Frank Karp

Isolation of Secretory Cells from Plant Glandular Trichomes and Their Use in Biosynthetic Studies of Monoterpenes and Other Gland Products

The natural products that accumulate in or exude from plant glandular trichomes are biosynthesized by secretory cells located at the apex of the trichome. To investigate the formation of glandular trichome constituents in several species of mints (Lamiaceae), a new procedure was developed for isolating large numbers of highly purified secretory cells. In this method, the leaf surface is gently abraded with glass beads in a way that fragments the glandular trichomes and yields clusters of intact secretory cells. The isolated, intact secretory cells and cell-free preparations derived from them are very active in monoterpene biosynthesis and provide useful starting materials for the purification of several key enzymes of monoterpene metabolism. The procedure described is adaptable to a broad range of plant species and should find wide application in the preparation of whole cell and cell-free systems for biosynthetic studies of plant natural products found in glandular trichomes.

Monoterpene biosynthesis: specificity of the hydroxylations of (-)-limonene by enzyme preparations from peppermint (Mentha piperita), spearmint (Mentha spicata), and perilla (Perilla frutescens) leaves

Microsomal preparations from the epidermal oil glands of Mentha piperita, Mentha spicata, and Perilla frutescens leaves catalyze the NADPH- and O2-dependent allylic hydroxylation of the monoterpene olefin (-)-limonene at C-3, C-6, and C-7, respectively, to produce the corresponding alcohols, (-)-trans-isopiperitenol, (-)-trans-carveol, and (-)-perillyl alcohol. These transformations are the key steps in the biosynthesis of oxygenated monoterpenes in the respective species, and the responsible enzyme systems meet most of the established criteria for cytochrome P450-dependent mixed function oxygenases. The reactions catalyzed are completely regiospecific and, while exhibiting only a modest degree of enantioselectivity, are highly specific for limonene as substrate. Of numerous monoterpene olefins tested, including several positional isomers of limonene, only the 8,9-dihydro analog served as an alternate substrate for ring (C-3 and C-6) hydroxylation, but not side chain (C-7) hydroxylation. In addition to the regiospecificity of the allylic hydroxylation, these enzymes are also readily distinguishable based on differential inhibition by substituted imidazoles.

Biosynthesis of monoterpenes: Preliminary characterization of bornyl pyrophosphate synthetase from sage (Salvia officinalis) and demonstration that geranyl pyrophosphate is the preferred substrate for cyclization

Abstract Previous studies with soluble enzyme preparations from sage ( Salvia officinalis ) demonstrated that the monoterpene ketone (+)-camphor was synthesized by the cyclization of neryl pyrophosphate to (+)-bornyl pyrophosphate followed by hydrolysis of this unusual intermediate to (+)-borneol and then oxidation of the alcohol to camphor (R. Croteau, and F. Karp, 1977, Arch. Biochem. Biophys. 184 , 77–86) . Preliminary investigation of the (+)-bornyl pyrophosphate synthetase in crude preparations indicated that both neryl pyrophosphate and geranyl pyrophosphate could be cyclized to (+)-bornyl pyrophosphate, but the presence of high levels of phosphatases in the extract prevented an accurate assessment of substrate specificity. The competing phosphatases were removed by combination of gel filtration on Sephadex G-150, chromatography on hydroxylapatite, and chromatography on O -(diethylaminoethyl)-cellulose. In these fractionation steps, activities for the cyclization of neryl pyrophosphate and geranyl pyrophosphate to bornyl pyrophosphate were coincident, and on the removal of competing phosphatases, the synthetase was shown to prefer geranyl pyrophosphate as substrate ( V K m for geranyl pyrophosphate was 20-fold that of neryl pyrophosphate). No interconversion of geranyl and neryl pyrophosphates was detected. The partially purified bornyl pyrophosphate synthetase had an apparent molecular weight of 95,000, and required Mg 2+ for catalytic activity ( K m for Mg 2+ ~ 3.5 m m ). Mn 2+ and other divalent cations were ineffective in promoting the formation of bornyl pyrophosphate. The enzyme exhibited a pH optimum at 6.2 and was strongly inhibited by both p -hydroxymercuribenzoate and diisopropylfluorophosphate. Bornyl pyrophosphate synthetase is the first monoterpene synthetase to be isolated free from competing phosphatases, and the first to show a strong preference for geranyl pyrophosphate as substrate. A mechanism for the cyclization of geranyl pyrophosphate to bornyl pyrophosphate is proposed.

