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

Abstract

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.

FIGURE 1.

Biosynthesis of prenylated chalcone and isoflavone in plants. CHI, chalcone isomerase; IFS, 2-hydroxyisoflavanone synthase; HID, 2-hydroxyisoflavanone dehydratase.

FIGURE 2.

Phylogenetic relationship of plant prenyltransferases accepting aromatic substrates. A rooted phylogram was generated using a ClustalW alignment. The abbreviations are: PF, prenylated flavonoid; VE, vitamin E; PQ, plastoquinone. Species abbreviations are: Ap, Allium porrum; At, Arabidopsis thaliana; Cp, Cuphea pulcherrima; Gm, G. max; Hl, H. lupulus; Hv, Hordeum vulgare; Os, Oryza sativa; Ta, Triticum aestivum. Homogentisate phytyltransferases (VTE2-1) and homogentisate geranylgeranyltransferases (HGGT) are involved in vitamin E biosynthesis, and homogentisate solanesyltransferases (VTE2-2) are involved in plastoquinone biosynthesis. Accession numbers are: ApVTE2-1, DQ231057; AtVTE2-1, AY089963; AtVTE2-2; DQ231060, CpVTE2-1, DQ231058; GmG4DT, AB434690; GmVTE2-1, DQ231059; GmVTE2-2, DQ231061; HlPT1, AB543053; HvHGGT, AY222860; OsHGGT, AY222862; SfG6DT, AB604224; SfiLDT, AB604223; SfN8DT1, AB325579; SfN8DT2, AB370330; SfN8DT3, AB604222; TaHGGT, AY222861.

FIGURE 3.

LC/ESI-MS analysis of enzymatic reaction products. A and C, ethyl acetate-soluble portion of enzyme incubation mixture of flavonoids and DMAPP with recombinant protein expressed in yeast (A, isoliquiritigenin dimethylallyltransferase assay using recombinant SfiLDT; C, genistein dimethylallyltransferase assay using recombinant SfG6DT). Upper, total ion chromatogram (TIC); middle, selected ion monitoring for substrate [M + H]⁺; lower, selected ion monitoring for prenylated products [M + H]⁺. B and D, MS spectra of dimethylallylated products formed from flavonoids by recombinant protein (B, dimethylallyl isoliquiritigenin; D, 6-dimethylallyl genistein).

FIGURE 4.

Substrate specificity of recombinant SfiLDT (A and C) and SfG6DT (B and D). A and B, relative enzyme activity for various flavonoid substrates as prenyl acceptors. The abbreviations are: isoliq, isoliquiritigenin; nari-chal, naringenin chalcone; 2-hydro-chal, 2-hydroxychalcone; 2′-hydro-chal, 2′-hydroxychalcone; 4-hydro-chal, 4-hydroxychalcone; penta-hydro-chal, 2′,4′,6′,3,4-pentahydroxychalcone; chal, chalcone; nari, naringenin; liq, liquiritigenin; LG, leachianone G; kae, kaempferol; api, apigenin; tax, taxifolin; geni, genistein; bioch, biochanin A; daid, daidzein; form, formononetin; and maa, maackiain. C and D, relative enzyme activity with various prenyl diphosphates of different chain length as substrates, DMAPP, GPP, FPP, and GGPP. N.D. indicates not detected. All experiments were replicated three times, and relative activity is shown as a percentage where S.D. is shown.

FIGURE 5.

Flavonoid dimethylallyltransferase activity of chimeric enzymes between SfN8DT-1 and SfG6DT. A, amino acid sequences of SfN8DT-1 and SfG6DT. Arrowheads indicate the junction to make chimeras between SfN8DT-1 and SfG6DT. TM indicates transmembrane α-helices predicted by the TMHMM program. B, schematic drawing of chimeric enzymes and their flavonoid prenyltransferase activities. N.D., not detected. C, HPLC chromatograms of enzymatic reaction products. Nari, naringenin; Geni, genistein; mAu, milliabsorbance units; Inset, UV spectrum of enzymatic products. 8DN, 8-dimethylallyl naringenin; 6DG, 6-dimethylallyl genistein.

FIGURE 6.

Transient expression of the SfG6DT-TP-GFP fusion protein in onion epidermal peels. A and C, SfG6DT-TP-GFP and WxTP-DsRed plasmids were co-transformed into onion epidermal peels by particle bombardment. B, For the control, modified pGWB5 (CaMV35S promoter + GFP) and WxTP-DsRed plasmids were double-transformed into onion epidermal peels. The images were obtained at 24 h after bombardment. WxTP-DsRed (red fluorescence) was used as a control for plastid targeting. Scale bars show 100 μm (A and B), and 25 μm (C).