Abstract
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.
Highlights
Prenylated flavonoids and isoflavonoids possess antimicrobial activity against fungal pathogens of plants
From a total of about 8,000 white lupin EST sequences obtained from our own EST collection (Tian et al, 2009) and the National Center for Biotechnology Information database, two candidate ESTs were selected based on their sequence similarities with known flavonoid and isoflavonoid prenyltransferases
One EST (FG092841), obtained from developing roots of white lupin ’Netherland’ (Tian et al, 2009), shared 35.7% and 35.7% sequence identities with SfN8DT-1 and G4DT, respectively, but higher sequence identity (96.2%) with VET2-2
Summary
From a total of about 8,000 white lupin EST sequences obtained from our own EST collection (Tian et al, 2009) and the National Center for Biotechnology Information database, two candidate ESTs were selected based on their sequence similarities with known flavonoid and isoflavonoid prenyltransferases. The Km of full-length and truncated LaPT1 variants for genistein was calculated to be 0.3, 0.5, and 38.8 mM, respectively (Supplemental Fig. S7), compared with 55 mM for SfN8DT-1 and 68 mM for G4DT for their preferred flavanone and pterocarpan prenyl acceptor substrates, respectively. To address the prenyl acceptor specificity of LaPT1, flavanones (naringenin, liquiritigenin, hesperetin), flavone (apigenin), chalcones (2-hydroxychalcone, 29-hydroxychalcone, isoliquiritigenin), isoflavones (daidzein, formononetin, genistein, 29-hydroxygenistein, biochanin A, 7-hydroxyisoflavone), and flavonols (kaempferol, quercetin, taxifolin) were incubated with the yeast microsomal membrane fraction containing recombinant LaPT1 protein, with DMAPP as prenyl donor, and products were analyzed by reverse-phase HPLC. Because the retention times of isowighteone and wighteone are so close, the product was further confirmed by supplementing the methanolic extract of a 35S::LaPT1 transgenic line with authentic standards of wighteone and isowighteone This analysis clearly showed that the new product comigrated with isowighteone rather than wighteone (Fig. 7D). LaPT1 can convert endogenous genistein to isowighteone in planta, providing proof of concept for the metabolic engineering of antimicrobial prenylated isoflavone derivatives in transgenic plants
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