Elicitor‐induced biosynthesis of psoralens in Ammi majus L. suspension cultures

Abstract

Suspension cultures of Ammi majus L. cells produce various linear furanocoumarins in response to treatment with elicitor preparations from either Alternaria carthami Chowdhury or Phytophthora megasperma f.sp. glycinea. Microsomes which were isolated from these cells 14 h after addition of the elicitor eficiently catalyzed the conversion of demethyl[3 -''C]suberosin into labelled (+)marmesin in the presence of NADPH and oxygen. In contrast to the chemical cyclization of demethylsuberosin by rn-chloroperoxybenzoic acid, the reaction catalyzed by the marmesin synthase proceeded rapidly and no intermediate demethylsuberosin epoxide could be recovered. Significant blue-light-reversible inhibition by carbon monoxide and inhibition by various chemicals known to inhibit reactions dependent on cytochrome P450 suggested that the marmesin synthase is a cytochrome-P450-dependent monooxygenase.Upon prolonged incubation, a subsequent major labelled product originated from (+)marmesin, which was identified as psoralen. The psoralen synthase was also characterized as a cytochrome-P4,0-dependent monooxygenase. Both the marmesin synthase and the psoralen synthase, as well as enzymes catalyzing the formation of demethylsuberosin and 0-prenylumbelliferone from umbelliferone and dimethylallyl diphosphate, were associated with the endoplasmic reticulum in Ammi majus cells and their activities were concomitantly induced by elicitor treatment of the cells. We propose that in vivo these enzymes are active in the lumen of the endoplasmic reticulum from where the furanocoumarin phytoalexins are excreted into the cell culture fluid.

Molecular Cloning and Functional Characterization of Psoralen Synthase, the First Committed Monooxygenase of Furanocoumarin Biosynthesis

“…5). Overall, the data demonstrated that CYP71AJ1 encodes psoralen synthase, which is probably too labile for detection in isolated yeast microsomes, in accordance with previous observations on A. majus microsomes (36,42). The activity might be stabilized by adjustment of the N-terminal anchor sequence.…”

“…Three consecutive cytochrome P450-dependent reactions converting demethylsuberosin to bergaptol (Fig. 1) and identified in Ammi microsomes (35,36) provided the basis for the differential cloning approach, which fully confirmed the previous proposal of separate CYP entities (31,35,36). Psoralen synthase represents the first cloned coumarin-committed monooxygenase, and CYP71AJ1 gives access to a new CYP71 subfamily of enzymes.…”

“…It is conceivable that psoralen synthase activity essentially requires the tight association with the membrane and the reductase, which is supported by the beneficial effect of replacing the membrane insertion region by that of CYP73A1, although the activity of isolated microsomes remained unstable upon storage. Recombinant psoralen synthase showed narrow specificity and high affinity (K m 1.5 M) for (ϩ)-marmesin (Table 1), compatible with the observation of (ϩ)-marmesin as an intermediate of psoralen and bergaptol synthesis in elicited A. majus cells (36). An alternate pathway to bergaptol, however, appeared possible based on 5-hydroxylation of (ϩ)-marmesin and subsequent cyclization.…”

“…Isoproturon and chlortoluron were from CIL (Paris, France). Demethylsuberosin was synthesized as described previously (36) Cell Cultures and Separation of Coumarins-Cell suspensions of A. majus L. (40-ml B5 cultures in 250-ml flasks) were propagated continuously in the dark at 21-24°C, and 7-day-old cultures were treated with crude Phytophthora sojae cell wall elicitor (5 mg/40 ml, sterilized in 1 ml of water) as described earlier (35,36). Control cultures received sterile distilled water only.…”

“…All subsequent steps converting demethylsuberosin to psoralen or osthenol to angelicin are supposedly catalyzed by highly analogous enzymes (34), but only the linear series has been studied in detail. Enzyme studies conducted mostly with elicited A. majus or P. crispum cells (35)(36)(37)(38) identified marmesin and psoralen synthases as well as psoralen 5-monooxygenase as separate cytochrome P450 (CYP) 3 entities for the consecutive conversion of demethylsuberosin to bergaptol, which then is methylated to bergapten (39). These studies became possible, because furanocoumarins are completely lacking from the cell suspensions propagated in the dark but accumulate rapidly in the cells and culture fluid upon treatment with fungal elicitor.…”