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In addition to the psychoactive constituents that are typically associated with Cannabis sativa L., there exist numerous other specialized metabolites in this plant that are believed to contribute to its medicinal versatility. This study focused on two such compounds, known as cannflavin A and cannflavin B. These prenylated flavonoids specifically accumulate in C. sativa and are known to exhibit potent anti-inflammatory activity in various animal cell models. However, almost nothing is known about their biosynthesis. Using a combination of phylogenomic and biochemical approaches, an aromatic prenyltransferase from C. sativa (CsPT3) was identified that catalyzes the regiospecific addition of either geranyl diphosphate (GPP) or dimethylallyl diphosphate (DMAPP) to the methylated flavone, chrysoeriol, to produce cannflavins A and B, respectively. Further evidence is presented for an O-methyltransferase (CsOMT21) encoded within the C. sativa genome that specifically converts the widespread plant flavone known as luteolin to chrysoeriol, both of which accumulate in C. sativa. These results therefore imply the following reaction sequence for cannflavins A and B biosynthesis: luteolin ► chrysoeriol ► cannflavin A and cannflavin B. Taken together, the identification of these two unique enzymes represent a branch point from the general flavonoid pathway in C. sativa and offer a tractable route towards metabolic engineering strategies that are designed to produce these two medicinally relevant Cannabis compounds.

Prenylation plays a major role in the diversification of aromatic natural products, such as phenylpropanoids, flavonoids, and coumarins. This biosynthetic reaction represents the crucial coupling process of the shikimate or polyketide pathway providing an aromatic moiety and the isoprenoid pathway derived from the mevalonate or methyl erythritol phosphate (MEP) pathway, which provides the prenyl (isoprenoid) chain. In particular, prenylation contributes strongly to the diversification of flavonoids, due to differences in the prenylation position on the aromatic rings, various lengths of prenyl chain, and further modifications of the prenyl moiety, e.g., cyclization and hydroxylation, resulting in the occurrence of ca. 1000 prenylated flavonoids in plants. Many prenylated flavonoids have been identified as active components in medicinal plants with biological activities, such as anti-cancer, anti-androgen, anti-leishmania, and anti-nitric oxide production. Due to their beneficial effects on human health, prenylated flavonoids are of particular interest as lead compounds for producing drugs and functional foods. However, the gene coding for prenyltransferases that catalyze the key step of flavonoid prenylation have remained unidentified for more than three decades, because of the membrane-bound nature of these enzymes. Recently, we have succeeded in identifying the first prenyltransferase gene SfN8DT-1 from Sophora flavescens, which is responsible for the prenylation of the flavonoid naringenin at the 8-position, and is specific for flavanones and dimethylallyl diphosphate (DMAPP) as substrates. Phylogenetic analysis showed that SfN8DT-1 has the same evolutionary origin as prenyltransferases for vitamin E and plastoquinone. A prenyltransferase GmG4DT from soybean, which is involved in the formation of glyceollin, was also identified recently. This enzyme was specific for pterocarpan as its aromatic substrate, and (-)-glycinol was the native substrate yielding the direct precursor of glyceollin I. These enzymes are localized to plastids and the prenyl chain is derived from the MEP pathway. Further relevant genes involved in the prenylation of other types of polyphenol are expected to be cloned by utilizing the sequence information provided by the above studies.

Cistus creticus ssp. creticus is an indigenous shrub of the Mediterranean area. The glandular trichomes covering its leaf surfaces secrete a resin called ‘‘ladanum”, which among others contains a number of specific labdane-type diterpenes that exhibit antibacterial and antifungal action as well as in vitro and in vivo cytotoxic and cytostatic activity against human cancer cell lines. In view of the properties and possible future exploitation of these metabolites, it was deemed necessary to study the geranylgeranyl diphosphate synthase enzyme (GGDPS, EC 2.5.1.30), a short chain prenyltransferase responsible for the synthesis of the precursor molecule of all diterpenes. In this work, we present the cloning, functional characterisation and expression profile at the gene and protein levels of two differentially expressed C. creticus full-length cDNAs, CcGGDPS1 and CcGGDPS2. Heterologous yeast cell expression system showed that these cDNAs exhibited GGDPS enzyme activity. Gene and protein expression analyses suggest that this enzyme is developmentally and tissue-regulated showing maximum expression in trichomes and smallest leaves (0.5–1.0 cm). This work is the first attempt to study the terpenoid biosynthesis at the molecular level in C. creticus ssp. creticus. 2008 Elsevier Ltd. All rights reserved.

