RESUMO
Treatment of etiolated Vicia sativa seedlings by the plant hormone methyl jasmonate (MetJA) led to an increase of cytochrome P450 content. Seedlings that were treated for 48 h in a 1 mM solution of MetJA stimulated omega-hydroxylation of 12:0 (lauric acid) 14-fold compared with the control (153 versus 11 pmol min-1 mg-1 protein, respectively). Induction was dose dependent. The increase of activity (2.7-fold) was already detectable after 3 h of treatment. Activity increased as a function of time and reached a steady level after 24 h. Northern-blot analysis revealed that the transcripts coding for CYP94A1, a fatty acid omega-hydroxylase, had already accumulated after 1 h of exposure to MetJA and was maximal between 3 and 6 h. Under the same conditions, a study of the enzymatic hydrolysis of 9,10-epoxystearic acid showed that both microsomal and soluble epoxide hydrolase activities were not affected by MetJA treatment.
RESUMO
Cytochrome P450-dependent enzymes from wheat catalyze the oxidation of endogenous compounds (lauric and oleic acids) and of several herbicides (diclofop, chlortoluron, bentazon). Treatment of wheat seedlings with the safener, naphthalic anhydride and with phenobarbital increases dramatically several P450-dependent enzyme activities including diclofop and lauric acid hydroxylation. The parallel induction of lauric acid (omega-1)-hydroxylase and diclofop hydroxylase activities suggests that both compounds proceeds from the same or very similar forms of P450. To test whether either one or multiple P450 forms are involved in these oxidations, we have designed selective irreversible inhibitors of lauric acid (omega-1)-hydroxylase. Results of in vivo and in vitro experiments with acetylenic analogs of lauric acid (10- and 11-dodecynoic acids) strongly suggest that a single P450 catalyzes both laurate and diclofop hydroxylation. Treatment of wheat seedlings with these acetylenes results in a strong inhibition of the in vivo metabolism of diclofop although oxidation of chlortoluron and bentazon are not affected. Our results suggest that at least three distinct P450 forms are involved in the detoxification process of the three herbicides. Interestingly, we also demonstrate that herbicides themselves are potent inducers of the amount of total P450 and laurate/diclofop hydroxylase activies. This increased capacity of wheat to detoxify the herbicide through the induction of P450 enzymes seems to be for a large extend the mechanism which confers a tolerance on various herbicides.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Herbicidas/metabolismo , Herbicidas/toxicidade , Triticum/enzimologia , Triticum/genética , Benzotiadiazinas/metabolismo , Benzotiadiazinas/toxicidade , Cromatografia em Camada Fina , Inibidores das Enzimas do Citocromo P-450 , Resistência a Medicamentos , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Éteres Difenil Halogenados , Microssomos/enzimologia , Oxirredução , Fenobarbital/farmacologia , Éteres Fenílicos/metabolismo , Éteres Fenílicos/toxicidade , Compostos de Fenilureia/metabolismo , Compostos de Fenilureia/toxicidade , SementesAssuntos
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
NADPH-cytochrome P-450 (cytochrome c) reductase (EC 1.6.2.4) was solubilized by detergent from microsomal fraction of wounded Jerusalem-artichoke (Helianthus tuberosus L.) tubers and purified to electrophoretic homogeneity. The purification was achieved by two anion-exchange columns and by affinity chromatography on 2',5'-bisphosphoadenosine-Sepharose 4B. An Mr value of 82,000 was obtained by SDS/polyacrylamide-gel electrophoresis. The purified enzyme exhibited typical flavoprotein redox spectra and contained equimolar quantities of FAD and FMN. The purified enzyme followed Michaelis-Menten kinetics with Km values of 20 microM for NADPH and 6.3 microM for cytochrome c. In contrast, with NADH as substrate this enzyme exhibited biphasic kinetics with Km values ranging from 46 microM to 54 mM. Substrate saturation curves as a function of NADPH at fixed concentration of cytochrome c are compatible with a sequential type of substrate-addition mechanism. The enzyme was able to reconstitute cinnamate 4-hydroxylase activity when associated with partially purified tuber cytochrome P-450 and dilauroyl phosphatidylcholine in the presence of NADPH. Rabbit antibodies directed against plant NADPH-cytochrome c reductase affected only weakly NADH-sustained reduction of cytochrome c, but inhibited strongly NADPH-cytochrome c reductase and NADPH- or NADH-dependent cinnamate hydroxylase activities from Jerusalem-artichoke microsomal fraction.
