Biosynthesis of justicidin B and diphyllin in cell cultures of Linum perenne L. Himmelszelt

The arylnaphthalene lignans justicidin B (JusB) and diphyllin (Diph) exhibit several pharmaceutically interesting properties as antimicrobial, anti-inflammatory and anticancer activity. Cell suspension cultures of Linum perenne L. Himmelszelt accumulate JusB as the main component together with Diph-glycosides. A hypothetical biosynthetic pathway for these compounds is suggested. A cDNA encoding a pinoresinol-lariciresinol reductase (PLR-Lp1) which catalyses two early steps in the lignan biosynthesis was cloned from a cell culture of L. corymbulosum. Recombinant PLR-Lp1 prefers 8R, 8'R-pinoresinol in the first reaction step, but 8S, 8'S-lariciresinol in the second. We could prove its involvement in arylnaphthalene lignan biosynthesis in hairy roots of L. corymbulosum by an RNAi approach. Justicidin B 7-hydroxylase (JusB7H) catalyzes the last step in the biosynthesis of Diph by introducing a hydroxyl group in position 7 of JusB. This enzyme was characterized from a microsomal fraction prepared from a Linum perenne H suspension culture for the first time. The hydroxylase activity was strongly inhibited by cytochrome C as well as other cytochrome P450 inhibitors like clotrimazole indicating the involvement of a cytochrome P450-dependent monooxygenase. Justicidin B was the only substrate accepted by JusB7H with an apparent K _m of 3.9±1.3µM. NADPH is predominantly accepted as the electron donor, but NADH was a weak cosubstrate. A synergistic effect of NADPH and NADH was not observed. The apparent K _m for NADPH is 102±10µM.

Acknowledgements: Financial support from the Deutsche Forschungsgemeinschaft and from the „Gesellschaft von Freunden und Förderern der Heinrich-Heine-Universität Düsseldorf“ is gratefully acknowledged.

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