Biosynthesis of monoterpenes: Enzymatic conversion of neryl pyrophosphate to 1,8-cineole, α-terpineol, and cyclic monoterpene hydrocarbons by a cell-free preparation from sage (Salvia officinalis)☆

Abstract The volatile oil of sage ( Salvia officinalis ) leaf contains primarily cyclic monoterpenes, and a cell-free enzyme system prepared from immature sage leaves catalyzed the conversion of [1- 3 H]neryl pyrophosphate to cyclic monoterpenes, including 1,8-cineole, α-terpineol, and limonene. The identity of these products was confirmed by the preparation of derivatives, chemical degradation studies, and radiochromatographic analyses. Enzymatic activity capable of 1,8-cineole and hydrocarbon formation was located primarily in the soluble fraction of the leaf homogenate, whereas activity responsible for α-terpineol formation, and phosphatase activity, was distributed among the soluble fraction and the 3000g particles. The formation of all cyclic products was stimulated in the presence of MnCl 2 (1–2 m m ) and MgCl 2 (5–10 m m ) and by inclusion of phosphatase inhibitors, such as NaF, in the incubation medium. Enzymatic activity increased linearly with protein concentration and time and was maximum at about pH 6.5. As α-terpineol could give rise to limonene by dehydration and yield 1,8-cineole by intramolecular attack of the hydroxyl at the double bond, the possible intermediary role of α-terpineol was examined. Inclusion of 1 m m (±)-α-terpineol in the incubation medium suppressed 1,8-cineole and hydrocarbon formation from neryl pyrophosphate by approximately 35%, suggesting that this alcohol could be an intermediate. However, the inhibition appeared to be nonspecific as several other monoterpene alcohols were similarly effective. In order to provide a more direct examination of the pathway, (±)-[3- 3 H]α-terpineol was prepared and tested as a substrate with the enzyme system. (±)-[3- 3 H]α-Terpineol was not converted to 1,8-cineole or to cyclic hydrocarbons, indicating that this alcohol was not an intermediate in the biosynthesis of the structurally related compounds. Furthermore, labeled α-terpineol was not incorporated into other monoterpenes in sage leaf slices, whereas the acyclic alcohols, [1- 3 H]nerol and [1- 3 H]geraniol, were readily incorporated into the characteristic cyclic monoterpenes of sage leaf. Similar lines of evidence excluded 1,8-cineole, limonene, and terpinolene as intermediates in the formation of cyclic products from neryl pyrophosphate. Thus, all of these results suggest that 1,8-cineole, α-terpineol, and limonene are derived independently from neryl pyrophosphate, rather than as free intermediates of a common reaction sequence. Additionally, this is the first report on the biosynthesis of 1,8-cineole in a cell-free system.

Mechanized techniques for the selective extraction of enzymes from plant epidermal glands

Many plant products are biosynthesized and accumulated in epidermal glands. For investigations on the metabolism of these compounds it is most convenient to obtain cell-free preparations enriched in gland contents. Two simple mechanized procedures have been developed for gently abrading the plant surface in order to efficiently extract glandular enzymes in high purity. These methods allow rapid processing of large quantities of plant material and yield extracts largely uncontaminated with materials from underlying tissue. The use of these procedures for isolating several enzymes of terpenoid metabolism is described. These techniques work especially well for microsomal enzymes and may be useful not only for enzymes found in epidermal glands but also for other enzymes localized in or near the epidermis. With simple modification, these procedures can be adapted for use with a variety of different types of plant tissues.

Origin of Natural Odorants

Only substances that have a molecular weight below about 400 and an appreciable vapor pressure at room temperature can be perceived as having odor. The spectrum of odorants is thus limited to relatively small, neutral organic compounds, including undissociated acids and nitrogenous bases.1 Relatively few organic acids are sufficiently volatile to contribute to natural aromas. Acetic (vinegary), propionic (goaty), butyric (spoiled butter), and lactic (sauerkraut) acids are odorous at relatively high concentration.

Biosynthesis of monoterpenes: Partial purification and characterization of 1,8-cineole synthetase from Salvia officinalis☆

The heterocyclic monoterpene 1,8-cineole is one of the major components of the volatile oil produced by sage (Salvia officinalis), and soluble enzyme extracts prepared from young sage leaves catalyzed the anaerobic conversion of the acyclic precursor neryl pyrophosphate to 1,8-cineole. This enzymatic activity was partially purified by a combination of ammonium sulfate precipitation and chromatography on hydroxylapatite, and the bulk of the competing activities, including phosphatases, were removed from the preparation. Cineole synthetase activity had a pH optimum at 6.1. The rate of 1,8-cineole formation was linear up to 1 h, and up to a protein concentration of 450 μg/ml. A divalent cation was required for catalysis, and maximum activity was obtained with MnCl2 (1 mm). ZnCl2 was nearly as effective as MnCl2, and MgCl2 could substitute for MnCl2 only at tenfold higher concentrations. The apparent Km and V of the enzyme were 10−5m and 5.6 nmol/h-mg-ml, respectively. Inhibition of activity was observed at neryl pyrophosphate concentrations above 2 × 10−4m. Nerol, neryl phosphate, 6,7-dihydroneryl pyrophosphate, citronellyl pyrophosphate, and 3,7-dimethyloctyl pyrophosphate were inactive as substrates for 1,8-cineole biosynthesis, indicating that the pyrophosphate and both double bonds of neryl pyrophosphate were required for catalysis. Geranyl pyrophosphate and linaloyl pyrophosphate were converted to 1,8-cineole at only 9 and 15%, respectively, of the rate of neryl pyrophosphate. Thus, the enzyme was highly specific for neryl pyrophosphate. α-Terpineol and its phosphorylated derivatives were not converted to 1,8-cineole, and this observation, coupled with the resolution of cineole synthetase activity from α-terpineol synthetase activity, proved conclusively that α-terpineol was not an intermediate in 1,8-cineole biosynthesis. p-Hydroxymercuribenzoate strongly inhibited the conversion of neryl pyrophosphate to 1,8-cineole (90% inhibition at 4 × 10−5m); however, other thiol-directed reagents such as N-ethylmaleimide were much less effective. The enzyme was insensitive to NaF and to several other metabolic inhibitors. This is the first report on the properties of cineole synthetase, a novel enzyme which catalyzes both a carbocyclization and a heterocyclization.