This review deals with some bioorganic chemical aspects of isoflavonoids. Its highlights are rather restricted to the structural modifications and the diverse oxygenated side attachments originating from the isoprenoid substituents, 3,3-dimethylallyl (prenyl), 1,1-dimethylallyl and geranyl groups. Some aspects of naturally occurring isoflavonoids, their new classes and biological functions are also described.

Microsomal preparations of white lupin ( Lupinus albus ) radicles and cell suspension cultures catalyse the prenylation of positions 6, 8 and 3′ of the isoflavones genistein and 2′-hydroxygenistein. Both the substrates and the enzyme reaction products are natural constituents of lupin tissues. Enzymatic prenylation of isoflavones required dimethylallyl pyrophosphate (DMAPP) and 12 mM Mn 2+ with optimum activity at pH 7.5 in Tris-HCl buffer. The apparent K m values for DMAPP and the prenyl acceptors were 4 and 5 μM, respectively. Isopentenyl pyrophosphate was a competitive inhibitor of the prenylation reaction, with an apparent K i of 5 μM. The bulk of enzymatic activity was associated with the membrane fraction and could only be solubilized in the presence of a detergent. The differences observed in prenyltransferase activity ratios, in relation to the source of the enzyme and the type of detergent used, suggest that prenylation at positions 6, 8 and 3′ of isoflavones is catalysed by a number of distinct enzymes.

Aromatic prenyltransferases transfer prenyl moieties onto aromatic acceptor molecules, catalyzing an electrophilic substitution of the aromatic ring under formation of carbon-carbon bonds. They give rise to an astounding diversity of primary and secondary metabolites in plants, fungi and bacteria. This review describes a recently discovered family of aromatic prenyltransferases. The structure of these enyzmes shows a type of beta/alpha fold with antiparallel beta strands. Due to the alpha-beta-beta-alpha architecture of this fold, this group of enzymes was designated as ABBA prenyltransferases. They lack the (N/D)DxxD motif which is characteristic for many other prenyltransferases. At present, 14 genes with sequence similarity to ABBA prenyltransferases can be identified in the database. A phylogenetic analysis of these genes separates them into two clades. One of them comprises the 4-hydroxyphenylpyruvate 3-dimethylallyltransferases CloQ and NovQ involved in aminocoumarin antibiotic biosynthesis in Streptomyces strains, as well as four genes of unknown function from fungal genomes. The other clade comprises genes involved in the biosynthesis of prenylated naphthoquinones and prenylated phenazines in different streptomycetes. ABBA prenyltransferases are soluble biocatalysts which can easily be obtained as homogeneous proteins in significant amounts. Their substrates are accommodated in a surprisingly spacious central cavity which explains their promiscuity for different aromatic substrates. Therefore, the enzymes of this family represent attractive tools for the chemoenzymatic synthesis of bioactive molecules.

A series of putative indole prenyltransferase genes could be identified in the genome sequences of different fungal strains including Aspergillus fumigatus and Neosartorya fischeri. The gene products show significant sequence similarities to dimethylallyltryptophan synthases from different fungi. We have cloned and overexpressed seven of these genes, fgaPT1, fgaPT2, ftmPT1, ftmPT2, 7-dmats, cdpNPT and anaPT in Escherichia coli and Saccharomyces cerevisiae. The overproduced enzymes were characterised biochemically. Three additional indole prenyltransferases, DmaW-Cs, TdiB and MaPT were also identified and characterised in the last years. Sequence analysis and comparison with known aromatic prenyltransferases as well as biochemical investigation revealed that these enzymes belong to a group of aromatic prenyltransferases. The characterised prenyltransferases are soluble proteins, catalyse different prenyl transfer reactions on indole moieties of various substrates and do not require divalent metal ions for their prenyl transfer reactions. In addition, indole prenyltransferases carry tryptophan aminopeptidase activity, which strengths their relationship in the evolution. These properties differ clearly from membrane-bound aromatic prenyltransferases from different sources and soluble prenyltransferases from bacteria. All of the indole prenyltransferases accepted only dimethylallyl diphosphate as prenyl donor. On the other hand, they showed broad substrate specificity towards their aromatic substrates. Diverse simple tryptophan derivatives and tryptophan-containing cyclic dipeptides were accepted by these enzymes, providing a strategy for convenient production of biologically active substances, e.g. by chemoenzymatic synthesis.

