Molecular characterization of a feruloyl-CoA 6'-hydroxylase involved in coumarin biosynthesis in Clematis terniflora DC.

Coumarin is an important secondary metabolite that affects plant physiology. It is a lactone of cis-o-hydroxycinnamic acid and widely exists in medicinal plants. Clematis terniflora DC. is a plant belonging to Ranunculaceae and is rich in variety of coumarins. Feruloyl-CoA 6'-hydroxylase has been reported as a key enzyme in the formation of coumarin basic skeleton only in some common plants, however, its evidence in other species is still lacking especially for the biosynthesis of coumarins in C. terniflora. In the present study, we identified a feruloyl-CoA 6'-hydroxylase CtF6'H in C. terniflora, and functional characterization indicated that CtF6'H could hydroxylate feruloyl-CoA to 6-hydroxyferuloyl-CoA. Furthermore, the expression level of CtF6'H was differed among different tissues in C. terniflora, while under UV-B radiation, the level of CtF6'H was increased in the leaves. Biochemical characteristics and subcellular location showed that CtF6'H was mainly present in the cytosol. The crystal structure of CtF6'H was simulated by homology modeling to predict the potential residues affecting enzyme activity. This study provides the additional evidence of feruloyl-CoA 6'-hydroxylase in different plant species and enriches our understanding of biosynthetic mechanism of coumarin in C. terniflora.