RESUMO
In recent years, genome sequencing has revealed that cytochromes P450 (P450s) constitute the largest family of enzymatic proteins in higher plants. P450s are mono-oxygenases that insert one atom of oxygen into inert hydrophobic molecules to make them more reactive and hydrosoluble. Besides their physiological functions in the biosynthesis of hormones, lipids and secondary metabolites, P450s help plants to cope with harmful exogenous chemicals including pesticides and industrial pollutants, making them less phytotoxic. The recovery of an increasing number of plant P450 genes in recombinant form has enabled their use in experimentation, which has revealed their extraordinary potential for engineering herbicide tolerance, biosafening, bioremediation and green chemistry.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Resistência a Medicamentos/genética , Herbicidas/farmacologia , Oxigenases/genética , Engenharia de Proteínas/métodos , Herbicidas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Especificidade por SubstratoRESUMO
Piperonylic acid (PA) is a natural molecule bearing a methylenedioxy function that closely mimics the structure of trans-cinnamic acid. The CYP73A subfamily of plant P450s catalyzes trans-cinnamic acid 4-hydroxylation, the second step of the general phenylpropanoid pathway. We show that when incubated in vitro with yeast-expressed CYP73A1, PA behaves as a potent mechanism-based and quasi-irreversible inactivator of trans-cinnamate 4-hydroxylase. Inactivation requires NADPH, is time dependent and saturable (KI = 17 &mgr;M, kinact = 0.064 min-1), and results from the formation of a stable metabolite-P450 complex absorbing at 427 nm. The formation of this complex is reversible with substrate or other strong ligands of the enzyme. In plant microsomes PA seems to selectively inactivate the CYP73A P450 subpopulation. It does not form detectable complexes with other recombinant plant P450 enzymes. In vivo PA induces a sharp decrease in 4-coumaric acid concomitant to cinnamic acid accumulation in an elicited tobacco (Nicotiana tabacum) cell suspension. It also strongly decreases the formation of scopoletin in tobacco leaves infected with tobacco mosaic virus.
RESUMO
trans-Cinnamate 4-hydroxylase (C4H) is a plant-specific cytochrome (P450) that is encoded by the gene CYP73A and catalyzes the second step of the multibranched phenylpropanoid pathway. Increases in C4H activity in response to physical and chemical stresses have been well documented, but the mechanism of these increases has never been studied in detail. This paper reports on the regulatory mechanism controlling C4H activity in Jerusalem artichoke (Helianthus tuberosus) tubers in response to wounding and chemical treatments. We compared induction of C4H and other P450-catalyzed activities. C4H was moderately induced by chemicals relative to other P450s. Increases in enzyme activity, C4H protein, and transcripts were quantified and compared in tuber tissue 48 h after wounding and chemical treatments. Our data suggest that induction of the enzyme activity results primarily from gene activation. Time-course experiments were performed after wounding and aminopyrine treatment. Compared with wounded tissues, aminopyrine triggered an additional and delayed peak of transcript accumulation. The timing of the induced changes in activity, protein, and transcripts confirms that C4H induction results primarily from an increase in CYP73A1 mRNA, in both wounded and aminopyrine-treated tissues. However, posttranscriptional mechanisms might also contribute to the regulation of C4H activity.
RESUMO
Cytochrome P450s (P450s) constitute one of the major classes of enzymes that are responsible for detoxification of exogenous molecules both in animals and plants. On the basis of its inducibility by exogenous chemicals, we recently isolated a new plant P450, CYP76B1, from Jerusalem artichoke (Helianthus tuberosus) and showed that it was capable of dealkylating a model xenobiotic compound, 7-ethoxycoumarin. In the present paper we show that CYP76B1 is more strongly induced by foreign compounds than other P450s isolated from the same plant, and metabolizes with high efficiency a wide range of xenobiotics, including alkoxycoumarins, alkoxyresorufins, and several herbicides of the class of phenylureas. CYP76B1 catalyzes the double N-dealkylation of phenylureas with turnover rates comparable to those reported for physiological substrates and produces nonphytotoxic compounds. Potential uses for CYP76B1 thus include control of herbicide tolerance and selectivity, as well as soil and groundwater bioremediation.
RESUMO
The phenylpropanoid pathway controls the synthesis of lignin, flower pigments, signalling molecules, and a large spectrum of compounds involved in plant defence against pathogens and UV light. More than 15 P450-dependent reactions have been characterised in this pathway. Several of these reactions constitute important regulatory branching points. Indirect and direct data indicate that distinct P450s catalyse the different reactions. The coding sequences of two enzymes have recently been determined. They belong to different P450 families, CYP73 and CYP75. The second enzyme of the main pathway, cinnamate 4-hydroxylase (C4H), is the most extensively studied plant P450 and is suspected of playing an essential role in the regulation of the whole pathway.
