Cloning and characterization of naringenin 8-prenyltransferase, a flavonoid-specific prenyltransferase of Sophora flavescens

Prenylated flavonoids are natural compounds that often represent the active components in various medicinal plants and exhibit beneficial effects on human health. Prenylated flavonoids are hybrid products composed of a flavonoid core mainly attached to either 5-carbon (dimethylallyl) or 10-carbon (geranyl) prenyl groups derived from isoprenoid (terpenoid) metabolism, and the prenyl groups are crucial for their biological activity. Prenylation reactions in vivo are crucial coupling processes of two major metabolic pathways, the shikimate-acetate and isoprenoid pathways, in which these reactions are also known as a rate-limiting step. However, none of the genes responsible for the prenylation of flavonoids has been identified despite more than 30 years of research in this field. We have isolated a prenyltransferase gene from Sophora flavescens, SfN8DT-1, responsible for the prenylation of the flavonoid naringenin at the 8-position, which is specific for flavanones and dimethylallyl diphosphate as substrates. Phylogenetic analysis shows that SfN8DT-1 has the same evolutionary origin as prenyltransferases for vitamin E and plastoquinone. The gene expression of SfN8DT-1 is strictly limited to the root bark where prenylated flavonoids are solely accumulated in planta. The ectopic expression of SfN8DT-1 in Arabidopsis thaliana resulted in the formation of prenylated apigenin, quercetin, and kaempferol, as well as 8-prenylnaringenin. SfN8DT-1 represents the first flavonoid-specific prenyltransferase identified in plants and paves the way for the identification and characterization of further genes responsible for the production of this large and important class of secondary metabolites.     

Prenylation of aromatic compounds,a key diversification of plant secondary metabolites

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.     

Molecular Characterization and Phylogenetic Analysis of Two Novel Regio-specific Flavonoid Prenyltransferases from Morus alba and Cudrania tricuspidata

Prenylated flavonoids are attractive specialized metabolites with a wide range of biological activities and are distributed in several plant families. The prenylation catalyzed by prenyltransferases represents a Friedel-Crafts alkylation of the flavonoid skeleton in the biosynthesis of natural prenylated flavonoids and contributes to the structural diversity and biological activities of these compounds. To date, all identified plant flavonoid prenyltransferases (FPTs) have been identified in Leguminosae. In the present study two new FPTs, Morus alba isoliquiritigenin 3′-dimethylallyltransferase (MaIDT) and Cudrania tricuspidata isoliquiritigenin 3′-dimethylallyltransferase (CtIDT), were identified from moraceous plants M. alba and C. tricuspidata, respectively. MaIDT and CtIDT shared low levels of homology with the leguminous FPTs. MaIDT and CtIDT are predicted to be membrane-bound proteins with predicted transit peptides, seven transmembrane regions, and conserved functional domains that are similar to other homogentisate prenyltransferases. Recombinant MaIDT and CtIDT were able to regioselectively introduce dimethylallyl diphosphate into the A ring of three flavonoids with different skeleton types (chalcones, isoflavones, and flavones). Phylogenetic analysis revealed that MaIDT and CtIDT are distantly related to their homologs in Leguminosae, which suggests that FPTs in Moraceae and Leguminosae might have evolved independently. MaIDT and CtIDT represent the first two non-Leguminosae FPTs to be identified in plants and could thus lead to the identification of additional evolutionarily varied FPTs in other non-Leguminosae plants and could elucidate the biosyntheses of prenylated flavonoids in various plants. Furthermore, MaIDT and CtIDT might be used for regiospecific prenylation of flavonoids to produce bioactive compounds for potential therapeutic applications due to their high efficiency and catalytic promiscuity.     

