Aryltetralin-lignan formation in two different cell suspension cultures of Linum album: deoxypodophyllotoxin 6-hydroxylase, a key enzyme for the formation of 6-methoxypodophyllotoxin.

ABSTRACT

Suspension cultures initiated from two different Linum album seedlings accumulate either podophyllotoxin (PTOX, 2.6 mg/g DW) or 6-methoxypodophyllotoxin (6MPTOX, 5.4 mg/g DW) as main lignans. Two molecules of coniferyl alcohol are dimerized to pinoresinol which is converted via several steps into deoxypodophyllotoxin (DOP) which seems to be the branching point to PTOX or 6MPTOX biosynthesis. DOP is hydroxylated at position 7 to give PTOX by deoxypodophyllotoxin 7-hydroxylase (DOP7H). In contrast, 6MPTOX biosynthesis is achieved by DOP hydroxylation at position 6 to beta-peltatin by the cytochrome P450 enzyme deoxypodophyllotoxin 6-hydroxylase (DOP6H). The following methylation to beta-peltatin-A-methylether is catalyzed by beta-peltatin 6-O-methyltransferase (betaP6OMT) from which 6MPTOX is formed by hydroxylation at position 7 by beta-peltatin-A-methylether 7-hydroxylase (PAM7H). DOP6H and betaP6OMT could be characterized in protein extracts from cell cultures of L. flavum and L. nodiflorum, respectively, and here in L. album for the first time. DOP7H and PAM7H activities could not yet be detected with protein extracts. Experiments of feeding DOP together with inhibitors of cytochrome P450 depending as well as dioxygenase enzymes were performed in order to shed light on the type of DOP7H and PAM7H. Growth parameters and specific activities of enzymes from the phenylpropane as well as the lignan specific biosynthetic pathway were measured during a culture period of 16 days. From the enzymes studied only the DOP6H showed a differential activity sustaining the hypothesis that this enzyme is responsible for the differential lignan accumulation in both cell lines.