Assuntos
Cumarínicos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Flavonoides/metabolismo , Plantas/enzimologia , Fenômenos Fisiológicos VegetaisRESUMO
The ability of a plant cytochrome P450 to bind and metabolise plant endogenous molecules and xenobiotics was investigated. The work was performed on the yeast-expressed CYP73A1, a cinnamate 4-hydroxylase isolated from Helianthus tuberosus. CYP73 controls the general phenylpropanoid pathway and is likely to be one of the most abundant sources of P450 in the biosphere. The enzyme shows a high selectivity toward plant secondary metabolites. Nevertheless, it oxygenates several small and planar xenobiotics with low efficiency, including an herbicide (chlorotoluron). One xenobiotic molecule, 2-naphthoic acid, is hydroxylated with an efficiency comparable to that of the physiological substrate. This reaction was used to devise a fluorimetric test for the rapid measurement of enzyme activity. A series of herbicidal molecules (hydroxybenzonitriles) are shown to bind the active site without being metabolised. These molecules behave as strong competitive inhibitors of CYP73 with a K(i) in the same micromolar range as the K(m) for the physiological substrate. It is proposed that their inhibition of the phenylpropanoid pathway reinforces their other phytotoxic effects at the level of the chloroplasts. All our results indicate a strong reciprocal interaction between plant P450s and xenobiotics.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Oxigenases de Função Mista/metabolismo , Plantas/enzimologia , Fracionamento Celular , Sistema Enzimático do Citocromo P-450/análise , Sistema Enzimático do Citocromo P-450/biossíntese , Indução Enzimática , Microssomos/enzimologia , Oxigenases de Função Mista/análise , Oxigenases de Função Mista/biossíntese , Ensaio Radioligante , Espectrometria de Fluorescência , Transcinamato 4-Mono-OxigenaseRESUMO
Cytochrome P450 mono-oxygenases play prominent roles in a diverse set of metabolic pathways, but the function of most of these enzymes remains obscure. A bottleneck in the functional genomics of this superfamily constitutes hypothesis generation to identify potential substrates (or substrate classes) individual P450s may act on. We used publicly available large-scale expression data to perform co-expression analysis comparing the expression matrix of each P450 with those from more than 4000 selected genes across thousands of microarrays. Based on functional annotations of co-expressed genes from a diverse set of databases, co-expressed pathways were thus identified for each P450. Using this approach, most P450s with known functions were placed into their respective pathways, thereby proofing the concept. As examples, pathway mapping results identifying novel P450s potentially acting on flower-specific monoterpenes and root-specific triterpenes are described. Co-expression results for all Arabidopsis P450s will be presented as a web resource on the 'CYPedia' web pages (http://ibmp.u-strasbg.fr/CYPedia/).
Assuntos
Arabidopsis/enzimologia , Sistema Enzimático do Citocromo P-450/genética , Proteínas de Arabidopsis/metabolismo , Sistema Enzimático do Citocromo P-450/classificação , Flores/enzimologia , Perfilação da Expressão Gênica , Raízes de Plantas/enzimologia , Plântula/enzimologia , Sementes/enzimologiaRESUMO
A lauric acid monooxygenase which catalyzes the formation of hydroxylaurate from lauric acid has been characterized in ageing tissues of Jerusalem artichoke (Helianthus tuberosus L.) tuber. Three reaction products have been identified from the mass fragmentation pattern of their methyltrimethylsilyl derivatives: 10-hydroxylauric acid, 9-hydroxylauric acid and 8-hydroxylauric acid. Enzyme activity is located on the microsomal fraction which also carries cytochrome P-450 and NADPH cytochrome-c reductase. The apparent Km of the enzyme for lauric acid is 0.97 micronM. Laurate monooxygenation is dependent upon O2 and inhibited by CO. The latter effect is light reversible. NADPH is the preferred electron donor although appreciable NADH-sustained activity was observed. NADPH cytochrome c reductase is involved in electron transfer as evidenced by the inhibitory effects of NADP+ and oxidized cytochrome c on laurate monooxygenation. Thus, the enzyme catalyzing laurate oxidation in Jerusalem artichoke tuber tissues appears to be a typical (cytochrome P-450)-linked monooxygenase.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos/enzimologia , Oxigenases de Função Mista/metabolismo , Plantas/enzimologia , Cinética , Ácidos LáuricosRESUMO
Ovaries from normal and hypophysectomized 16-day-old duck embryos were cultured in vitro using 14C-sodium acetate as a precursor and the synthesis level of both 14C-labelled oestrone and oestradiol was determined in each case. The results demonstrate that the synthesis of oestradiol is under hypophyseal control as early as 16 days of incubation, while oestrone synthesis seems not yet to be dependent on pituitary LH at this same age.