Metabolic engineering for the production of prenylated polyphenols in transgenic legume plants using bacterial and plant prenyltransferases

Prenylated polyphenols are secondary metabolites beneficial for human health because of their various biological activities. Metabolic engineering was performed using Streptomyces and Sophora flavescens prenyltransferase genes to produce prenylated polyphenols in transgenic legume plants. Three Streptomyces genes, NphB, SCO7190, and NovQ, whose gene products have broad substrate specificity, were overexpressed in a model legume, Lotus japonicus, in the cytosol, plastids or mitochondria with modification to induce the protein localization. Two plant genes, N8DT and G6DT, from Sophora flavescens whose gene products show narrow substrate specificity were also overexpressed in Lotus japonicus. Prenylated polyphenols were undetectable in these plants; however, supplementation of a flavonoid substrate resulted in the production of prenylated polyphenols such as 7-O-geranylgenistein, 6-dimethylallylnaringenin, 6-dimethylallylgenistein, 8-dimethylallynaringenin, and 6-dimethylallylgenistein in transgenic plants. Although transformants with the native NovQ did not produce prenylated polyphenols, modification of its codon usage led to the production of 6-dimethylallylnaringenin and 6-dimethylallylgenistein in transformants following naringenin supplementation. Prenylated polyphenols were not produced in mitochondrial-targeted transformants even under substrate feeding. SCO7190 was also expressed in soybean, and dimethylallylapigenin and dimethylallyldaidzein were produced by supplementing naringenin. This study demonstrated the potential for the production of novel prenylated polyphenols in transgenic plants. In particular, the enzymatic properties of prenyltransferases seemed to be altered in transgenic plants in a host species-dependent manner.     

Production of prenylated flavonoids in tomato fruits expressing a prenyltransferase gene from Streptomyces coelicolor A3(2)

Flavonoids are natural compounds found in many plants, including the important fruit crop, tomato. Prenylated flavonoids consist of a large group of compounds, which often exhibit antitumour, antibacterial and/or anti-androgen activities. In this study, we engineered the biosynthesis of prenylated flavonoids using a Streptomyces prenyltransferase HypSc (SCO7190) possessing broad-range substrate specificity, in tomato as a host plant. LC/MS/MS analysis demonstrated the generation of 3'-dimethylallyl naringenin in tomato fruits when recombinant HypSc protein was targeted to the plastids, whereas the recombinant protein hardly produced this compound in vitro. This is the first report confirming the accumulation of a prenylated flavonoid using a bacterial prenyltransferase in transgenic plants, and our results suggest that the product specificities of prenyltransferases can be significantly influenced by the host plant.     

Chemoenzymatic syntheses of prenylated aromatic small molecules using Streptomyces prenyltransferases with relaxed substrate specificities

NphB is a soluble prenyltransferase from Streptomyces sp. strain CL190 that attaches a geranyl group to a 1,3,6,8-tetrahydroxynaphthalene-derived polyketide during the biosynthesis of anti-oxidant naphterpin. Here we report multiple chemoenzymatic syntheses of various prenylated compounds from aromatic substrates including flavonoids using two prenyltransferases NphB and SCO7190, a NphB homolog from Streptomyces coelicolor A3(2), as biocatalysts. NphB catalyzes carbon-carbon-based and carbon-oxygen-based geranylation of a diverse collection of hydroxyl-containing aromatic acceptors. Thus, this simple method using the prenyltransferases can be used to explore novel prenylated aromatic compounds with biological activities. Kinetic studies with NphB reveal that the prenylation reaction follows a sequential ordered mechanism.     

