Justicidin B 7-hydroxylase, a cytochrome P450 monooxygenase from cell cultures of Linum perenne Himmelszelt involved in the biosynthesis of diphyllin

A biosynthetic pathway for the formation of arylnaphthalene lignans like diphyllin is suggested. Justicidin B 7-hydroxylase (JusB7H) which catalyzes the last step in the biosynthesis of diphyllin was characterized as a cytochrome P450 monooxygenase from suspension cultures of Linum perenne Himmelszelt. Cell suspension cultures of Linum perenne L. Himmelszelt accumulate justicidin B as the main component together with glycosides of 7-hydroxyjusticidin B (diphyllin). A hypothetical biosynthetic pathway for these compounds is suggested. Justicidin B 7-hydroxylase (JusB7H) catalyzes the last step in the biosynthesis of diphyllin by introducing a hydroxyl group in position 7 of justicidin B. This enzyme was characterized from a microsomal fraction prepared from a Linum perenne Himmelszelt 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. JusB7H has a pH optimum of 7.4 and a temperature optimum of 26 °C. Justicidin B was the only substrate accepted by JusB7H with an apparent K m of 3.9 ± 1.3 μM. NADPH is predominantly accepted as the electron donor, but NADH was a weak co-substrate. A synergistic effect of NADPH and NADH was not observed. The apparent K m for NADPH is 102 ± 10 μM.