Assuntos
Patos/embriologia , Estrona/biossíntese , Oviductos/embriologia , Hipófise/embriologia , Animais , Técnicas de Cultura , Embrião não Mamífero , Feminino , Hipofisectomia , Masculino , Oviductos/metabolismo , Hipófise/fisiologiaRESUMO
The substrate and product specifities of the (cytochrome P-450)-dependent laurate monooxygenase from tuber tissues of Jerusalem artichoke (Helianthus tuberosus L.) were investigated. The plant enzyme appeared strictly specific for the C12 free fatty acid and produced a mixture of C-8, C-9 and C-10 hydroxylated lauric acids, the C-9 derivative being predominant. No C-12 or C-11 hydroxylated laurates were detected. The activity of the enzyme, which was not detectable in the intact tuber, was induced by slicing and aging the tissues on water, and strongly superinduced by the addition of manganese and phenobarbital to the aging medium. Regulation of laurate hydroxylase was clearly independent from that of cinnamic acid 4-hydroxylase, another plant cytochrome P-450 enzyme.
Assuntos
Sistema Enzimático do Citocromo P-450 , Microssomos/enzimologia , Oxigenases de Função Mista/metabolismo , Plantas/enzimologia , Indução Enzimática , Oxigenases de Função Mista/biossíntese , Especificidade por SubstratoRESUMO
The mixed function oxidase trans-cinnamic acid 4-hydroxylase, cytochrome P-450, cytochrome b(5), and NADPH-cytochrome c (P-450) reductase were measured in microsomes from aging artichoke tuber slices exposed to manganese, ethanol, phenobarbital, and the herbicides Chloro-IPC, Dichlobenil, and Monuron. Although the microsomal hydroxylating complex is already induced by the slicing and aging process, 25 millimolar MnCl(2), 4 millimolar phenobarbital, and 300 millimolar ethanol caused a marked increase of hydroxylase activity and cytochrome P-450 content and shifted their time course. The herbicides, 200 micromolar Dichlobenil and 200 micromolar Monuron, were less effective. Chloro-IPC was slightly inhibitory. NADPH cytochrome c reductase was significantly increased only in phenobarbital-treated slices. Cytochrome b(5) was generally the least affected among the parameters being measured. The mechanisms by which these compounds increase cytochrome P-450 content and hydroxylase activity are not yet defined.
RESUMO
SUMMARY: Cytochrome P450 proteins, named for the absorption band at 450 nm of their carbon-monoxide-bound form, are one of the largest superfamilies of enzyme proteins. The P450 genes (also called CYP) are found in the genomes of virtually all organisms, but their number has exploded in plants. Their amino-acid sequences are extremely diverse, with levels of identity as low as 16% in some cases, but their structural fold has remained the same throughout evolution. P450s are heme-thiolate proteins; their most conserved structural features are related to heme binding and common catalytic properties, the major feature being a completely conserved cysteine serving as fifth (axial) ligand to the heme iron. Canonical P450s use electrons from NAD(P)H to catalyze activation of molecular oxygen, leading to regiospecific and stereospecific oxidative attack of a plethora of substrates. The reactions carried out by P450s, though often hydroxylation, can be extremely diverse and sometimes surprising. They contribute to vital processes such as carbon source assimilation, biosynthesis of hormones and of structural components of living organisms, and also carcinogenesis and degradation of xenobiotics. In plants, chemical defense seems to be a major reason for P450 diversification. In prokaryotes, P450s are soluble proteins. In eukaryotes, they are usually bound to the endoplasmic reticulum or inner mitochondrial membranes. The electron carrier proteins used for conveying reducing equivalents from NAD(P)H differ with subcellular localization. P450 enzymes catalyze many reactions that are important in drug metabolism or that have practical applications in industry; their economic impact is therefore considerable.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Animais , Catálise , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Evolução Molecular , Genes/genética , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Microsomes from apical buds of pea (Pisum sativum L. var. Téléphone à rames) seedlings hydroxylate lauric acid at the omega-position. This oxidation is catalyzed by a cytochrome P-450 enzyme which differs from laurate hydroxylases previously described in microorganisms and mammals by its strict substrate specificity and the ability of low NADH concentrations to support unusually high oxidation rates. The apparent K(m) for lauric acid was 20 micromolar. NADPH- and NADH-dependent laurate hydroxylation followed non-Michaelian kinetics with apparent K(m) values ranging from 0.2 to 28 micromolar for NADPH, and 0.2 to 318 micromolar for NADH. When induced by the photomorphogenic photoreceptor phytochrome, the time course for the enhancement of laurate omega-hydroxylase was totally different from that of the cinnamic acid 4-hydroxylase, providing evidence for the existence of multiple cytochrome P-450 species in pea microsomes.
