Identification moléculaire et caractérisation fonctionnelle d'une nouvelle sous-famille de cytochromes P450, CYP71AZ, impliquée dans la synthèse de furanocoumarines et coumarines chez Pastinaca sativa

Abstract : Furanocoumarins (FCs) are secondary metabolites mainly synthetized in four botanical families deriving from the phenylpropanoid biosynthetic pathway. These phytoalexins are involved in plant defense mechanisms and present strong therapeutic potential. Early studies in the 1960s based on cell cultures and the use of radiolabeled precursors have shown that many enzymes involved in this pathway belong to the cytochrome P450 family (P450s). Only two of them had been identified from a molecular point of view at the beginning of this thesis. In order to generate information regarding the genome of plants producing FCs, we sequenced the mRNA extracted from leaves of Pastinaca sativa, Ruta graveolens, and Cullen cinereum. In silico analysis of these three libraries identified nearly 800 cDNA fragments encoding for P450s. Previous studies in the laboratory and comparative transcriptome analysis of these three plants have led us to focus on the subfamily CYP71AZ through a detailed study of CYP71AZ3 and CYP71AZ4. Functional characterization of these enzymes was performed in an eukaryote heterologous expression system: Saccharomyces cerevisiae. The results showed that CYP71AZ4 had a broad substrate specificity enough as it could metabolize one FC and 4 coumarins. The analysis and comparison of the kinetic constants for each of these substrates indicate, however, that the preferred substrate is psoralen. The functional characterization of CYP71AZ3 showed that this enzyme could hydroxylate esculetin, a coumarin, but played no role in the synthesis of FCs. This study highlights the functional diversity within a single enzyme subfamily and allows to issue new hypotheses about the emergence of this biosynthetic pathway in Apiaceae on one hand, and among other botanical families on the other hand
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Célia Krieger. Identification moléculaire et caractérisation fonctionnelle d'une nouvelle sous-famille de cytochromes P450, CYP71AZ, impliquée dans la synthèse de furanocoumarines et coumarines chez Pastinaca sativa. Alimentation et Nutrition. Université de Lorraine, 2014. Français. ⟨NNT : 2014LORR0185⟩. ⟨tel-01751146⟩

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