SummaryWe investigated the P450 dependent¯avonoid hydroxylase from the ornamental plant Catharanthus roseus. cDNAs were obtained by heterologous screening with the CYP75 Hf1 cDNA from Petunia hybrida. The C. roseus protein shared 68±78% identity with other CYP75s, and genomic blots suggested one or two genes. The protein was expressed in Escherichia coli as translational fusion with the P450 reductase from C. roseus. Enzyme assays showed that it was a¯avonoid 3¢,5¢-hydroxylase, but 3¢-hydroxylated products were also detected. The substrate speci®city was investigated with the C. roseus enzyme and a fusion protein of the Petunia hybrida CYP75 with the C. roseus P450 reductase. Both enzymes accepted¯avanones as well as¯avones, dihydro¯avonols and¯avonols, and both performed 3¢-as well as 3¢5¢-hydroxylation. Kinetics with C. roseus cultures on the level of enzyme activity, protein and RNA showed that the F3¢5¢H was present in dark-grown cells and was induced by irradiation. The same results were obtained for cinnamic acid 4-hydroxylase and avanone 3b-hydroxylase. In contrast, CHS expression was strictly dependent on light, although CHS is necessary in the synthesis of the F3¢5¢H substrates. Immunohistochemical localization of F3¢5¢H had not been performed before. A comparison of CHS and F3¢5¢H in cotyledons and¯ower buds from C. roseus identi®ed CHS expression preferentially in the epidermis, while F3¢5¢H was only detected in the phloem. The cell-type speci®c expression suggests that intercellular transport may play an important role in the compartmentation of the pathways to the different¯avonoids.

Vinblastine and vincristine are two medically important bisindole alkaloids from Catharanthus roseus (Madagascar periwinkle). Attempts at production in cell cultures failed because a part of the complex pathway was not active, i.e. from tabersonine to vindoline. It starts with tabersonine 16-hydroxylase (T16H), a cytochrome P450-dependent enzyme. We now show that T16H is induced in the suspension culture by light and we report the cloning of the cDNA. The enzyme was expressed in Escherichia coli as translational fusion with the P450 reductase from C. roseus, and the reaction product was identified by mass spectrometry. The protein (CYP71D12) shares 47^52% identity with other members of the CYP71D subfamily with unknown function. The induction by light was strongly enhanced by a nutritional downshift (transfer into 8% aqueous sucrose). We discuss the possibility that the entire pathway to bisindoles can be expressed in suspension cultures.z 1999 Federation of European Biochemical Societies.