Assuntos
Microssomos/enzimologia , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Plantas/enzimologia , Cromatografia por Troca Iônica , Cinamatos/metabolismo , Eletroforese em Gel de Poliacrilamida , Flavoproteínas/metabolismo , Cinética , NADPH-Ferri-Hemoproteína Redutase/imunologia , NADPH-Ferri-Hemoproteína Redutase/isolamento & purificação , EspectrofotometriaRESUMO
Monoclonal antibodies (mAbs) against a plant NADPH-cytochrome P-450 (Cyt P-450) reductase from Jerusalem artichoke (Helianthus tuberosus) tuber were prepared. These antibodies were produced by hybridoma resulting from the fusion of spleen cells from a rat immunized with a purified preparation of the reductase and mouse myeloma cells. The mAbs thus obtained were screened for their interaction with the reductases, first in western dots and then in blots, and for their ability to inhibit the NADPH-cytochrome c (Cyt c) reductase activity from Jerusalem artichoke microsomes. Among the 11 clones giving a positive response on western blots, only 6 were also able to inhibit microsomal NADPH-Cyt c reductase activity, and the microsomal Cyt P-450 monooxygenase activities dependent upon electrons transferred by the reductase. Thus, two families of mAbs were characterized: a family of mAbs that interact with epitopes of the reductase implicated in the reduction of Cyt P-450 by NADPH (binding sites for NADPH, flavin mononucleotide, flavin adenine dinucleotide, and Cyt P-450), and a structural family, whose members recognize epitopes outside the active site of the reductases. These mAbs specifically recognize the reductase, and all of them interact with all of the isoforms, indicating that important primary or secondary structural analogies exist between the isoforms, not only at the active site, but also at the level of epitopes not directly associated with catalytic activity.
RESUMO
Polyclonal antibodies were prepared against NADPH-cytochrome P-450 reductase purified from Jerusalem artichoke. These antibodies inhibited efficiently the NADPH-cytochrome c reductase activity of the purified enzyme, as well as of Jerusalem artichoke microsomes. Likewise, microsomal NADPH-dependent cytochrome P-450 mono-oxygenases (cinnamate and laurate hydroxylases) were efficiently inhibited. The antibodies were only slightly inhibitory toward microsomal NADH-cytochrome c reductase activity, but lowered NADH-dependent cytochrome P-450 mono-oxygenase activities. The Jerusalem artichoke NADPH-cytochrome P-450 reductase is characterized by its high Mr (82,000) as compared with the enzyme from animals (76,000-78,000). Western blot analysis revealed cross-reactivity of the Jerusalem artichoke reductase antibodies with microsomes from plants belonging to different families (monocotyledons and dicotyledons). All of the proteins recognized by the antibodies had an Mr of approx. 82,000. No cross-reaction was observed with microsomes from rat liver or Locusta migratoria midgut. The cross-reactivity generally paralleled well the inhibition of reductase activity: the enzyme from most higher plants tested was inhibited by the antibodies; whereas Gingko biloba, Euglena gracilis, yeast, rat liver and insect midgut activities were insensitive to the antibodies. These results point to structural differences, particularly at the active site, between the reductases from higher plants and the enzymes from phylogenetically distant plants and from animals.
Assuntos
Helianthus/enzimologia , Imunoglobulina G/imunologia , NADPH-Ferri-Hemoproteína Redutase/imunologia , Reações Cruzadas , Técnicas Imunoenzimáticas , Microssomos/enzimologia , Plantas/enzimologiaRESUMO
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
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
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
Fatty acid omega-hydroxylation is involved in the biosynthesis of the plant cuticle, formation of plant defense signaling molecules, and possibly in the rapid catabolism of free fatty acids liberated under stress conditions. CYP94A2 is a cytochrome P450-dependent medium-chain fatty acid hydroxylase that was recently isolated from Vicia sativa. Contrary to CYP94A1 and CYP86A1, two other fatty acid hydroxylases previously characterized in V. sativa and Arabidopsis thaliana, CYP94A2 is not a strict omega-hydroxylase, but exhibits chain-length-dependent regioselectivity of oxidative attack. Sequence alignments of CYP94A2 with CYP94A1 and molecular modeling studies suggested that F494, located in SRS-6 (substrate recognition site) was involved in substrate recognition and positioning. Indeed, a conservative amino acid substitution at that position markedly altered the regiospecificity of CYP94A2. The observed shift from omega toward omega-1 hydroxylation was prominent with lauric acid as substrate and declined with increasing fatty acid chain length.