RESUMO
Chlorophyll and haem synthesis in illuminated Jerusalem artichoke tuber tissues were very efficiently inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid). This inhibition seems to be due specifically to a blockade of the pathway for 5-aminolaevulinate biosynthesis which used glutamate as a substrate (the so-called C5 pathway) since we could not detect any inhibition of protein synthesis in the treated tissues and there was no effect of gabaculine on the glycine-dependent yeast 5-aminolaevulinate synthase used as a model. In dark-aged artichoke tissues, gabaculine also effectively blocked cytochrome P-450 induction, peroxidase activity and 5-aminolaevulinic acid synthesis, thus suggesting the involvement of a C5 pathway in cytoplasmic and microsomal haemoprotein synthesis in this higher plant. Allylglycine and (2-amino-ethyloxyvinyl)glycine, two olefinic glycine analogues which are potential suicide inhibitors of pyridoxal phosphate enzymes, were also demonstrated to be effective blockers of chlorophyll synthesis in artichoke tuber and Euglena cells exposed to light.
Assuntos
Ácido Aminolevulínico/biossíntese , Sistema Enzimático do Citocromo P-450/biossíntese , Helianthus/enzimologia , Heme/biossíntese , Ácidos Levulínicos/biossíntese , Alilglicina/farmacologia , Clorofila/biossíntese , Ácidos Cicloexanocarboxílicos/farmacologia , Escuridão , Transporte de Elétrons/efeitos dos fármacos , Indução Enzimática , Helianthus/efeitos dos fármacos , Luz , Peroxidases/antagonistas & inibidores , Proteínas de Plantas/biossíntese , Fosfato de Piridoxal/metabolismoRESUMO
Cinnamic acid 4-hydroxylase (CA4H) was purified from microsomes of manganese-induced Jerusalem artichoke (Helianthus tuberosus L.) tuber tissues. The three-step purification procedure involved solubilization and phase partitioning in Triton X-114, followed by chromatography on DEAE-Trisacryl and hydroxylapatite columns. Purification was monitored using carbon monoxide and type I substrate binding properties of the enzyme. The protein, purified to electrophoretic homogeneity, showed an Mr of about 57,000 on SDS-PAGE. Polyclonal antibodies raised against this protein selectively reacted with a 57-kDa polypeptide on Western blots of induced Jerusalem artichoke microsomes. The antibody selectively and strongly inhibited CA4H activity from several plant species.
Assuntos
Sistema Enzimático do Citocromo P-450/isolamento & purificação , Oxigenases de Função Mista/isolamento & purificação , Plantas/enzimologia , Aminoácidos/análise , Especificidade de Anticorpos , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/imunologia , Imunoquímica , Microssomos/enzimologia , Oxigenases de Função Mista/química , Oxigenases de Função Mista/imunologia , Peso Molecular , Transcinamato 4-Mono-OxigenaseRESUMO
The O-dealkylating activities of 7-ethoxycoumarin O-de-ethylase (ECOD) and 7-ethoxyresorufin O-de-ethylase (EROD) have been fluorimetrically detected in microsomes prepared from manganese-induced Jerusalem artichoke tubers. Cytochrome P-450 dependence of the reactions was demonstrated by light-reversed CO inhibition, NADPH-dependence, NADH-NADPH synergism and by use of specific inhibitors: antibodies to NADPH-cytochrome P-450 reductase, mechanism-based inactivators and tetcyclasis. Apparent Km values of 161 microM for 7-ethoxycoumarin and 0.4 microM for 7-ethoxyresorufin were determined. O-De-ethylase activity was also detected in microsomes prepared from several other plant species, including wheat, maize, tulip, avocado and Vicia. ECOD and EROD were low or undetectable in uninduced plant tissues, and both activities were stimulated by wounding or by chemical inducers. Two distinct cytochrome P-450 isoforms are involved in ECOD and EROD activities since (1) they showed different distributions among plant species; (2) they showed contrasting inhibition and induction patterns; and (3) ECOD but not EROD activity was supported by cumene hydroperoxide.
