2-7-4--trihydroxyisoflavanone and liquiritigenin

Isoflavonoids are distributed predominantly in leguminous plants, and play pivotal roles in the interaction of host plants with biological environments. Isoflavones in the diet also have beneficial effects on human health as phytoestrogens. The isoflavonoid skeleton is constructed by the CYP93C subfamily of cytochrome P450s in plant cells. The reaction consists of hydroxylation of the flavanone molecule at C-2 and an intramolecular 1,2-aryl migration from C-2 to C-3 to yield 2-hydroxyisoflavanone. In this study, with the aid of alignment of amino acid sequences of CYP93 family P450s and a computer-generated putative stereo structure of the protein, candidates for key amino acid residues in CYP93C2 responsible for the unique aryl migration in 2-hydroxyisoflavanone synthase reaction were identified. Microsomes of recombinant yeast cells expressing mutant proteins of CYP93C2 were prepared, and their catalytic activities tested. The reaction with the mutant in which Ser 310 in the centre of the I-helix was converted to Thr yielded increased formation of 3-hydroxyflavanone, a by-product of the 2-hydroxyisoflavanone synthase reaction, in addition to the major isoflavonoid product. More dramatically, the mutant in which Lys 375 in the end of beta-sheet 1-4 was replaced with Thr produced only 3-hydroxyflavanone and did not yield the isoflavonoid any longer. The roles of these amino acid residues in the catalysis and evolution of isoflavonoid biosynthesis are discussed.

Topics: Amino Acid Sequence; Amino Acids; Catalysis; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Flavanones; Flavonoids; Gene Expression Regulation, Enzymologic; Hydrogen-Ion Concentration; Isoflavones; Kinetics; Microsomes; Mixed Function Oxygenases; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Protein Binding; Protein Conformation; Yeasts

The bioactive isoflavonoids of the Leguminosae often are methylated on the 4'-position of their B-rings. Paradoxically, reverse genetic evidence implicates alfalfa isoflavone O-methyltransferase (IOMT) in the biosynthesis of 4'-O-methylated isoflavonoids such as the phytoalexin medicarpin in vivo, whereas biochemical studies indicate that IOMT has strict specificity for methylation of the A-ring 7-hydroxyl of daidzein, the presumed substrate for O-methylation, in vitro. Radiolabeling and isotope dilution studies now confirm that daidzein is not an intermediate in isoflavonoid phytoalexin biosynthesis in alfalfa. Furthermore, protein gel blot analysis and confocal microscopy of a transiently expressed IOMT-green fluorescent protein fusion in alfalfa leaves show that the operationally soluble IOMT localizes to endomembranes after elicitation of the isoflavonoid pathway. We propose that IOMT colocalizes with the endoplasmic reticulum-associated isoflavone synthase cytochrome P450 to ensure rapid B-ring methylation of the unstable 2,4',7-trihydroxyisoflavanone product of isoflavone synthase, thereby preventing its dehydration to daidzein and subsequent A-ring methylation by free IOMT. In this way, metabolic channeling at the entry point into isoflavonoid phytoalexin biosynthesis protects an unstable intermediate from an unproductive metabolic conversion.

Topics: Benzopyrans; Cells, Cultured; Endoplasmic Reticulum; Flavanones; Flavonoids; Ion Channels; Isoflavones; Isotope Labeling; Medicago sativa; Methylation; Methyltransferases; Microsomes; Oxygenases; Phytoalexins; Plant Extracts; Plant Leaves; Plants, Genetically Modified; Pterocarpans; Sesquiterpenes; Terpenes