Assuntos
Sistema Enzimático do Citocromo P-450 , Oxigenases de Função Mista/metabolismo , Rosales/enzimologia , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência Conservada , Hidroxilação , Leucina/metabolismo , Oxigenases de Função Mista/genética , Modelos Moleculares , Dados de Sequência Molecular , Fenilalanina/genética , Fenilalanina/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Valina/metabolismoRESUMO
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.
Assuntos
Sistema Enzimático do Citocromo P-450/isolamento & purificação , Citocromos b5/isolamento & purificação , Temperatura Baixa , Glicerol , Proteínas de Membrana/isolamento & purificação , Microssomos/química , Octoxinol , Proteínas de Plantas/isolamento & purificação , PolietilenoglicóisRESUMO
We report on the presence of multiple forms of NADPH-cyt P450 reductase in microsomes from higher plants. This contrasts with the animal cyt P450 monooxygenases, where the numerous cyt P450 isoforms are reduced by a single form of reductase. Three NADPH-cyt c reductases have been resolved from Jerusalem artichoke tuber microsomes by chromatography on Reactive Red Agarose and Concanavalin A-Sepharose. Their molecular weights, determined by sodium dodecylsulfate-gel electrophoresis, are 80,000, 82,000 and 84,000. The three proteins share common epitopes and are dependent upon FMN for catalytic activity. They are highly selective for NADPH as electron donor, and allowed effective reconstitution of trans-cinnamic acid and 3,9-dihydroxypterocarpan 6a-hydroxylase activities with purified cyt P450 fractions from Helianthus tuberosus and Glycine max, respectively. As such, they appear as true isoenzyme forms of NADPH-cyt P-450 reductase.
Assuntos
Isoenzimas/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Plantas/enzimologia , Cromatografia de Afinidade/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Mononucleotídeo de Flavina/metabolismo , Isoenzimas/isolamento & purificação , Cinética , Microssomos/enzimologia , Peso Molecular , NADPH-Ferri-Hemoproteína Redutase/isolamento & purificaçãoRESUMO
The A. thaliana EST database was screened using consensus motifs derived from P450 families CYP52 and CYP4 catalyzing the omega-hydroxylation of fatty acids and alkanes in Candida and in mammals. One EST cDNA fragment was detected in this way and the corresponding full-length cDNA was cloned from a cDNA library of A. thaliana. This cDNA coded the first member of a new plant P450 family and was termed CYP86A1. The deduced peptide sequence showed highest homology with P450s from families 4 and 52. To confirm the catalytic function, CYP86A1 was expressed in a yeast overexpressing its own NADPH-P450 reductase. Efficient expression was evidenced by spectrophotometry, SDS-PAGE and catalytic activity. CYP86A1 was found to catalyze the omega-hydroxylation of saturated and unsaturated fatty acids with chain lengths from C12 to C18 but not of hexadecane. Genomic organization analyzed by Southern blot suggested a single gene encoding CYP86A1 in A. thaliana.
Assuntos
Arabidopsis/enzimologia , Sistema Enzimático do Citocromo P-450/genética , Genes de Plantas , Oxigenases de Função Mista/genética , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis , Bacillus megaterium/enzimologia , Sequência de Bases , Southern Blotting , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Ácidos Graxos/metabolismo , Hidroxilação , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Filogenia , Homologia de SequênciaRESUMO
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
A full length cDNA encoding a new cytochrome P450-dependent fatty acid hydroxylase (CYP94A2) was isolated from a Vicia sativa library. CYP94A2 displays 58% sequence identity with CYP94A1, a fatty acid omega-hydroxylase isolated from the same material. Heterologous expression of CYP94A2 in Saccharomyces cerevisiae yeast strain WAT11 shows that it catalyses the hydroxylation of myristic (C14) acid with a K(m(app)) of 4.0 microM and a turnover rate number of 80 min(-1). In addition, lauric (C12) and palmitic (C16) acids were hydroxylated at a ten-fold lower rate, while C18 fatty acids were not oxidized. Remarkably, the regiospecificity of hydroxylation is different for the C12, C14, and C16 fatty acids and appears to be correlated with the length of the carbon chain. Northern blot analysis showed a low level of constitutive expression of CYP94A2 in V. sativa seedlings. In contrast to CYP94A1, transcript accumulation of CYP94A2 was only weakly enhanced in seedlings treated with clofibrate or methyl jasmonate, indicating that both substrate range and gene regulation of the two fatty acid hydroxylases are different.
