RESUMO
In Apiaceae, furanocoumarins (FCs) are plant defence compounds that are present as linear or angular isomers. Angular isomers appeared during plant evolution as a protective response to herbivores that are resistant to linear molecules. Isomeric biosynthesis occurs through prenylation at the C6 or C8 position of umbelliferone. Here, we report cloning and functional characterization of two different prenyltransferases, Pastinaca sativa prenyltransferase 1 and 2 (PsPT1 and PsPT2), that are involved in these crucial reactions. Both enzymes are targeted to plastids and synthesize osthenol and demethylsuberosin (DMS) using exclusively umbelliferone and dimethylallylpyrophosphate (DMAPP) as substrates. Enzymatic characterization using heterologously expressed proteins demonstrated that PsPT1 is specialized for the synthesis of the linear form, demethylsuberosin, whereas PsPT2 more efficiently catalyses the synthesis of its angular counterpart, osthenol. These results are the first example of a complementary prenyltransferase pair from a single plant species that is involved in synthesizing defensive compounds. This study also provides a better understanding of the molecular mechanisms governing the angular FC biosynthetic pathway in apiaceous plants, which involves two paralogous enzymes that share the same phylogenetic origin.
Assuntos
Dimetilaliltranstransferase/metabolismo , Evolução Molecular , Furocumarinas/biossíntese , Pastinaca/metabolismo , Proteínas de Plantas/metabolismo , Catharanthus/genética , Membrana Celular/metabolismo , Clonagem Molecular , Cumarínicos/metabolismo , Dimetilaliltranstransferase/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Plastídeos/metabolismo , Especificidade por Substrato , Nicotiana/genética , Umbeliferonas/biossíntese , Umbeliferonas/metabolismoRESUMO
Coumarins are important compounds that contribute to the adaptation of plants to biotic or abiotic stresses. Among coumarins, umbelliferone occupies a pivotal position in the plant phenylpropanoid network. Previous studies indicated that umbelliferone is derived from the ortho-hydroxylation of p-coumaric acid by an unknown biochemical step to yield 2,4-dihydroxycinnamic acid, which then undergoes spontaneous lactonization. Based on a recent report of a gene encoding a 2-oxoglutarate-dependent dioxygenase from Arabidopsis thaliana that exhibited feruloyl CoA 6'-hydroxylase activity (Bourgaud et al., 2006), we combined a bioinformatic approach and a cDNA library screen to identify an orthologous ORF (Genbank accession number JF799117) from Ruta graveolens L. This ORF shares 59% amino acid identity with feruloyl CoA 6'-hydroxylase, was functionally expressed in Escherichia coli, and converted feruloyl CoA into scopoletin and p-coumaroyl CoA into umbelliferone with equal activity. Its bi-functionality was further confirmed in planta: transient expression of JF799117 in Nicotiana benthamiana yielded plants with leaves containing high levels of umbelliferone and scopoletin when compared to control plants, which contained barely detectable traces of these compounds. The expression of JF799117 was also tightly correlated to the amount of umbelliferone that was found in UV-elicited R. graveolens leaves. Therefore, JF799117 encodes a p-coumaroyl CoA 2'-hydroxylase in R. graveolens, which represents a previously uncharacterized step in the synthesis of umbelliferone in plants. Psoralen, which is an important furanocoumarin in R. graveolens, was found to be a competitive inhibitor of the enzyme, and it may exert this effect through negative feedback on the enzyme at an upstream position in the pathway.
Assuntos
Dioxigenases/metabolismo , Ruta/enzimologia , Umbeliferonas/biossíntese , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência Conservada , Cumarínicos/análise , Cumarínicos/isolamento & purificação , Cumarínicos/metabolismo , Dioxigenases/antagonistas & inibidores , Dioxigenases/genética , Dioxigenases/isolamento & purificação , Escherichia coli/enzimologia , Escherichia coli/genética , Furocumarinas/metabolismo , Furocumarinas/farmacologia , Expressão Gênica/genética , Dados de Sequência Molecular , Filogenia , Folhas de Planta/química , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Raízes de Plantas/química , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Caules de Planta/química , Caules de Planta/enzimologia , Caules de Planta/genética , RNA de Plantas/metabolismo , Ruta/química , Ruta/genética , Escopoletina/análise , Escopoletina/metabolismo , Alinhamento de Sequência , Nicotiana/enzimologia , Nicotiana/genética , Transgenes , Umbeliferonas/análise , Umbeliferonas/metabolismoRESUMO
Cytochrome P450 (P450) 2A6 mutants from randomized libraries generated in the substrate recognition sequence (SRS) regions were screened in Escherichia coli on the basis of indole metabolism. SRS 3 and 4 libraries yielded colonies that produced indigo at least as well as wild-type (WT) P450 2A6, and some colonies were consistently more blue upon replating. One mutant, F209T, showed indole 3-hydroxylation
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Indóis/química , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Mutagênese , Membrana Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Corantes/análise , Corantes/metabolismo , Cumarínicos/química , Cumarínicos/metabolismo , Citocromo P-450 CYP2A6 , Sistema Enzimático do Citocromo P-450/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Índigo Carmim , Indóis/análise , Indóis/metabolismo , Cinética , Oxigenases de Função Mista/metabolismo , Oxirredução , Espectrofotometria , Umbeliferonas/biossínteseRESUMO
A test designed to estimate the extent and rate of formation of 7-hydroxycoumarin by measuring the urinary excretion of the metabolite in humans after administering 5 mg coumarin was developed. Coumarin was rapidly metabolized after oral administration and more than 95% of the 7OHC formed was excreted in 4 h. The total amount of 7OHC formed was 64 +/- 15% (mean +/- SD, variation 20-100%) of the dose given. The percentage of 7OHC excreted in 2 h, as compared with the 7OHC excretion in 4 h, was found to be a constant and stable individual characteristic for the rate of the formation of 7OHC ('2 h coumarin test'). In 110 volunteers, there was a great interindividual variability in the extent and rate of 7OHC formation. Four individuals had relatively 'slow' coumarin test values (50-60%), but much larger populations would be needed for the demonstration of polymorphism.