Stewart A. Brown

Localization, purification, and characterization of dimethylallylpyrophosphate:Umbelliferone dimethylallyltransferase from Ruta graveolens

Abstract Fractionation of Ruta graveolens L. leaf extracts showed chloroplasts to be the major if not exclusive site of umbelliferone:dimethylallyl pyrophosphate dimethylallyl transferase (EC 2.5.1.3 tentative), which mediates the synthesis of demethylsuberosin, the first committed step in the formation of linear furanocoumarins. Tritium from [G 3 H]umbelliferone administered to leaves is concentrated in association with the chloroplasts, as demonstrated by autoradiography. Most of the enzyme associated with a particulate fraction sedimenting at 30,000g could be solubilized by washing in lowmolarity (5 m m ) Tris-HCl buffer, suggesting attachment to the membrane by weak ionic bonds. The solubilized enzyme from cultured cells of R. graveolens was purified 330-fold and characterized. It exhibits a broad pH optimum between 7.4 and 7.8. The apparent K m approximated 60 μ m for umbelliferone and 20 μ m for dimethylallyl pyrophosphate. Sulfhydryl-binding reagents were not inhibitory, indicating no involvement of thiol groups at the active site. Even at high concentrations, strong inhibition was not observed in the presence of demethylsuberosin or several biosynthetically related coumarins. The significance of these findings in the biosynthesis of furanocoumarins is discussed.

Separations of some coumarins of higher plants by liquid chromatography

Abstract The behaviour of 67 coumarins occurring in higher plants was examined by high-performance liquid chromatography. Resolutions of neutral coumarins were achieved on a normal-phase silica column (10-μm particles) in hexane—ethyl acetate solvent systems and on a C 18 reversed-phase column (5 μm) with aqueous acetonitrile or aqueous methanol. Phenolic coumarins were resolved by stepwise elution with increasing concentrations of aqueous methanol containing acetic acid, sometimes with the addition of tetrahydrofuran, on the reversed-phase column. A mixture of ten coumarins simulating the neutral coumarin fraction Citrus aurantifolia was fully resolved by the use of the two columns in sequence.

Seasonal changes of furanocoumarin concentrations in leaves ofHeracleum lanatum

Concentrations of three dermatitis-inducing furanocoumarins— xanthotoxin, bergapten and psoralen—were measured in whole leaves ofHeracleum lanatum and in extracts of the leaf surface over an entire vegetative season. The concentrations of surface furanocoumarins, localized by extraction involving brief dipping in almost-boiling water followed by HPLC quantitative analysis, increased until the middle of May and decreased until maturity. The concentration on autumn leaves (new growth) was 20–100 times as high as the ones in May, or those of similar size in April. Furanocoumarin concentrations in the whole leaf at different stages of leaf development varied, being the highest April 25, then decreasing sharply with rapid leaf enlargement. Again, in the small autumn leaves the coumarin concentration was two to three times that in April. Seasonal changes in surface furanocoumarins may be important in that these compounds are postulated to form the first defense barrier of the plant.

Dermatitis-inducing furanocoumarins on leaf surfaces of eight species of rutaceous and umbelliferous plants

Eight species of Rutaceae or Umbelliferae, known to cause or suspected of causing photophytodermatitis, had the linear furanocoumarins psoralen, bergapten, and xanthotoxin on their leaf surfaces, in concentrations varying from 0.014 to 1800 /gmg/g fresh weight, equivalent to 0.17–56% of the total leaf concentration. The higher percentage generally observed for spring leaves compared to autumn leaves suggests a higher rate of transfer of these furanocoumarins to the surface in the younger leaves. Among the plants studied,Ruta graveolens had the highest surface concentrations of all three furanocoumarins. The relatively high effectiveness in causing dermatitis of some species with low surface concentrations may be explained by a more effective mechanism of transfer of the furanocoumarins to the skin. A role in the defense of the plant is suggested by their accumulation on the plant surface.

Furanocoumarin biosynthesis in Ruta graveolens cell cultures

Abstract The biosynthetic routes to four linear furanocoumarins—psoralen, xanthotoxin, bergapten. isopimpinellin-co-occurring in Ruta graveolens cell cultures have been investigated with six 14 C-labelled compounds. Mevalonic acid was only poorly incorporated, in contrast to umbelliferone. In support of previous suggestions, 7-demethylsuberosin and (±)-marmesin were very good precursors of the linear furanocoumarins. 7- O -Prenylumbelliferone also was fairly well utilized, but this was probably owing to a prior ether cleavage yielding umbelliferone. Psoralen was well incorporated into bergapten and xanthotoxin, but not into the dimethoxylated isopimpinellin. Differences exist between the organized plant and its cell culture in terms of metabolic products and, by implication, precursor utilization. S (+)-Marmesin was obtained in small quantity from an acid-hydrolysable conjugate present in the culture medium. Syntheses of [2- 14 C]7-demethylsuberosin, [2- 14 C]osthenol, [2- 14 C]7- O -prenylumbelliferone, [3- 14 C] (±)-marmesin, and [3- 14 C]psoralen are described, as well as an improved method for separation of furanocoumarin mixtures by TLC and GLC.