Characterization of leachianone G 2"-dimethylallyltransferase, a novel prenyl side-chain elongation enzyme for the formation of the lavandulyl group of sophoraflavanone G in Sophora flavescens Ait. cell suspension cultures

Leachianone G (LG) 2"-dimethylallyltransferase, a novel prenyl side-chain elongation enzyme, was identified in Sophora flavescens Ait. cultured cells. The enzyme transfers a dimethylallyl group to the 2" position of another dimethylallyl group attached at position 8 of LG to form sophoraflavanone G, a branched monoterpenoid-conjugated flavanone characteristic to this plant. This membrane-bound dimethylallyltransferase required Mg2+ (optimum concentration was 10 mm) for the reaction and had an optimum pH of 8.8. It utilized dimethylallyl diphosphate as the sole prenyl donor, and the 2'-hydroxy function in LG was indispensable to the activity. The apparent Km values for dimethylallyl diphosphate and LG were 59 and 2.3 microm, respectively. Subcellular localization of three enzymes that participated in the formation of the lavandulyl group was also investigated by sucrose density gradient centrifugation. Two prenyltransferases, naringenin 8-dimethylallyltransferase and LG 2"-dimethylallyltransferase, were localized in the plastids, whereas 8-dimethylallylnaringenin 2'-hydroxylase, which catalyzes the crucial step in the lavandulyl-group formation, was associated with the endoplasmic reticulum. These results suggest the close cooperation between the plastids and the endoplasmic reticulum in the formation of lavandulyl groups.     

Prenyl transfer to aromatic substrates in the biosynthesis of aminocoumarins,meroterpenoids and phenazines: The ABBA prenyltransferase family

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 β/α fold with antiparallel β strands. Due to the α-β-β-α 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.     

Efficient production and capture of 8-prenylnaringenin and leachianone G-biosynthetic intermediates of sophoraflavanone G--by the addition of cork tissue to cell suspension cultures of Sophora flavescens

It has previously been demonstrated that the addition of cork tissue to cell suspension cultures of Sophora flavescens stimulates the production of sophoraflavanone G, most of which has been recovered from the added cork tissue. In the present study, it was found that two precursors of sophoraflavanone G, 8-prenylnaringenin (sophoraflavanone B) and leachianone G, both of which have never been detected either in cultured cells or in the original plants, also accumulated in the added cork tissue. Thirteen minor flavonoids including three prenylated flavonoids, in addition to 8-prenylnaringenin and leachianone G, were isolated from the cork tissue co-incubated with S. flavescens cells. The new compounds flavescenones A, B and C, were determined to be (3R)-5, 7, 2'-trihydroxy-6-gamma, gamma-dimethylallyl-4', 5'-methylenedioxyisoflavanone; 5, 7, 2'-trihydroxy-6-gamma, gamma-dimethylallyl-4', 5'-methylenedioxyisoflavone and 2-[2',4'-dihydroxy-3'-(gamma-hydroxymethyl-gamma-methylallyl)phenyl]-5,6-methylenedioxybenzofuran, respectively, by means of spectroscopic analyses that included 2D-NMR techniques.     

Aromatic Prenylation in Phenazine Biosynthesis: DIHYDROPHENAZINE-1-CARBOXYLATE DIMETHYLALLYLTRANSFERASE FROM STREPTOMYCES ANULATUS*S?

