agrobacterium tumefaciens strain GV3101, and introduced into root explants of centaury (Centaurium erythraea Rafn.). The integration of each transgene was confirmed by genomic PCR. Of the total transformed explants, 30 and 28.2 % of the transformants carried AtCKX1 and AtCKX2 transgenes, respectively. Of these transformants, 50 % exhibited expression of the AtCKX1 transgene, while 64 % of transformants exhibited expression of the AtCKX2 transgene. For all analysed AtCKX transgenic centaury lines, as well as for untransformed control plants, CKX activity was higher in roots than in shoots. Expression of AtCKX in most transgenic lines contributed to enhanced levels of CKX activity in root tissues; whereas, only a few lines demonstrated increased CKX activity in shoot tissues compared to those of control plants. Moreover, overexpression of AtCKX resulted in reduced morphogenetic potential in transgenic plants, but did not significantly affect biomass production in comparison to untransformed control plants. 相似文献
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The content of cytokinins (CKs), the plant inhibitors of the final phase of plant development, senescence, is effectively
controlled by irreversible degradation catalysed by cytokinin oxidase/dehydrogenase (CKX). In transgenic tobacco, denoted
as AtCKX, with over-expressed CKX causing lowered CK content, we investigated changes in the time courses of chlorophyll (Chl)
and xanthophyll (violaxanthin, antheraxanthin, zeaxanthin, neoxanthin, and lutein) contents. We also determined parameters
of slow Chl fluorescence kinetics such as minimum Chl fluorescence yield in the darkadapted state F0, maximum quantum yield of PS2 photochemistry (Fv/Fm), maximum ratio of quantum yields of photochemical and concurrent non-photochemical processes in photosystem 2 (PS2), Fv/F0, non-photochemical quenching (NPQ), and effective quantum yield of photochemical energy conversion in PS2 (Φ2). We used three different developmental leaf stages, old, mature, and young, and compared this with time courses of these
characteristics in leaves with natural CK levels. The parameters Fv/Fm, Fv/F0, and Φ2 were unchanged during ageing in AtCKX plants in contrast to control ones where a significant decrease in old leaves was found.
In control plants F0 increased during ageing, but in the oldest leaf a considerable decrease was observed. This could indicate progressive damage
to PS2 reaction centres and then detachment and rapid degradation of Chl. This is in agreement with time course of Chl content.
NPQ decreased with age and was similar in both plant types. We observed a decline of xanthophyll contents in the oldest leaves
in both plant types, but the contents were enhanced in AtCKX compared to control plants, especially of neoxanthin. The higher
xanthophyll contents in the transgenic plants contribute to a better photoprotection and the fluorescence parameters indicated
that photosynthetic apparatus was in better condition compared to control and it consequently postponed the onset of leaf
senescence. 相似文献
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Following a study of the relationship between cytokinin oxidase/dehydrogenase (CKX) and senescence in darkened barley leaf segments, we have now investigated the influence of light on the in vitro activity of CKX. Seedlings of Hordeum vulgare L. were grown for 8 d under a light/dark regime of 18 h white light and 6 h darkness. Then apical parts of 7 cm length were cut from the first foliage leaves and their bases were placed in water. In segments kept in the dark, the CKX activity measured by cleavage of N6-(Δ2-isopentenyl)adenine rose from 0.1 pkat (g FW)−1 to 0.8 pkat (g initial FW)−1 within the first 4 d of incubation. In contrast, in segments kept under the light/dark regime it reached a value of 8.6 pkat (g initial FW)−1 over the same time period. The chlorophyll a content declined slightly slower during light/dark cycling than in darkness. In contrast to segments and isolated laminae, corresponding attached laminae exhibited less CKX activity after 2 d under light/dark conditions than after 2 d in the dark. The activity in attached laminae of first foliage leaves of plants growing in light/dark cycling increased strongly only when the plants were older than 4 weeks. In line with this, the CKX activity in attached laminae of flag leaves of barley growing in fields increased in a late developmental state. The senescence of darkened isolated laminae of Zea mays L. and Phragmites australis (Cav.) Trin. ex Steudel was associated with an enhancement of CKX activity too. Because in most cases a positive correlation between CKX activity and senescence was found, it is likely that the enzyme promotes senescence by destroying cytokinins, which help to keep Poaceae leaves green. Light may promote not only cytokinin degradation but also the formation of bioactive cytokinins in leaf segments. 相似文献
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A cytokinin oxidase in Zea mays 总被引:10,自引:0,他引:10
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We studied changes in physiological parameters of whole leaves and in antioxidant protection of chloroplasts during ageing
and senescence of tobacco (Nicotiana tabacum L. cv. Samsun NN) leaves with enhanced cytokinin oxidase/dehydrogenase activity (CKX) or without it (WT). Old leaves of CKX
plants maintained higher pigment content and photosystem 2 activity compared to WT leaves of the same age. Chloroplasts of
old CKX plants showed better antioxidant capacity represented by higher superoxide dismutase, dehydroascorbate reductase and
glutathione reductase activities. 相似文献
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Stirk WA Novák O Zižková E Motyka V Strnad M van Staden J 《Journal of plant physiology》2012,169(7):696-703
Tagetes minuta L. achenes are thermoinhibited at temperatures above 35°C and have accelerated radicle emergence (germination) when subsequently transferred to an optimal temperature (25°C). Endogenous cytokinins and cytokinin oxidase/dehydrogenase (CKX) activity were compared in normally germinating (25°C) and thermoinhibited (72h at 36°C then transferred to 25°C) T. minuta achenes. Following imbibition, endogenous cytokinin concentrations changed in normally germinating T. minuta achenes, with a gradual decrease in dihydrozeatin-type (DHZ) cytokinins, a large increase in cis-zeatin-type (cZ) cytokinins, a smaller increase in N?-(2-isopentenyl)adenine-type (iP) cytokinins and a peak of trans-zeatin-type (tZ) cytokinins at 13 h. These changes in the isoprenoid cytokinin profile were similar in the thermoinhibited achenes imbibed at 36°C, despite the thermal block preventing radicle emergence. The exception was the iP-type cytokinins that only increased when transferred to 25°C. Profiles of the physiologically active free bases showed an increase in tZ prior to radical emergence in both normally germinating (13 h) and thermoinhibited achenes. A large transient peak in aromatic cytokinins [N?-benzyladenine-type (BA)] occurred during early seedling establishment in normally germinating achenes (40 h) while a transient maximum in BA-type cytokinins was found prior to radicle emergence in the thermoinhibited achenes (24 h). The CKX activity was enhanced in normally germinating achenes as the cytokinin concentration increased following imbibition. In thermoinhibited achenes, an elevated temperature negatively affected the CKX activity that only increased when the achenes were transferred to 25°C, corresponding to an increase in iP-type cytokinins. However, the favored cytokinin deactivation pathway in T. minuta appears to be 9-glycosylation, as 9-glucosides accounted for over 50% of the total cytokinin pool in both normal and thermoinhibited achenes. 相似文献