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.     

Molecular characterization of a membrane-bound prenyltransferase specific for isoflavone from Sophora flavescens

Prenylated isoflavones are secondary metabolites that are mainly distributed in legume plants. They often possess divergent biological activities such as anti-bacterial, anti-fungal, and anti-oxidant activities and thus attract much attention in food, medicinal, and agricultural research fields. Prenyltransferase is the key enzyme in the biosynthesis of prenylated flavonoids by catalyzing a rate-limiting step, i.e. the coupling process of two major metabolic pathways, the isoprenoid pathway and shikimate/polyketide pathway. However, so far only two genes have been isolated as prenyltransferases involved in the biosynthesis of prenylated flavonoids, namely naringenin 8-dimethylallyltransferase from Sophora flavescens (SfN8DT-1) specific for some limited flavanones and glycinol 4-dimethylallyltransferase from Glycine max (G4DT), specific for pterocarpan substrate. We have in this study isolated two novel genes coding for membrane-bound flavonoid prenyltransferases from S. flavescens, an isoflavone-specific prenyltransferase (SfG6DT) responsible for the prenylation of the genistein at the 6-position and a chalcone-specific prenyltransferase designated as isoliquiritigenin dimethylallyltransferase (SfiLDT). These prenyltransferases were enzymatically characterized using a yeast expression system. Analysis on the substrate specificity of chimeric enzymes between SfN8DT-1 and SfG6DT suggested that the determinant region for the specificity of the flavonoids was the domain neighboring the fifth transmembrane α-helix of the prenyltransferases.     

Isoprenoids and Related Pharmacological Interventions: Potential Application in Alzheimer’s Disease

Two major isoprenoids, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, serve as lipid donors for the posttranslational modification (known as prenylation) of proteins that possess a characteristic C-terminal motif. The prenylation reaction is catalyzed by prenyltransferases. The lipid prenyl group facilitates to anchor the proteins in cell membranes and mediates protein-protein interactions. A variety of important intracellular proteins undergo prenylation, including almost all members of small GTPase superfamilies as well as heterotrimeric G protein subunits and nuclear lamins. These prenylated proteins are involved in regulating a wide range of cellular processes and functions, such as cell growth, differentiation, cytoskeletal organization, and vesicle trafficking. Prenylated proteins are also implicated in the pathogenesis of different types of diseases. Consequently, isoprenoids and/or prenyltransferases have emerged as attractive therapeutic targets for combating various disorders. This review attempts to summarize the pharmacological agents currently available or under development that control isoprenoid availability and/or the process of prenylation, mainly focusing on statins, bisphosphonates, and prenyltransferase inhibitors. Whereas statins and bisphosphonates deplete the production of isoprenoids by inhibiting the activity of upstream enzymes, prenyltransferase inhibitors directly block the prenylation of proteins. As the importance of isoprenoids and prenylated proteins in health and disease continues to emerge, the therapeutic potential of these pharmacological agents has expanded across multiple disciplines. This review mainly discusses their potential application in Alzheimer's disease.     

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 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.     

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.     

芳香族异戊烯转移酶的研究进展

异戊烯基转移酶(prenyltransferase)催化异戊烯基转移至异戊烯单元、芳香环或蛋白质上。芳香族异戊烯基转移酶将异戊烯单元融入含有芳环的化合物, 从而形成具有重要生物学功能的各类活性分子, 如泛醌、质体醌、维生素E、异戊烯黄酮类以及真菌代谢物等。该文综述了近年来植物和真菌芳香族异戊烯转移酶的分子生物学研究进展, 包括膜结合的参与质体醌生物合成的homogentisate solanesyltransferase、参与维生素E生物合成的homogentisate phytyltransferase、类黄酮异戊烯转移酶(flavonoid prenyltransferase)和可溶性的真菌吲哚异戊烯转移酶等。     

芳香族异戊烯转移酶的研究进展

异戊烯基转移酶（prenyltransferase）催化异戊烯基转移至异戊烯单元、芳香环或蛋白质上。芳香族异戊烯基转移酶将异戊烯单元融入含有芳环的化合物,从而形成具有重要生物学功能的各类活性分子,如泛醌、质体醌、维生素E、异戊烯黄酮类以及真菌代谢物等。该文综述了近年来植物和真菌芳香族异戊烯转移酶的分子生物学研究进展,包括膜结合的参与质体醌生物合成的homogentisate solanesyltransferase、参与维生素E生物合成的homogentisate phytyltransferase、类黄酮异戊烯转移酶（flavonoid prenyltransferase）和可溶性的真菌吲哚异戊烯转移酶等。     

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.     