Biosynthesis of monoterpenes: hydrolysis of bornyl pyrophosphate, an essential step in camphor biosynthesis, and hydrolysis of geranyl pyrophosphate, the acyclic precursor of camphor, by enzymes from sage (Salvia officinalis).

Abstract Soluble enzyme preparations from sage ( Salvia officinalis ) leaves catalyze the hydrolysis of (+)-bornyl pyrophosphate to (+)-borneol, which is an essential step in the biosynthesis of the cyclic monoterpene (+)-camphor [(1 R ,4 R )-bornan-2-one] in this tissue. Chromatography of the preparation on Sephadex G-150 allowed the separation of two regions of bornyl pyrophosphate hydrolase activity. One region was further separated into a pyrophosphate hydrolase and a monophosphate hydrolase by chromatography on hydroxylapatite, but the other contained pyrophosphate and monophosphate hydrolase activities which were inseparable by this or any other chromatographic technique tested. Each phosphatase and pyrophosphatase activity was characterized with respect to molecular weight, pH optimum, response to inhibitors, K m for bornyl phosphate or bornyl pyrophosphate, and substrate specificity, and each activity was distinctly different with regard to these properties. One pyrophosphatase activity was specific for pyrophosphate esters of sterically hindered monoterpenols such as bornyl pyrophosphate. The other preferred pyrophosphate esters of primary allylic alcohols such as geranyl pyrophosphate and neryl pyrophosphate, which are precursors of cyclic monoterpenes, and it hydrolyzed geranyl pyrophosphate at faster rates than neryl pyrophosphate. The monophosphate hydrolase activities were similar in substrate specificity, showing a preference for phosphate esters of primary allylic alcohols. The terpenyl pyrophosphate hydrolase exhibiting specificity for bornyl pyrophosphate may be involved in camphor biosynthesis in vivo , while the terpenyl pyrophosphate hydrolase more specific for geranyl pyrophosphate was shown to be a source of potential interference in studies on monoterpene cyclization processes.

Metabolism of monoterpenes: Demonstration of the hydroxylation of (+)-sabinene to (+)- cis-sabinol by an enzyme preparation from sage (Salvia officinalis) leaves

A microsomal preparation from the epidermis of Salvia officinalis leaves catalyzed the NADPH- and O2-dependent hydroxylation of the monoterpene olefin (+)-sabinene to (+)-cis-sabinol. The reaction catalyzed is a key step in the biosynthesis of C3-oxygenated thujane monoterpenes, and the hydroxylase is highly specific for (+)-sabinene as substrate. The hydroxylase from leaf homogenates was solubilized and characterized with regard to reaction conditions, inhibitors, and activators. Activity was partially inhibited by rabbit anti-rat cytochrome P-450 and by CO, and the latter inhibition was reversed by 450 nm light. A CO-difference spectrum and type I substrate binding spectrum were obtained. The hydroxylase meets most of the established criteria for a cytochrome P-450-dependent mixed function oxygenase and represents one of very few enzyme systems of this type to be isolated from leaves of a higher plant.

Demonstration of a cyclic pyrophosphate intermediate in the enzymatic conversion of neryl pyrophosphate to borneol

A soluble enzyme preparation obtained from young sage (Salvia officinalis) leaves catalyzes the conversion of neryl pyrophosphate to (+)-borneol and the oxidation of (+)-borneol to (+)-camphor. Attempts to purify the borneol synthetase activity by gel permeation column chromatography resulted in the apparent loss of catalytic capability; however, subsequent recombination of column fractions demonstrated that two separable enzymatic activities were required for the conversion of neryl pyrophosphate to borneol. Several lines of evidence indicated that a water-soluble, dialyzable intermediate was involved in this transformation. The intermediate was isolated and subsequently identified as bornyl pyrophosphate by direct chromatographic analysis and by the preparation of derivatives and chromatographic analysis of both the hydrogenolysis (LiAlH4) and enzymatic hydrolysis products of bornyl pyrophosphate. The results presented indicate that borneol is derived by cyclization of neryl pyrophosphate to bornyl pyrophosphate, followed by hydrolysis. This is the first demonstration of a cyclic pyrophosphorylated intermediate in the biosynthesis of bicyclic monoterpenes.