The bacterium Streptomyces anulatus 9663, isolated from the intestine of different arthropods, produces prenylated derivatives of phenazine 1-carboxylic acid. From this organism, we have identified the prenyltransferase gene ppzP. ppzP resides in a gene cluster containing orthologs of all genes known to be involved in phenazine 1-carboxylic acid biosynthesis in Pseudomonas strains as well as genes for the six enzymes required to generate dimethylallyl diphosphate via the mevalonate pathway. This is the first complete gene cluster of a phenazine natural compound from streptomycetes. Heterologous expression of this cluster in Streptomyces coelicolor M512 resulted in the formation of prenylated derivatives of phenazine 1-carboxylic acid. After inactivation of ppzP, only nonprenylated phenazine 1-carboxylic acid was formed. Cloning, overexpression, and purification of PpzP resulted in a 37-kDa soluble protein, which was identified as a 5,10-dihydrophenazine 1-carboxylate dimethylallyltransferase, forming a C–C bond between C-1 of the isoprenoid substrate and C-9 of the aromatic substrate. In contrast to many other prenyltransferases, the reaction of PpzP is independent of the presence of magnesium or other divalent cations. The K_m value for dimethylallyl diphosphate was determined as 116 μm. For dihydro-PCA, half-maximal velocity was observed at 35 μm. K_cat was calculated as 0.435 s^-1. PpzP shows obvious sequence similarity to a recently discovered family of prenyltransferases with aromatic substrates, the ABBA prenyltransferases. The present finding extends the substrate range of this family, previously limited to phenolic compounds, to include also phenazine derivatives.The transfer of isoprenyl moieties to aromatic acceptor molecules gives rise to an astounding diversity of secondary metabolites in bacteria, fungi, and plants, including many compounds that are important in pharmacotherapy. However, surprisingly little biochemical and genetic data are available on the enzymes catalyzing the C-prenylation of aromatic substrates. Recently, a new family of aromatic prenyltransferases was discovered in streptomycetes (1), Gram-positive soil bacteria that are prolific producers of antibiotics and other biologically active compounds (2). The members of this enzyme family show a new type of protein fold with a unique α-β-β-α architecture (3) and were therefore termed ABBA prenyltransferases (1). Only 13 members of this family can be identified by sequence similarity searches in the data base at present, and only four of them have been investigated biochemically (3–6). Up to now, only phenolic compounds have been identified as aromatic substrates of ABBA prenyltransferases. We now report the discovery of a new member of the ABBA prenyltransferase family, catalyzing the transfer of a dimethylallyl moiety to C-9 of 5,10-dihydrophenazine 1-carboxylate (dihydro-PCA).² Streptomyces strains produce many of prenylated phenazines as natural products. For the first time, the present paper reports the identification of a prenyltransferase involved in their biosynthesis.Streptomyces anulatus 9663, isolated from the intestine of different arthropods, produces several prenylated phenazines, among them endophenazine A and B (Fig. 1A) (7). We wanted to investigate which type of prenyltransferase might catalyze the prenylation reaction in endophenazine biosynthesis. In streptomycetes and other microorganisms, genes involved in the biosynthesis of a secondary metabolite are nearly always clustered in a contiguous DNA region. Therefore, the prenyltransferase of endophenazine biosynthesis was expected to be localized in the vicinity of the genes for the biosynthesis of the phenazine core (i.e. of PCA).Open in a separate windowFIGURE 1.A, prenylated phenazines from S. anulatus 9663. B, biosynthetic gene cluster of endophenazine A.In Pseudomonas, an operon of seven genes named phzABCDEFG is responsible for the biosynthesis of PCA (8). The enzyme PhzC catalyzes the condensation of phosphoenolpyruvate and erythrose-4-phosphate (i.e. the first step of the shikimate pathway), and further enzymes of this pathway lead to the intermediate chorismate. PhzD and PhzE catalyze the conversion of chorismate to 2-amino-2-deoxyisochorismate and the subsequent conversion to 2,3-dihydro-3-hydroxyanthranilic acid, respectively. These reactions are well established biochemically. Fewer data are available about the following steps (i.e. dimerization of 2,3-dihydro-3-hydroxyanthranilic acid, several oxidation reactions, and a decarboxylation, ultimately leading to PCA via several instable intermediates). From Pseudomonas, experimental data on the role of PhzF and PhzA/B have been published (8, 9), whereas the role of PhzG is yet unclear. Surprisingly, the only gene cluster for phenazine biosynthesis described so far from streptomycetes (10) was found not to contain a phzF orthologue, raising the question of whether there may be differences in the biosynthesis of phenazines between Pseudomonas and Streptomyces.Screening of a genomic library of the endophenazine producer strain S. anulatus now allowed the identification of the first complete gene cluster of a prenylated phenazine, including the structural gene of dihydro-PCA dimethylallyltransferase.     