A chain-length-specific geranyl diphosphate synthase was detected in leaves of Pelargonium roseum. The enzyme systems, farnesyl diphosphate synthase [EC 2.5.1.1], geranylgeranyl diphosphate synthase and isopentenyl diphosphate isomerase [EC 5.3.2.2], were also detected. Furthermore, geranyl diphosphate synthase was separated from farnesyl diphosphate synthase by hydrophobic chromatography on Butyl-Toyopearl 650. This separation demonstrates that there are two specific syntheses, geranyl diphosphate synthase and farnesyl diphosphate synthase, in this higher plant. The geranyl diphosphate synthase was localized in a subcellular membrane fraction rather than in the soluble fraction. The synthase was activated by Mn2+ rather than Mg2+.

A refined enzyme assay for the membrane bound dimethylallylpyrophosphate: trihydroxypterocarpan dimethylallyl transferase (prenyltransferase) enabled us to study further properties not previously reported. Isopentenylpyrophosphate (IPP) acts as a competitive inhibitor with a Ki of 7.5 μM. Aminophenethylpyrophosphate (APP), a structural analogue of IPP inhibits the reaction only slightly. Mn2+ ions in the standard assay can be replaced by Co2+ (both 12 mM). Furthermore, the optimized enzyme assay allowed us to investigate the solubilization and chromatographic behaviour of the soluble enzyme. Solubilization was only achieved by using detergents. Three detergents, Chaps, dodecylmaltoside (DOM) and 1-dodecyl-2-deoxy-phosphatidylcholine (ES12H) were analysed in detail. Their influence on enzyme activity and stability, their solubilization properties and the optimal detergent/protein ratios were determined. A narrow optimal ratio of about 3 was observed only with ES12H. In gel permeation chromatography of solubilized prenyltransferase the detergent-protein micelles containing enzyme activity eluted corresponding to approximate Mrs of 440 000 in DOM, 300 000 in ES12H and above 1 500 000 in Chaps. A separation of bulk protein from enzyme activity by ion exchange chromatography was only possible with ES12H. This detergent proved to be most suited for purification procedures.

Geranyl diphosphate (GPP), the universal precursor of monoterpenes, is formed from isopentenyl diphosphate and dimethylallyl diphosphate by the action of geranyl diphosphate synthase, one of the key branchpoint enzymes of terpene biosynthesis. Three types of GPP synthase can be distinguished in plants based on sequence similarity and subunit architecture, but until now individual species have been reported to contain only one of these types. Here we show that the conifer, Norway spruce (Picea abies), contains two different types of GPP synthase belonging to two separate groups of homodimeric proteins. One enzyme, designated PaIDS2 (P. abies isoprenyl diphosphate synthase 2), has high sequence similarity to other gymnosperm GPP synthases. It produces solely GPP in in vitro assays after expression in Escherichia coli and likely participates in monoterpene biosynthesis accompanying induced oleoresin formation, based on dramatic increases in transcript level after methyl jasmonate application. The other enzyme, designated PaIDS3, has highest similarity to the previously reported Arabidopsis thaliana GPP synthase and several other angiosperm sequences, and is not associated with induced oleoresin formation in Norway spruce. In vitro assay of this protein and one encoded by a similar gene sequence from Quercus robur gave substantial amounts of the larger prenyl diphosphates, FPP and GGPP, in addition to GPP. Hence these proteins may not be involved in monoterpene formation and could conceivably form products in addition to GPP in planta.

The conifer Picea abies (Norway spruce) employs terpenoid-based oleoresins as part of its constitutive and induced defense responses to herbivores and pathogens. The isoprenyl diphosphate synthases are branch-point enzymes of terpenoid biosynthesis leading to the various terpene classes. We isolated three genes encoding isoprenyl diphosphate synthases from P. abies cDNA libraries prepared from the bark and wood of methyl jasmonate-treated saplings and screened via a homology-based PCR approach using degenerate primers. Enzyme assays of the purified recombinant proteins expressed in Escherichia coli demonstrated that one gene (PaIDS 4) encodes a farnesyl diphosphate synthase and the other two (PaIDS 5 and PaIDS 6) encode geranylgeranyl diphosphate synthases. The sequences have moderate similarity to those of farnesyl diphosphate and geranylgeranyl diphosphate synthases already known from plants, and the kinetic properties of the enzymes are not unlike those of other isoprenyl diphosphate synthases. Of the three genes, only PaIDS 5 displayed a significant increase in transcript level in response to methyl jasmonate spraying, suggesting its involvement in induced oleoresin biosynthesis.

