Cloning and Characterization of Naringenin 8-Prenyltransferase, a Flavonoid-Specific Prenyltransferase of Sophora flavescens    

Abstract

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.The prenylation of aromatic compounds is a major contributor to the diversity of plant secondary metabolites due to differences in prenylation position on the aromatic ring, various lengths of prenyl chain, and further modifications of the prenyl moiety, e.g. cyclization and hydroxylation, resulting in the occurrence of more than 1,000 prenylated compounds in plants (Tahara and Ibrahim, 1995;Barron and Ibrahim, 1996). In particular, prenylated flavonoids in higher plants protect them by exhibiting strong antibacterial and antifungal activities (Sohn et al., 2004). Many prenylated flavonoids have been identified as active components in medicinal plants with biological activities, such as anticancer, anti-androgen, anti-leishmania, and anti-nitric oxide production (De Naeyer et al., 2004;Ahmed-Belkacem et al., 2005;Han et al., 2006). Due to the beneficial effects for human health, prenylated flavonoids are of particular interest as lead compounds for producing new drugs and functional foods. The prenylation of the flavonoid core increases the lipophilicity and the membrane permeability, which is one of the proposed reasons for the enhanced biological activities of prenylated flavonoids (Wang et al., 1997;Maitrejean et al., 2000;Murakami et al., 2000). However, none of the genes responsible for the prenylation reactions has been identified d...

Molecular Characterization of a Membrane-bound Prenyltransferase Specific for Isoflavone from Sophora flavescens

“…flavescens Cells-Our previous discovery of SfN8DT-1 indicated that plant prenyltransferases for flavonoids belong to the homogentisate prenyltransferase family involved in vitamin E and plastoquinone biosynthesis (14). Thus, the sequence information of SfN8DT-1 was expected to provide a very powerful tool for isolating cDNAs encoding prenyltransferase that recognized other flavonoids, e.g.…”

“…A phylogenic tree was made with these candidates and flavonoid prenyltransferases previously reported (SfN8DT-1, SfN8DT-2 isolated from S. flavescens, G4DT from Glycine max) (14,15), in addition to homogentisate prenyltransferases involved in vitamin E and plastoquinone biosynthesis (Fig. 2).…”

“…The microsomes prepared from the transgenic yeast were incubated with 14 C-labeled DMAPP and various flavonoids of different groups (naringenin, leachianone G, liquiritigenin, kaempferol, apigenin, taxifolin, naringenin chalcone, isoliquiritigenin, genistein, and maackiain; supplemental Fig. S1) as substrates in the presence of Mg 2ϩ at 30°C for 12 h. The enzymatic reaction products were detected using autoradiography, which showed that all three clones revealed clear dimethylallyltransferase activity for either flavonoid in a Mg 2ϩ -dependent manner.…”

“…Radioactive Assay of Flavonoid Prenyltransferase with TLCThree candidate clones and 10 chimeric enzymes of flavonoid prenyltransferases were expressed in the yeast strain W303-1A-delta-coq2, in which the p-hydroxybenzoate prenyltransferase gene (coq2) was disrupted (19), by culturing in SD (selective dropout without uracil) liquid medium (180 ml) to reach mid-log phase, and the microsomal fraction of the transformed yeast was prepared as described previously (14). The resuspended membrane fraction from each transformant was used as an enzyme solution for the flavonoid prenyltransferase assay using various flavonoids (final concentration 1 mM) and [1-14 C]DMAPP (final concentration 4.5 M, specific activity 55 mCi/mmol, American Radiolabeled Chemicals, Inc.) as sub- 3 The abbreviations used are: DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; EST, expressed sequence tag; ESI, electrospray mass ionization; N8DT, naringenin 8-dimethylallyltransferase; G6DT, genistein 6-dimethylallyltransferase; iLDT, isoliquiritigenin dimethylallyltransferase; G4DT, glycinol 4-dimethylallyltransferase.…”

“…In our recent study, the first cDNA encoding flavanone-specific plant prenyltransferase, naringenin 8-dimethylallyltransferase (SfN8DT-1) from Sophora flavescens, was reported (14). After the discovery of SfN8DT-1, it was expected that prenyltransferases recognizing more divergent substrates such as flavones and isoflavones could be cloned, but thus far only a pterocarpan-specific prenyltransferase has been reported from soybeans (15).…”