A prenylated flavonol,sophoflavescenol: a potent and selective inhibitor of cGMP phosphodiesterase 5

During the search for naturally occurring cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5) inhibitors, it was found that the extracts from Sophora flavescens exhibit potent inhibitory activity against cGMP PDE5 prepared from rat diaphragm. Therefore, the inhibitory activities of five flavonoids, kushenol H (1), kushenol K (2), kurarinol (3), sophoflavescenol (4) and kuraridine (5), isolated from S. flavescens were measured against cGMP PDE5 to identify potent cGMP PDE5 inhibitory constituents. Among tested compounds, sophoflavescenol (4), a C-8 prenylated flavonol, showed the most potent inhibitory activity (IC(50)=0.013 microM) against cGMP PDE5 with 31.5- and 196.2-fold selectivity over PDE3 and PDE4, respectively. Kinetic analysis revealed that sophoflavescenol was a mixed inhibitor of PDE5 with a K(i) value of 0.005 microM.     

HlPT-1, a membrane-bound prenyltransferase responsible for the biosynthesis of bitter acids in hops

Female flowers of hop (Humulus lupulus L.) develop a large number of glandular trichomes called lupulin glands that contain a variety of prenylated compounds such as α- and β-acid (humulone and lupulone, respectively), as well as xanthohumol, a chalcone derivative. These prenylated compounds are biosynthesized by prenyltransferases catalyzing the transfer of dimethylallyl moiety to aromatic substances. In our previous work, we found HlPT-1 a candidate gene for such a prenyltransferase in a cDNA library constructed from lupulin-enriched flower tissues. In this study, we have characterized the enzymatic properties of HlPT-1 using a recombinant protein expressed in baculovirus-infected insect cells. HlPT-1 catalyzed the first transfer of dimethylallyl moiety to phloroglucinol derivatives, phlorisovalerophenone, phlorisobutyrophenone and phlormethylbutanophenone, leading to the formation of humulone and lupulone derivatives. HlPT-1 also recognized naringenin chalcone as a flavonoid substrate to yield xanthohumol, and this broad substrate specificity is a unique character of HlPT-1 that is not seen in other reported flavonoid prenyltransferases, all of which show strict specificity for their aromatic substrates. Moreover, unlike other aromatic substrate prenyltransferases, HlPT-1 revealed an exclusive requirement for Mg(2+) as a divalent cation for its enzymatic activity and also showed exceptionally narrow optimum pH at around pH 7.0.     

Isoprenoids and protein prenylation: implications in the pathogenesis and therapeutic intervention of Alzheimer’s disease

The mevalonate–isoprenoid–cholesterol biosynthesis pathway plays a key role in human health and disease. The importance of this pathway is underscored by the discovery that two major isoprenoids, farnesyl and geranylgeranyl pyrophosphate, are required to modify an array of proteins through a process known as protein prenylation, catalyzed by prenyltransferases. The lipophilic prenyl group facilitates the anchoring of proteins in cell membranes, mediating protein–protein interactions and signal transduction. Numerous essential intracellular proteins undergo prenylation, including most members of the small GTPase superfamily as well as heterotrimeric G proteins and nuclear lamins, and are involved in regulating a plethora of cellular processes and functions. Dysregulation of isoprenoids and protein prenylation is implicated in various disorders, including cardiovascular and cerebrovascular diseases, cancers, bone diseases, infectious diseases, progeria, and neurodegenerative diseases including Alzheimer’s disease (AD). Therefore, isoprenoids and/or prenyltransferases have emerged as attractive targets for developing therapeutic agents. Here, we provide a general overview of isoprenoid synthesis, the process of protein prenylation and the complexity of prenylated proteins, and pharmacological agents that regulate isoprenoids and protein prenylation. Recent findings that connect isoprenoids/protein prenylation with AD are summarized and potential applications of new prenylomic technologies for uncovering the role of prenylated proteins in the pathogenesis of AD are discussed.     