Structure and mechanism of the magnesium-independent aromatic prenyltransferase CloQ from the clorobiocin biosynthetic pathway

CloQ is an aromatic prenyltransferase from the clorobiocin biosynthetic pathway of Streptomyces roseochromogenes var. oscitans. It is involved in the synthesis of the prenylated hydroxybenzoate moiety of the antibiotic, specifically catalyzing the attachment of a dimethylallyl moiety to 4-hydroxyphenylpyruvate. Herein, we report the crystal structure of CloQ and use it as a framework for interpreting biochemical data from both wild-type and variant proteins. CloQ belongs to the aromatic prenyltransferase family, which is characterized by an unusual core fold comprising five consecutive ααββ elements that form a central 10-stranded anti-parallel β-barrel. The latter delineates a solvent-accessible cavity where substrates bind and catalysis takes place. This cavity has well-defined polar and nonpolar regions, which have distinct roles in substrate binding and facilitate a Friedel-Crafts-type mechanism. We propose that the juxtaposition of five positively charged residues in the polar region circumvents the necessity for a Mg²⁺, which, by contrast, is a strict requirement for the majority of prenyltransferases characterized to date. Our structure of CloQ complexed with 4-hydroxyphenylpyruvate reveals the formation of a covalent link between the substrate and Cys215 to yield a thiohemiketal species. Through site-directed mutagenesis, we show that this link is not essential for enzyme activity in vitro. Furthermore, we demonstrate that CloQ will accept alternative substrates and, therefore, has the capacity to generate a range of prenylated compounds. Since prenylation is thought to enhance the bioactivity of many natural products, CloQ offers considerable promise as a biocatalyst for the chemoenzymatic synthesis of novel compounds with therapeutic potential.     

Evolution of aromatic prenyltransferases in the biosynthesis of indole derivatives

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.     

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.     

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.     

Engineering Escherichia coli as a platform for the in vivo synthesis of prenylated aromatics

Prenylated aromatics (PAs) are an important class of natural products with valuable pharmaceutical applications. To address current limitations of their sourcing from plants, here, we present a microbial platform for the in vivo synthesis of PAs based on the aromatic prenyltransferase NphB from Streptomyces sp. strain CL190. As proof of concept, we targeted the prenylation of phenolic/phenolcarboxylic acids, including orsellinic (OSA), divarinolic (DVA), and olivetolic (OLA) acids, whose prenylated products have important biopharmaceutical applications. Although the ability of wild-type NphB to catalyze the prenylation reaction with each acid was validated by in vitro characterization, improvement of product titers in vivo required protein modeling and rational design to engineer NphB variants with increased activity and product selectivity. When a designed NphB variant with eightfold improved catalytic efficiency toward OSA was expressed in an Escherichia coli host engineered to generate geranyl pyrophosphate at high flux through the mevalonate pathway, we observed up to 300 mg/L prenylated products by exogenously supplying OSA. The improved properties of engineered NphB were also utilized to demonstrate the diversification of this in vivo platform by using both different aromatic acceptors and different prenyl donors to generate various PA compounds, including medicinally important compounds such as cannabigerovarinic, cannabigerolic, and grifolic acids.     

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.     

Xanthohumol and related prenylflavonoids from hops and beer: to your good health!

Xanthohumol (3'-[3,3-dimethyl allyl]-2',4',4-trihydroxy-6'-methoxychalcone) is the principal prenylated flavonoid of the female inflorescences of the hop plant ('hops'), an ingredient of beer. Human exposure to xanthohumol and related prenylflavonoids, such as 8-prenylnaringenin and isoxanthohumol, is primarily through beer consumption. Xanthohumol has been characterized a 'broad-spectrum' cancer chemopreventive agent in in vitro studies, while 8-prenylnaringenin enjoys fame as the most potent phytoestrogen known to date. These biological activities suggest that prenylflavonoids from hops have potential for application in cancer prevention programs and in prevention or treatment of (post-)menopausal 'hot flashes' and osteoporosis. Xanthohumol and 8-prenylnaringenin are metabolized into many flavonoid derivatives with modified 3,3-dimethyl allyl (prenyl) moieties. Xanthohumol is formed in lupulin glands by a specialized branch of flavonoid biosynthesis that involves prenylation and O-methylation of the polyketide intermediate chalconaringenin. Although a lupulin gland-specific chalcone synthase is known, the aromatic prenyltransferase and O-methyltransferase participating in xanthohumol have not been identified. The prenylflavonoid pathway is a possible target for breeding or biotechnological modification of hops with the aim of increasing xanthohumol levels for beer brewing and 8-prenylnaringenin levels for pharmaceutical production.     