7-Demethylsuberosin and osthenol as intermediates in furanocoumarin biosynthesis

Abstract 14 C-Labelled 7-demethylsuberosin (DMS) was a precursor of linear furanocoumarins in Conium maculatum and Heracleum lanatum (Umbelliferae), and in Ruta graveolens (Rutaceae), but not in Coronilla glauca (Leguminosae). Trapping experiments with 14 C-umbelliferone over 3- to 24-hr periods revealed that DMS is rapidly metabolized, and that in short experiments its specific activity relative to the linear furano- coumarins′ is that of an intermediate. The specific activity of umbelliferone exhibited anomalies attributed to pool compartmentation. Analogous but less extensive experiments established that osthenol is an intermediate in angular furanocoumarin biosynthesis, but is much less rapidly metabolized than DMS. No significant incorporation into furanocoumarins of 14 C from labelled l -valine, l -leucine, acetate, 2,3-di-hydroxyisovaleric acid, senecioic acid, isopentenylpyrophosphate, or mevalonate could be demonstrated. A synthesis of [1- 14 C]2,3-dihydroxyisovaleric acid is described.

Identification of eight coumarins occurring with psoralen, xanthotoxin, and bergapten on leaf surfaces

Surface extracts of the leaves of five species in the Umbelliferae,Citrus limon (Rutaceae), andPsoralea bituminosa (Leguminosae) were examined for the presence of coumarins, after a previous study had shown the presence of three psoralens. In the current investigation eight more coumarins were identified by mass spectrometric techniques: the simple coumarins scopoletin, scoparone, and osthol, the linear furanocoumarins imperatorin and phellopterin, the angular furanocoumarins angelicin and pimpinellin, and the pyranocoumarin seselin. Five of these occur inApium graveolens, and scopoletin, scoparone, and imperatorin were each found in three of the species examined. The co-occurrence of all these coumarins on the surface may be significant in communication between the plant and its environment.

O-methyltransferases of furanocoumarin biosynthesis

Extracts of the furanocoumarin-synthesizing plants, Ruta graveolens L. and Heracleum lanatum Michx., contain a soluble O-methyltransferase system mediating the methylation of the 5- and 8-hydroxyls of linear furanocoumarins by S-adenosyl- l -methionine. Additive effects observed in mixed-substrate experiments and variations in specific activity ratios during purification, supported by differences in the patterns of effects of divalent cations, stability, and molecular weights, demonstrate the presence in Ruta cell culture extracts of two discrete enzymes acting at the two different positions. One enzyme probably methylates the 5-hydroxyls of both bergaptol and 5-hydroxyxanthotoxin, and the other enzyme the 8-hydroxyls of xanthotoxol and 8-hydroxybergapten. The former, which is highly substrate specific, acts ortho to the side chain—a type previously unreported. The latter, acting meta to the side chain, appears to be less specific, as crude extracts show some activity against an 8-hydroxylated simple coumarin; it is, however, distinct from a caffeic acid meta-O-methyltransferase in the same extract. A 50-fold purification of the 5-methyltransferase and a 16-fold purification of the 8-O-methyltransferase have been achieved by affinity chromatography utilizing an S-adenosyl- l -homocysteine ligand.

Influence of low-intensity ultraviolet radiation on extrusion of furanocoumarins to the leaf surface

Exposure ofRuta graveolens leaves to low intensity 366-nm radiation led to a ca. 20% increase in concentrations of the furanocoumarins psoralen, xanthotoxin and bergapten, as compared to leaves kept in darkness. Both direct and, even more, scattered UV radiation produced increases in total concentrations. Changes in the concentrations of individual coumarins were generally parallel. Extrusion to the surface was increased, especially in lower, older leaves exposed to the scattered radiation, where it exceeded the control by factors of eight or nine. It is suggested that this response could enhance shielding of leaves against penetration of UV into the cells and that irradiation, by exciting the furanocoumarins, could augment protection against potential microbial invaders.

Biosynthesis of furanocoumarins in parsnips

Abstract Labelling experiments have shown that umbelliferone, and to a lesser degree p -coumaric acid, are precursors of bergapten, imperatorin, isopimpinellin and xanthotoxin in Pastinaca sativa L. C-2 and C-5 of mevalonate, which has been proposed as a precursor of two furan ring carbons and of dimethylallyl substituents, were both incorporated into furanocoumarins with low efficiency—poorer than that of acetate. Feeding experiments with [2- 14 C]5-methoxy-7-hydroxycoumarin provided no evidence that it is a major intermediate in bergapten formation, supporting the theory that carbon side-chain attachment precedes further oxygenation of umbelliferone in furanocoumarin biosynthesis.