Applications of dimethylallyltryptophan synthases and other indole prenyltransferases for structural modification of natural products

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 various fungi. These genes belong to different gene clusters and are involved in the biosynthesis of secondary metabolites. Ten of them were cloned and overexpressed in Escherichia coli and Saccharomyces cerevisiae and proven to be soluble proteins. They catalyse different prenyl transfer reactions onto indole moieties of various substrates and do not require divalent metal ions for their prenyl transfer reactions. These enzymes showed broad substrate specificities towards their aromatic substrates. Diverse simple tryptophan derivatives and tryptophan-containing cyclic dipeptides were accepted by several prenyltransferases as substrates and converted to prenylated derivatives. This feature of substrate flexibility was successfully used for regiospecific and stereospecific synthesis of different indole derivatives.     

Ubiquinone. Biosynthesis of quinone ring and its isoprenoid side chain. Intracellular localization

Ubiquinone, known as coenzyme Q, was shown to be the part of the metabolic pathways by Crane et al. in 1957. Its function as a component of the mitochondrial respiratory chain is well established. However, ubiquinone has recently attracted increasing attention with regard to its function, in the reduced form, as an antioxidant. In ubiquinone synthesis the para-hydroxybenzoate ring (which is the derivative of tyrosine or phenylalanine) is condensed with a hydrophobic polyisoprenoid side chain, whose length varies from 6 to 10 isoprene units depending on the organism. para-Hydroxybenzoate (PHB) polyprenyltransferase that catalyzes the condensation of PHB with polyprenyl diphosphate has a broad substrate specificity. Most of the genes encoding (all-E)-prenyltransferases which synthesize polyisoprenoid chains, have been cloned. Their structure is either homo- or heterodimeric. Genes that encode prenyltransferases catalysing the transfer of the isoprenoid chain to para-hydroxybenzoate were also cloned in bacteria and yeast. To form ubiquinone, prenylated PHB undergoes several modifications such as hydroxylations, O-methylations, methylations and decarboxylation. In eukaryotes ubiquinones were found in the inner mitochondrial membrane and in other membranes such as the endoplasmic reticulum, Golgi vesicles, lysosomes and peroxisomes. Still, the subcellular site of their biosynthesis remains unclear. Considering the diversity of functions of ubiquinones, and their multistep biosynthesis, identification of factors regulating their cellular level remains an elusive task.     

Molecular and biochemical characterization of tomato farnesyl-protein transferase.

The prenylation of membrane-associated proteins involved in the regulation of eukaryotic cell growth and signal transduction is critically important for their subcellular localization and biological activity. In contrast to mammalian cells and yeast, however, the function of protein prenylation in plants is not well understood and only a few prenylated proteins have been identified. We partially purified and characterized farnesyl-protein transferase from tomato (Lycopersicon esculentum, LeFTase) to analyze its biochemical and molecular properties. Using Ras- and G gamma-specific peptide substrates and competition assays we showed that tomato protein extracts have both farnesyl-protein transferase and geranylgeranyl-protein transferase 1 activities. Compared with the heterologous synthetic peptide substrates, the plant-specific CaaX sequence of the ANJ1 protein is a less efficient substrate for LeFTase in vitro. LeFTase activity profiles and LeFTase beta-subunit protein (LeFTB) levels differ significantly in various tissues and are regulated during fruit development. Partially purified LeFTase requires Zn2+ and Mg2+ for enzymatic activity and has an apparent molecular mass of 100 kD Immunoprecipitation experiments using anti-alpha LeFTB antibodies confirmed that LeFTB is a component of LeFTase but not of tomato geranylgeranyl-protein transferase 1. Based on their conserved bio-chemical activities, we expect that prenyltransferases are likely integrated with the sterol biosynthesis pathway in the control of plant cell growth.     