Assuntos
Fabaceae/enzimologia , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Plantas Medicinais , Acetatos/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Clofibrato/farmacologia , Clonagem Molecular , Ciclopentanos/farmacologia , Fabaceae/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hidroxilação , Cinética , Oxigenases de Função Mista/química , Oxigenases de Função Mista/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Ácido Mirístico/metabolismo , Oxilipinas , Saccharomyces cerevisiae/genética , Sementes/efeitos dos fármacos , Sementes/enzimologia , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The C(18) fatty acid derivatives 9,10-epoxystearic acid and 9,10-dihydroxystearic acid were hydroxylated on the terminal methyl by microsomes of yeast expressing CYP94A1 cloned from Vicia sativa. The reactions did not occur in incubations of microsomes from yeast transformed with a void plasmid or in the absence of NADPH. After incubation of a synthetic racemic mixture of 9,10-epoxystearic acid, the chirality of the residual epoxide was shifted to 66:34 in favour of the 9S,10R enantiomer. Both the 9S,10R and 9R,10S enantiomers were incubated separately. We determined respective K(m) and V(max) values of 1.2+/-0.1 microM and 19.2+/-0.3 nmol/min per nmol of cytochrome P450 for the 9R,10S enantiomer and of 5.9+/-0.1 microM and 20.2+/-1.0 nmol/min per nmol of cytochrome P450 for the 9S,10R enantiomer. This demonstrated that CYP94A1 is enantioselective for the 9R,10S, which is preferentially formed in V. sativa microsomes. Cutin analysis of V. sativa seedlings revealed that it is mainly constituted of derivatives of palmitic acid, a C(16) fatty acid. Our results suggest that CYP94A1 might play a minor role in cutin synthesis and could be involved in plant defence. Indeed, 18-hydroxy-9,10-epoxystearic acid and 9,10,18-trihydroxystearic acid have been described as potential messengers in plant-pathogen interactions.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Hidroxiácidos/metabolismo , Oxigenases de Função Mista/metabolismo , Ácido Oleico/metabolismo , Rosales/enzimologia , Clonagem Molecular , Epóxido Hidrolases/metabolismo , Hidroxilação , Cinética , Lipídeos de Membrana/metabolismo , Microssomos/enzimologia , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Especificidade por SubstratoRESUMO
CYP86B1 was cloned from a cDNA library and the protein expressed in E. coli. The protein gave the expected carbon monoxide difference spectrum. Using in vitro import assays with isolated pea chloroplasts, CYP86B1 was shown to be associated with the outer chloroplastic envelope membrane. This study provides the first direct evidence for a chloroplast-localized cytochrome P450-dependent monooxygenase.
Assuntos
Cloroplastos/química , Sistema Enzimático do Citocromo P-450/análise , Pisum sativum/química , Sequência de Aminoácidos , Proteínas de Arabidopsis , Cloroplastos/genética , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , DNA Complementar , DNA de Plantas , Escherichia coli , Membranas Intracelulares/química , Dados de Sequência Molecular , Pisum sativum/genética , Proteínas de Plantas/análise , Proteínas de Plantas/genéticaRESUMO
A full length cDNA encoding a new cytochrome P450-dependent fatty acid hydroxylase (CYP94A5) was isolated from a tobacco cDNA library. CYP94A5 was expressed in S. cerevisiae strain WAT11 containing a P450 reductase from Arabidopsis thaliana necessary for catalytic activity of cytochrome P450 enzymes. When incubated for 10 min in presence of NADPH with microsomes of recombinant yeast, 9,10-epoxystearic acid was converted into one major metabolite identified by GC/MS as 18-hydroxy-9,10-epoxystearic acid. The kinetic parameters of the reaction were Km,app = 0.9 +/- 0.2 microM and Vmax,app = 27 +/- 1 nmol x min(-1) x nmol(-1) P450. Increasing the incubation time to 1 h led to the formation of a compound identified by GC/MS as 9,10-epoxy-octadecan-1,18-dioic acid. The diacid was also produced in microsomal incubations of 18-hydroxy-9,10-epoxystearic acid. Metabolites were not produced in incubations with microsomes of yeast transformed with a control plasmid lacking CYP94A5 and their production was inhibited by antibodies raised against the P450 reductase, demonstrating the involvement of CYP94A5 in the reactions. The present study describes a cytochrome P450 able to catalyze the complete set of reactions oxidizing a terminal methyl group to the corresponding carboxyl. This new fatty acid hydroxylase is enantioselective: after incubation of a synthetic racemic mixture of 9,10-epoxystearic acid, the chirality of the residual epoxide was 40/60 in favor of 9R,10S enantiomer. CYP94A5 also catalyzed the omega-hydroxylation of saturated and unsaturated fatty acids with aliphatic chain ranging from C12 to C18.