In vitro isolation, elicitation of psoralen in callus cultures of Psoralea corylifolia and cloning of psoralen synthase gene.

Psoralen, an important furanocoumarin occurring abundantly in seeds of Psoralea corylifolia is used as an anticancerous compound against leukemia and other cancer cell lines. Evaluation and isolation of psoralen from the calluses derived from different plant parts, viz. cotyledons, nodes, leaves and roots have been done in the present case for the first time. Amongst all, a maximum of 1934.75 µg/g f.w. of psoralen was recorded in callus derived from cotyledons, followed by 1875.50 and 1465.75 µg/g f.w. of psoralen in node and leaf derived calluses, respectively. Amount of psoralen enhanced further when cotyledonary calluses were exposed to different concentrations of organic elicitors (yeast extract, proline, inositol, casein hydrolyzate (CH), glycine, glutamine and sucrose) and precursors of psoralen (umbelliferone, cinnamic acid and NADPH). Isolation of psoralen was done using methanol as solvent through column chromatography and TLC. FT-IR and NMR further characterized and confirmed the structure of psoralen. In addition, the putative gene, psoralen synthase involved in psoralen synthesis pathway has been isolated, cloned and sequenced which comprised 1237 bp length. BLAST analysis of the gene sequence of psoralen synthase revealed that its nucleotide sequence showed 93% homology with psoralen synthase isolated from Ammi majus. This is the first report of isolation, cloning and characterization of psoralen synthase from Psoralea corylifolia.

Molecular cloning and functional characterization of psoralen synthase, the first committed monooxygenase of furanocoumarin biosynthesis.

Ammi majus L. accumulates linear furanocoumarins by cytochrome P450 (CYP)-dependent conversion of 6-prenylumbelliferone via (+)-marmesin to psoralen. Relevant activities, i.e. psoralen synthase, are induced rapidly from negligible background levels upon elicitation of A. majus cultures with transient maxima at 9-10 h and were recovered in labile microsomes. Expressed sequence tags were cloned from elicited Ammi cells by a nested DD-RT-PCR strategy with CYP-specific primers, and full-size cDNAs were generated from those fragments correlated in abundance with the induction profile of furanocoumarin-specific activities. One of these cDNAs representing a transcript of maximal abundance at 4 h of elicitation was assigned CYP71AJ1. Functional expression in Escherichia coli or yeast cells initially failed but was accomplished eventually in yeast cells after swapping the N-terminal membrane anchor domain with that of CYP73A1. The recombinant enzyme was identified as psoralen synthase with narrow substrate specificity for (+)-marmesin. Psoralen synthase catalyzes a unique carbon-chain cleavage reaction concomitantly releasing acetone by syn-elimination. Related plants, i.e. Heracleum mantegazzianum, are known to produce both linear and angular furanocoumarins by analogous conversion of 8-prenylumbelliferone via (+)-columbianetin to angelicin, and it was suggested that angelicin synthase has evolved from psoralen synthase. However, (+)-columbianetin failed as substrate but competitively inhibited psoralen synthase activity. Analogy modeling and docked solutions defined the conditions for high affinity substrate binding and predicted the minimal requirements to accommodate (+)-columbianetin in the active site cavity. The studies suggested that several point mutations are necessary to pave the road toward angelicin synthase evolution.

Cations modulate the substrate specificity of bifunctional class I O-methyltransferase from Ammi majus.

Caffeoyl-coenzyme A O-methyltransferase cDNA was cloned from dark-grown Ammi majus L. (Apiaceae) cells treated with a crude fungal elicitor and the open reading frame was expressed in Escherichia coli. The translated polypeptide of 27.1-kDa shared significant identity to other members of this highly conserved class of proteins and was 98.8% identical to the corresponding O-methyltransferase from parsley. For biochemical characterization, the recombinant enzyme could be purified to apparent homogeneity by metal-affinity chromatography, although the recombinant enzyme did not contain any affinity tag. Based on sequence analysis and substrate specificity, the enzyme classifies as a cation-dependent O-methyltransferase with pronounced preference for caffeoyl coenzyme A, when assayed in the presence of Mg2+-ions. Surprisingly, however, the substrate specificity changed dramatically, when Mg2+ was replaced by Mn2+ or Co2+ in the assays. This effect could point to yet unknown functions and substrate specificities in situ and suggests promiscuous roles for the lignin specific cluster of plant O-methyltransferases.

