Induction of two prenyltransferases for the accumulation of coumarin phytoalexins in elicitor-treated Ammi majus cell suspension cultures

Hamerski, Daria; Schmitt, D; Matern, Ulrich

doi:10.1016/0031-9422(90)85417-e

Cited by 62 publications

(32 citation statements)

References 17 publications

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“…The enzymic product of 6-methyltryptophan was identified unequivocally as 4-dimethylallyl-6-methyltryptophan (6-methyl-DMAT) by 1 H-NMR (Table 1) . There was no product formation (<0?2 %) observed with 4-hydroxybenzoic acid, the substrate of the prenyltransferase of ubiquinone biosynthesis (Melzer & Heide 1994), or with 4-hydroxyphenylpyruvic acid or umbelliferone, the substrates of previously identified prenyltransferases of secondary metabolism (Hamerski et al, 1990;Pojer et al, 2003). These results are comparable with those obtained with DMATS from C. purpurea (Lee et al, 1976).…”

Section: Biochemical Properties Of Dmats Fgapt2supporting

confidence: 79%

Overproduction, purification and characterization of FgaPT2, a dimethylallyltryptophan synthase from Aspergillus fumigatus

2005

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A putative dimethylallyltryptophan synthase gene, fgaPT2, was identified in the genome sequence of Aspergillus fumigatus. fgaPT2 was cloned and overexpressed in Saccharomyces cerevisiae. The protein FgaPT2 was purified to near homogeneity and characterized biochemically. This enzyme was found to convert L-tryptophan to 4-dimethylallyltryptophan, a reaction known to be the first step in ergot alkaloid biosynthesis. FgaPT2 is a soluble, dimeric protein with a subunit size of 52 kDa, and contains no putative prenyl diphosphate binding site (N/D)DXXD. K m values for L-tryptophan and dimethylallyl diphosphate (DMAPP) were determined as 8 and 4 mM, respectively. Metal ions, such as Mg 2+ and Ca 2+ , enhance the reaction velocity, but are not essential for the enzymic reaction. FgaPT2 showed a relatively strict substrate specificity for both tryptophan and DMAPP. FgaPT2 is the first enzyme in the biosynthesis of ergot alkaloids to be purified and characterized in homogeneous form after heterologous overproduction.

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Section: Biochemical Properties Of Dmats Fgapt2supporting

confidence: 79%

Overproduction, purification and characterization of FgaPT2, a dimethylallyltryptophan synthase from Aspergillus fumigatus

2005

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“…1). Prenylation reactions at C-6 and O-7, but no C-8-prenylation, of umbelliferone were observed in A. majus (37), and thus more than one hurdle must be overcome to enter the route to angular furanocoumarins. These aspects cannot be studied in A. majus because of lack of angular furanocoumarins, but might be conducted in Citrus sinensis RPPLI GSGKF TYNYS DIA CYP71A9 RPSLY AANRL GYG-S TVS CYP71A10 RRQPT AAKIF GYGCK DVA CYP71D8 RPQLL APQFM VYGAT DIA CYP71D9 RPYVL AAEIM DYDFK GVA CYP71D10 RPDFV LSRIV …”

Section: Discussionmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Larbat

Kellner

Specker

et al. 2007

Journal of Biological Chemistry

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135

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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.Furanocoumarins are produced by many plants, mostly of the Apiaceae, Rutaceae, Moraceae, or the Coronilla and Psoralea genera of the Fabaceae (1-3). Multiple pharmacological effects have been ascribed to several of these metabolites (4 -6), which were included in clinical screenings but received attention also for their inhibitory effect on monooxygenases involved in drug metabolism (7-9) and potential toxicity (10). The (dihydro)furan-substituted 2H-1-benzopyran-2-one forms the characteristic core structure, and the annulation type distinguishes the linear furanocoumarins or psoralens from the angular furanocoumarins (Fig.

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“…tissues and conditions for precursors of metabolic pathways branched at truns-cinnamic acid, e.g. salicylate (Yalpani et al, 1993;Mauch-Mani and Slusarenko, 1996) and coumarin biosynthesis (Hamerski et al, 1990). In other words, not all PAL isozymes may be physiologically linked to C4H, although a11 of these isoforms are apparently indistinguishable based on their enzyme-kinetic properties (Appert et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

Isolation of a cDNA and a Genomic Clone Encoding Cinnamate 4-Hydroxylase from Arabidopsis and Its Expression Manner in Planta

1997

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We have isolated a cDNA for a cytochrome P450, cinnamate 4-hydroxylase (C4H), of Arabidopsis tha/iana using a C4H cDNA from mung bean as a hybridization probe. The deduced amino acid sequence is 84.7% identical to that of mung bean C4H and therefore was designated CYP73A5. The CYP73A5 protein was expressed in insect cells using the baculovirus expression system and when reconstituted with lipid and NADPH-cytochrome P450 reductase resulted in C4H activity with a specific activity of 68 nmol min-' nmol-' P450. Southern blot analysis revealed that CYP73A5 is a single-copy gene in Arabidopsis. C4H (CYP73A5) expression was apparently coordinated in Arabidopsis with both PALl and 4CL in response to light and wounding. Although the light induction of CHS followed a time course similar to that observed with C4H, no induction of CHS was detected upon wounding. On the other hand, the C4H expression patterns exhibited no significant coordination with those of PALZ and PAL3. A C4H promoter region of 907 bp contained all of the three cis-acting elements (boxes P, A, and L) conserved among the PAL and 4CL genes so far reported as controlling expression.

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Induction of two prenyltransferases for the accumulation of coumarin phytoalexins in elicitor-treated Ammi majus cell suspension cultures

Cited by 62 publications

References 17 publications

Overproduction, purification and characterization of FgaPT2, a dimethylallyltryptophan synthase from Aspergillus fumigatus

Overproduction, purification and characterization of FgaPT2, a dimethylallyltryptophan synthase from Aspergillus fumigatus

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

Isolation of a cDNA and a Genomic Clone Encoding Cinnamate 4-Hydroxylase from Arabidopsis and Its Expression Manner in Planta

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