Molecular Cloning and Characterization of a cDNA for Pterocarpan 4-Dimethylallyltransferase Catalyzing the Key Prenylation Step in the Biosynthesis of Glyceollin,a Soybean Phytoalexin

Glyceollins are soybean (Glycine max) phytoalexins possessing pterocarpanoid skeletons with cyclic ether decoration originating from a C₅ prenyl moiety. Enzymes involved in glyceollin biosynthesis have been thoroughly characterized during the early era of modern plant biochemistry, and many genes encoding enzymes of isoflavonoid biosynthesis have been cloned, but some genes for later biosynthetic steps are still unidentified. In particular, the prenyltransferase responsible for the addition of the dimethylallyl chain to pterocarpan has drawn a large amount of attention from many researchers due to the crucial coupling process of the polyphenol core and isoprenoid moiety. This study narrowed down the candidate genes to three soybean expressed sequence tag sequences homologous to genes encoding homogentisate phytyltransferase of the tocopherol biosynthetic pathway and identified among them a cDNA encoding dimethylallyl diphosphate: (6aS, 11aS)-3,9,6a-trihydroxypterocarpan [(−)-glycinol] 4-dimethylallyltransferase (G4DT) yielding the direct precursor of glyceollin I. The full-length cDNA encoding a protein led by a plastid targeting signal sequence was isolated from young soybean seedlings, and the catalytic function of the gene product was verified using recombinant yeast microsomes. Expression of the G4DT gene was strongly up-regulated in 5 to 24 h after elicitation of phytoalexin biosynthesis in cultured soybean cells similarly to genes associated with isoflavonoid pathway. The prenyl part of glyceollin I was demonstrated to originate from the methylerythritol pathway by a tracer experiment using [1-¹³C]Glc and nuclear magnetic resonance measurement, which coincided with the presumed plastid localization of G4DT. The first identification of a pterocarpan-specific prenyltransferase provides new insights into plant secondary metabolism and in particular those reactions involved in the disease resistance mechanism of soybean as the penultimate gene of glyceollin biosynthesis.Typical phytoalexins of the Leguminosae are isoflavonoid derivatives with characteristic species-specific modifications in both their skeletons and their decoration, e.g. prenylation (Dixon, 1999). Isoflavonoids are formed through an early branching pathway in flavonoid metabolism. The most abundantly found isoflavonoid skeleton of leguminous phytoalexins is pterocarpan, and more than one-half of these pterocarpanoids are decorated in a complex manner mainly by isoprenoid-derived substituents (Tahara and Ibrahim, 1995). Glyceollin is the collective name for soybean (Glycine max) phytoalexins with pterocarpanoid skeletons and cyclic ether decoration originating from C₅ prenyl substitutions (Fig. 1). The biosynthesis mechanism of soybean phytoalexins has been studied extensively during the 1970s to 1990s, most actively by Grisebach et al. (Ebel and Grisebach, 1988), and the pathway and biosynthetic enzymes involved have been characterized intensively at the biochemical level (Ebel, 1986; Dixon, 1999). More recent studies with leguminous plants such as alfalfa (Medicago sativa), licorice (Glycyrrhiza echinata), Lotus japonicus, and Medicago truncatula in addition to soybean have resulted in the identification of many genes encoding enzymes involved in isoflavonoid formation (Dixon, 1999; Shimada et al., 2007; Veitch, 2007). However, some genes encoding enzymes of the later stages of glyceollin biosynthesis, especially the crucial prenylation step, have remained uncharacterized until now.Open in a separate windowFigure 1.Biosynthesis of glyceollin isomers in soybean. Abbreviations not defined in the text: HID, 2-hydroxyisoflavanone dehydratase; IFS, 2-hydroxyisoflavanone synthase; P6aH, pterocarpan 6a-hydroxylase; G2DT, dimethylallyl diphosphate: (−)-glycinol 2-dimethylallyltransferase.During glyceollin biosynthesis, a dimethylallyl group is introduced at either C-4 or C-2 of the pterocarpan skeleton (C-8 or C-6 by isoflavone numbering, respectively). A prenyltransferase activity catalyzing the dimethylallylation of (6aS, 11aS)-3,9,6a-trihydroxypterocarpan, (−)-glycinol, has been demonstrated in microsomal fractions of soybean cotyledons and cell cultures treated with a glucan elicitor derived from the cell walls of Phytophthora sojae (Zähringer et al., 1979). An increased toxicity of the prenylated pterocarpans against a phytopathogenic fungus was also demonstrated (Zähringer et al., 1981). An important finding was that the prenylation activity was localized to the chloroplast fraction of cotyledon cells in contrast to the endoplasmic reticulum (ER) where many of the cytochrome P450s (P450s) for glyceollin formation are localized (Welle and Grisebach, 1988; Biggs et al., 1990; Ayabe and Akashi, 2006). Efficient solubilization of the activity and partial purification of the enzyme have also been reported (Welle and Grisebach, 1991), but no complete purification was achieved to sequence the amino acids, and thus the gene responsible remains unidentified.Recently, plant cDNAs of aromatic substrate prenyltransferases have been characterized, and their nucleotide sequence information has become available (Yazaki et al., 2002; Sasaki et al., 2008). In view of the potential benefits of understanding the molecular mechanism underlying the phytopathogen resistance of soybean for the future disease-resistance breeding, studies toward the complete identification of the enzymes involved in glyceollin biosynthesis are important. Thus, this study undertook the molecular cloning and biochemical characterization of a soybean prenyltransferase involved in the glyceollin biosynthetic pathway.     

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.     

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.