Furanocoumarin biosynthesis in Ammi majus L. Cloning of bergaptol O-methyltransferase.

Plants belonging to the Apiaceae or Rutaceae accumulate methoxylated psoralens, such as bergapten or xanthotoxin, as the final products of their furanocoumarin biosynthesis, and the rate of accumulation depends on environmental and other cues. Distinct O-methyltransferase activities had been reported to methylate bergaptol to bergapten and xanthotoxol to xanthotoxin, from induced cell cultures of Ruta graveolens, Petroselinum crispum and Ammi majus. Bergaptol 5-O-methyltransferase (BMT) cDNA was cloned from dark-grown Ammi majus L. cells treated with a crude fungal elicitor. The translated polypeptide of 38.7 kDa, composed of 354 amino acids, revealed considerable sequence similarity to heterologous caffeic acid 3-O-methyltransferases (COMTs). For homologous comparison, COMT was cloned from A. majus plants and shown to share 64% identity and about 79% similarity with the BMT sequence at the polypeptide level. Functional expression of both enzymes in Escherichia coli revealed that the BMT activity in the bacterial extracts was labile and rapidly lost on purification, whereas the COMT activity remained stable. Furthermore, the recombinant AmBMT, which was most active in potassium phosphate buffer of pH 8 at 42 degrees C, showed narrow substrate specificity for bergaptol (Km SAM 6.5 micro m; Km Bergaptol 2.8 micro m) when assayed with a variety of substrates, including xanthotoxol, while the AmCOMT accepted 5-hydroxyferulic acid, esculetin and other substrates. Dark-grown A. majus cells expressed significant BMT activity which nevertheless increased sevenfold within 8 h upon the addition of elicitor and reached a transient maximum at 8-11 h, whereas the COMT activity was rather low and did not respond to the elicitation. Complementary Northern blotting revealed that the BMT transcript abundance increased to a maximum at 7 h, while only a weak constitutive signal was observed for the COMT transcript. The AmBMT sequence thus represents a novel database accession specific for the biosynthesis of psoralens.

Functional expression of cinnamate 4-hydroxylase from Ammi majus L.

Total RNA was isolated from dark-grown cell suspension cultures of Ammi majus L. that had been induced with fungal elicitor or treated with water for control and used as template with cytochrome P450-specific primers for DD-RT-PCR amplifications. A cDNA clone was generated from the elicited transcripts and assigned to cinnamate 4-monooxygenase based on sequence alignments and functional expression in yeast cells. Comparison of the translated polypeptide with database accessions of heterologous cytochrome P450 monooxygenases revealed a high degree of similarity (99.6%) with 98.6% identity to cinnamic acid 4-hydroxylase from parsley, documenting the close evolutionary relationship within the Apiaceae family. Maximal activity of the Ammi hydroxylase in yeast microsomes was determined at 25 degrees C and in the pH range of 6.5-7.0 reaching 2.5 pkat/mg on average. An apparent K(m) of 8.9 microM was determined for cinnamate. Preincubations with psoralen or 8-methoxypsoralen added up to 100 microM in the presence or absence of NADPH hardly affected the turnover rate. A. majus cell cultures accumulate sets of O-prenylated umbelliferones and linear furanocoumarins besides lignin-like compounds upon treatment with elicitor, and cinnamic acid 4-hydroxylase catalyzes the initial reaction leading from the general into the various phenylpropanoid branch pathways. Correspondingly, the hydroxylase transcript abundance was induced in the elicited cells.