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Ácidos Graxos/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Nicotiana/enzimologia , Oxigênio/metabolismo , Plantas Tóxicas , Álcoois/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Catálise , Cromatografia em Camada Fina , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , DNA Complementar/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Biblioteca Gênica , Cinética , Microssomos/metabolismo , Oxigenases de Função Mista/genética , Dados de Sequência Molecular , Saccharomyces cerevisiae/metabolismo , Ácidos Esteáricos/metabolismo , Especificidade por Substrato , Fatores de TempoRESUMO
The major C(18) cutin monomers are 18-hydroxy-9,10-epoxystearic and 9,10,18-trihydroxystearic acids. These compounds are also known messengers in plant-pathogen interactions. We have previously shown that their common precursor 9,10-epoxystearic acid was formed by the epoxidation of oleic acid in Vicia sativa microsomes (Pinot, Salaün, Bosch, Lesot, Mioskowski and Durst (1992) Biochem. Biophys. Res. Commun. 184, 183-193). Here we determine the chirality of the epoxide produced as (9R,10S) and (9S,10R) in the ratio 90:10 respectively. We further show that microsomes from yeast expressing the cytochrome P450 CYP94A1 are capable of hydroxylating the methyl terminus of 9,10-epoxystearic and 9,10-dihydroxystearic acids in the presence of NADPH to form the corresponding 18-hydroxy derivatives. The reactions were not catalysed by microsomes from yeast transformed with a void plasmid or in absence of NADPH. After incubation of a synthetic racemic mixture of 9,10-epoxystearic acid with microsomes of yeast expressing CYP94A1, the chirality of the residual epoxide was shifted to 66:34 in favour of the (9S,10R) enantiomer. Both enantiomers were incubated separately and V(max)/K(m) values of 16 and 3.42 ml/min per nmol of P450 for (9R, 10S) and (9S,10R) respectively were determined, demonstrating that CYP94A1 is enantioselective for the (9R,10S) enantiomer, which is preferentially formed in V. sativa microsomes. Compared with the epoxide, the diol 9,10-dihydroxystearic acid was a much poorer substrate for the omega-hydroxylase, with a measured V(max)/K(m) of 0.33 ml/min per nmol of P450. Our results indicate that the activity of CYP94A1 is strongly influenced by the stereochemistry of the 9, 10-epoxide and the nature of substituents on carbons 9 and 10, with V(max)/K(m) values for epoxide>>oleic acid>diol.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Fabaceae/enzimologia , Lipídeos de Membrana/metabolismo , Oxigenases de Função Mista/metabolismo , Plantas Medicinais , Ácidos Esteáricos/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Citocromo P-450 CYP4A , Sistema Enzimático do Citocromo P-450/genética , Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Fabaceae/genética , Interações Hospedeiro-Parasita , Hidroxilação , Cinética , Lipídeos de Membrana/química , Microssomos/enzimologia , Microssomos/metabolismo , Oxigenases de Função Mista/genética , NADP/metabolismo , Ácidos Oleicos/química , Ácidos Oleicos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Transdução de Sinais , Ácidos Esteáricos/química , Estereoisomerismo , Especificidade por SubstratoRESUMO
The chemical tagging of a cytochrome P-450-dependent lauric acid omega-hydroxylase from clofibrate-treated Vicia sativa seedlings with [1-14C]11-dodecynoic acid allowed the isolation of a full-length cDNA designated CYP94A1. We describe here the functional expression of this novel P-450 in two Saccharomyces cerevisiae strains overproducing their own NADPH-cytochrome P-450 reductase or a reductase from Arabidopsis thaliana. The results show a much higher efficiency of the yeast strain overproducing the plant reductase compared with the yeast strain overproducing its own reductase for expressing CYP94A1. The methyl end of saturated (from C-10 to C-16) and unsaturated (C18:1, C18:2 and C18:3) fatty acids was mainly oxidized by CYP94A1. Both E/Z and Z/E configurations of 9, 12-octadecadienoic acids were omega-hydroxylated. Lauric, myristic and linolenic acids were oxidized with the highest turnover rate (24 min-1). The strong regioselectivity of CYP94A1 was clearly shifted with sulphur-containing substrates, since both 9- and 11-thia laurate analogues were sulphoxidized. Similar to animal omega-hydroxylases, this plant enzyme was strongly induced by clofibrate treatment. Rapid CYP94A1 transcript accumulation was detected less than 20 min after exposure of seedlings to the hypolipidaemic drug. The involvement of CYP94A1 in the synthesis of cutin monomers and fatty acid detoxification is discussed.