A recombinant alpha-farnesene synthase from apple (Malus x domestica), expressed in Escherichia coli, showed features not previously reported. Activity was enhanced 5-fold by K(+) and all four isomers of alpha-farnesene, as well as beta-farnesene, were produced from an isomeric mixture of farnesyl diphosphate (FDP). Monoterpenes, linalool, (Z)- and (E)-beta-ocimene and beta-myrcene, were synthesised from geranyl diphosphate (GDP), but at 18% of the optimised rate for alpha-farnesene synthesis from FDP. Addition of K(+) reduced monoterpene synthase activity. The enzyme also produced alpha-farnesene by a reaction involving coupling of GDP and isoprenyl diphosphate but at1% of the rate with FDP. Mutagenesis of active site aspartate residues removed sesquiterpene, monoterpene and prenyltransferase activities suggesting catalysis through the same active site. Phylogenetic analysis clusters this enzyme with isoprene synthases rather than with other sesquiterpene synthases, suggesting that it has evolved differently from other plant sesquiterpene synthases. This is the first demonstration of a sesquiterpene synthase possessing prenyltransferase activity.

Two dimethylallyl diphosphate:umbelliferone dimethylallyltransferase (prenyltransferase) activities, catalysing the 6-prenylation and the 7-O-prenylation, respectively, of umbelliferone in the course of phytoalexin synthesis, increased in Ammi majus cell suspension cultures in response to elicitor treatment. Both enzyme activities were dependent on Mg2+ or Mn2+ with significant preference for Mg2+ in the 6-prenylation reaction. Whereas dark-grown cells did not contain these activities, both prenyltransferase activities were induced rapidly by the addition of elicitor reaching a first maximum after 10-14 hr and a second maximum beyond 30 hr. Other coumarin specific, elicitor-induced enzyme activities of A. majus cells, in contrast, showed only one maximum of activity within the 50 hr experimental period, while the pattern of induction of phenylalanine ammonia-lyase activity resembled that of the prenyltransferases with maxima at ca 8 hr and 20-30 hr. Preliminary data suggest that the apparent biphasic induction of these enzyme activities is due to post-translational enzyme modifications.

Prenyltransferase (EC 2.5.1.1; assayed as farnesyl pyrophosphate synthetase)was purified 106-fold from an homogenate of 3-day-old seedlings of Pisum sativum . Some of the properties of the purified enzyme were determined and these differed in several significant respects from those reported for preparations from other sources, e.g. the apparent MW was 96000 ± 4000 and the preparation could be dissociated into two subunits of MW 45000 ± 3000. The total activity of the extractable enzyme went through a sharp maximum (in the range 1 to 28 days) 3 days after germination. Farnesyl pyrophosphate was formed in cell-free extracts of peas from either isopentenyl pyrophosphate alone, or this together with geranyl pyrophosphate (optimum yields 1.2 and 10% respectively). Use of [1- 14 C]- and [4- 14 C]-isopentenyl pyrophosphates as the sole substrates and degradation of the products showed that the crude extracts contained a pool of the biogenetic equivalent of 3,3-dimethylallyl pyrophosphate. No analogous pool of geranyl pyrophosphate could be detected.

A polyprenyltransferase catalysing the formation of Z-double bonds was partially purified from mulberry leaves, Morus bombysis . The enzyme catalysed a consecutive condensation of isopentenyl diphosphate with geranyl- geranyl diphosphate as an allylic primer to produce a series of ficaprenol-type Z , E -mixed polyprenyl diphosphates with carbon chain length ranging from C 40 to C 60 . Not only all-E -geranylgeranyl diphosphate but also E , E -farnesyl- and geranyl diphosphates were accepted as substrates. Addition of Triton X-100 stimulated the enzymatic activity. The stereochemistry of hydrogen elimination from the 2-position of isopentenyl diphosphate during the Z -prenyl chain formation was examined directly by experiments using this synthase and ( S )-[1- 14 C, 2- 3 H]isopentenyl diphosphate and it was demonstrated that the 2-pro- S hydrogen was lost. Feeding experiments of stereospecifically 3 H-labelled mevalonic acid with intact mulberry leaves, however, showed that mevalonic acid was incorporated into polyprenols with elimination of the 4-pro- S hydrogen of the acid.

The biosynthesis of monoterpenes in higher plants is reviewed, with particular emphasis on recent studies of the enzymology of biosynthesis.