Assuntos
O-Dealquilase 7-Alcoxicumarina/metabolismo , Hidrocarboneto de Aril Hidroxilases , Cumarínicos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Oxazinas/metabolismo , Oxirredutases/metabolismo , Plantas/enzimologia , Triazóis/metabolismo , Derivados de Benzeno/metabolismo , Citocromo P-450 CYP1A1 , Citocromo P-450 CYP2A6 , Inibidores das Enzimas do Citocromo P-450 , Concentração de Íons de Hidrogênio , Cinética , Oxigenases de Função Mista/metabolismo , TemperaturaRESUMO
Two cytochrome P450 enzymes, cinnamate 4-hydroxylase (C4H) and 3,9-dihydroxypterocarpan 6a-hydroxylase (D6aH), were isolated from elicitor-challenged soybean (Glycine max) cell cultures (G. Kochs and H. Grisebach, 1989, Arch. Biochem. Biophys. 273, 543-553). An earlier purification protocol was improved by the use of new chromatographic media, leading to a higher yield of enzymatic activity. After separation of C4H from D6aH on hydroxyapatite, the C4H was identified using anti-C4H antibody from Jerusalem artichoke (Helianthus tuberosus) (B. Gabriac et al., 1991, Arch. Biochem. Biophys. 288, 302-309). The two proteins show molecular weights of about 58,000 for C4H and about 55,000 for D6aH on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both enzyme activities are dependent on NADPH:cytochrome P450 reductase and cross-react with their respective antibodies. Both cytochrome P450 subspecies show substrate binding and CO-difference spectra typical for cytochrome P450 and were found to be glycoproteins by their cross-reaction with biotinylated lectins in Western blot. The N-terminal sequence of C4H from soybean shows high similarity to the N-terminus of C4H from Jerusalem artichoke.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Glycine max/enzimologia , Oxigenases de Função Mista/metabolismo , Extratos Vegetais/biossíntese , Western Blotting , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Glicosilação , Oxigenases de Função Mista/isolamento & purificação , Sesquiterpenos , Proteínas de Soja , Análise Espectral , Terpenos , Transcinamato 4-Mono-Oxigenase , FitoalexinasRESUMO
Lauric acid in-chain hydroxylation is inhibited in microsomes from Jerusalem artichoke tubers (Helianthus tuberosus L.) incubated with 9-decenoic, 11-dodecenoic, or 11-dodecynoic acids. 9-Decenoic acid is at best a weak competitive inhibitor of the in-chain hydroxylase, but inactivates the enzyme in a time-dependent, pseudo-first-order process with a rate constant of approximately 1.1 X 10(-3) s-1. In contrast, 11-dodecenoic acid causes a slower, time-dependent loss of the hydroxylase activity, but is a potent competitive inhibitor of the enzyme (Ki = 2 microM). Neither agent decreases the microsomal concentration of cytochrome b5, NADH-cytochrome b5 reductase, or NADPH cytochrome P-450 reductase. Cinnamic acid 4-hydroxylation, catalyzed by a cytochrome P-450 enzyme, is not affected by concentrations of 9-decenoic acid that suppress lauric acid hydroxylation. 11-Dodecenoic acid is much less specific and, at higher concentrations, markedly reduces the microsomal cytochrome P-450 content, and the hydroxylation of both lauric and cinnamic acids.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Ácidos Láuricos/farmacologia , Oxigenases de Função Mista/antagonistas & inibidores , Plantas/enzimologia , Ligação Competitiva , Catálise , Sistema Enzimático do Citocromo P-450 , Helianthus/enzimologia , Cinética , Microssomos/enzimologia , NADP/metabolismo , Especificidade por SubstratoRESUMO
The potentiality of the Triton X-114 phase separation technique for the purification of proteins from plant microsomal membranes has been investigated. It was shown that glycerol significantly lowers the cloud point of Triton X-114 solutions in water and of Triton X-114 solubilizates from microsomal membranes. It was also established that solubilized membrane components decrease the temperature of Triton X-114 micellar aggregation. Solubilization of microsomal membrane using detergent to protein ratios lower than 3.5, however, resulted in complete inhibition of detergent phase separation. Phase partitioning of Triton X-114 microsomal solubilizates, performed at low temperature (4 degrees C), in the presence of glycerol, provided a very fast and efficient step for the purification of cytochromes P-450 and b5. Conditions allowing optimal recoveries of these cytochromes have been defined.