Diversity of ABBA Prenyltransferases in Marine Streptomyces sp. CNQ-509: Promiscuous Enzymes for the Biosynthesis of Mixed Terpenoid Compounds

Terpenoids are arguably the largest and most diverse family of natural products, featuring prominently in e.g. signalling, self-defence, UV-protection and electron transfer. Prenyltransferases are essential players in terpenoid and hybrid isoprenoid biosynthesis that install isoprene units on target molecules and thereby often modulate their bioactivity. In our search for new prenyltransferase biocatalysts we focused on the marine-derived Streptomyces sp. CNQ-509, a particularly rich source of meroterpenoid chemistry. Sequencing and analysis of the genome of Streptomyces sp. CNQ-509 revealed seven putative phenol/phenazine-specific ABBA prenyltransferases, and one putative indole-specific ABBA prenyltransferase. To elucidate the substrate specificity of the ABBA prenyltransferases and to learn about their role in secondary metabolism, CnqP1 –CnqP8 were produced in Escherichia coli and incubated with various aromatic and isoprenoid substrates. Five of the eight prenyltransferases displayed enzymatic activity. The efficient conversion of dihydroxynaphthalene derivatives by CnqP3 (encoded by AA958_24325) and the co-location of AA958_24325 with genes characteristic for the biosynthesis of THN (tetrahydroxynaphthalene)-derived natural products indicates that the enzyme is involved in the formation of debromomarinone or other naphthoquinone-derived meroterpenoids. Moreover, CnqP3 showed high flexibility towards a range of aromatic and isoprenoid substrates and thus represents an interesting new tool for biocatalytic applications.     

Characterization of an isoflavonoid-specific prenyltransferase from Lupinus albus

Prenylated flavonoids and isoflavonoids possess antimicrobial activity against fungal pathogens of plants. However, only a few plant flavonoid and isoflavonoid prenyltransferase genes have been identified to date. In this study, an isoflavonoid prenyltransferase gene, designated as LaPT1, was identified from white lupin (Lupinus albus). The deduced protein sequence of LaPT1 shared high homologies with known flavonoid and isoflavonoid prenyltransferases. The LaPT1 gene was mainly expressed in roots, a major site for constitutive accumulation of prenylated isoflavones in white lupin. LaPT1 is predicted to be a membrane-bound protein with nine transmembrane regions and conserved functional domains similar to other flavonoid and isoflavonoid prenyltransferases; it has a predicted chloroplast transit peptide and is plastid localized. A microsomal fraction containing recombinant LaPT1 prenylated the isoflavone genistein at the B-ring 3' position to produce isowighteone. The enzyme is also active with 2'-hydroxygenistein but has no activity with other flavonoid substrates. The apparent K(m) of recombinant LaPT1 for the dimethylallyl diphosphate prenyl donor is in a similar range to that of other flavonoid prenyltransferases, but the apparent catalytic efficiency with genistein is considerably higher. Removal of the transit peptide increased the apparent overall activity but also increased the K(m). Medicago truncatula hairy roots expressing LaPT1 accumulated isowighteone, a compound that is not naturally produced in this species, indicating a strategy for metabolic engineering of novel antimicrobial compounds in legumes.     

Anti-atherosclerotic and anti-inflammatory activities of catecholic xanthones and flavonoids isolated from Cudrania tricuspidata

The catecholic xanthones and flavonoids 1-13 were isolated from the root bark of Cudrania tricuspidata. Compounds 1 and 3-8 exhibited significant antioxidant activity against low-density lipoprotein (LDL) oxidation in the thiobarbituric acid-reactive substance (TBARS) assay. Among them, prenylated flavonoids 10-12 showed an inhibitory effect on the NO production and iNOS expression in RAW264.7 cells. Also, compounds 1, 2, 5, 7, 9, and 11 preferentially inhibited hACAT-2 than hACAT-1, whereas compounds 3, 4, 6, and 8 showed a similar specificity against hACAT-1 and -2. However, flavonoids 10, 12, and 13 dominantly inhibited hACAT-2, not hACAT-1.