Untersuchungen zur Rolle von Cytochrom P450-Enzymen in der Biosynthese von Aryltetralin-Lignanen in Zellkulturen von Linum spec.

Lignans, phenolic compounds of plant secondary metabolism, are derived from the aminoacid phenylalanin and are widely distributed throughout the plant kingdom. Podophyllotoxin (PTOX), an aryltetralin-lignan, is highly cytotoxic and of medicinal interest, because semisynthetic glycosides like etopiside and teniposide are derived from it. Podophyllum plants, collected in the wild, are the most important source of podophyllotoxin today. Podophyllotoxin-like lignans were also found in some linum species. In this thesis, studies about the biosynthesis of lignans like PTOX and 6-mezhoxypodophyllotoxin (MPTOX)in suspension cultures of linum nodiflorum, linum flavum and linum album are described. The main focus is on the role of cytochrome p450 monooxygenases, a class of enzymes that can catalyze a lot of oxidative reactions in plant secondary metabolism. Biotransformation experiments in l. nodiflorum gave hints, that the formation of PTOX from desoxypodophyllotoxin (DOP), catalyzed by a DOP7H, could be cytochrome p450 dependent, because it was specifically inhibited by tetcyclacis. In microsomal preparations from l. nodiflorum DOP7H activity was not detectable. The desoxypodophyllotoxin 6-hydroxylase (DOP6H), recently described in l. flavum, could be detected in suspension cultures of l. nodiflorum and characterised in cell free extracts. Temperature and pH-optima were at 35 °C and pH 7.4, respectively. The inhibitory effect of different substances, like the known cytochrome p450-specific inhibitors cytochrome c and tetcyclacis, the substrate-analogon 4'-demethyl-DOP and divalent cations, espacially Mn2+ was shown. The Km-values of DOP and NADPH were determined as 8,3 µM and 47 µM, respectively. No synergistic effect, as known from animal and human systems, could be found with the alternative cosubstrate NADH. This could be due to a possible participation of cytochrome b5 in the electron transport chain to DOP6H in vitro. The biotechnological potential of DOP6H was investigated by the use of Zn/Co(III)-sepulchrate as alternative electron donor. A purification strategy via affinity chromatography with 4'-demethyl-DOP is discussed. Yatein is a possible precursor of DOP in lignan-biosynthesis. A turnover of Yatein to DOP, as described in podophyllum spec., could not be detected in the examined cell cultures. This may be due to the fact, that the biosynthetic routes in the genera podophyllum and linum are different. The possible role of yatein is discussed. Additionally, cloning of NADPH:cytochrome P450-reductases (CPRs) from the mentioned cell cultures was started. Via RT-PCR subclones of CPRs from l. album, l. flavum and l. nodiflorum were isolated. DOP seems to be a branching point in lignan-biosynthesis, because it is a precursor of PTOX and MPTOX alike. A better understanding of enzymatic turmover of DOP allows for an estimation of the biotechnological potential of the lignan-biosynthesis in linum cell cultures.