Cloning and functional expression of a cytochrome P450 cDNA encoding 2-hydroxyisoflavanone synthase involved in biosynthesis of the isoflavonoid skeleton in licorice

Abstract

Isoflavonoids are distributed predominantly in leguminous plants and play critical roles in plant physiology. A cytochrome P450 (P450), 2-hydroxyisoflavanone synthase, is the key enzyme in their biosynthesis. In cultured licorice (Glycyrrhiza echinata L., Fabaceae) cells, the production of both an isoflavonoid-derived phytoalexin (medicarpin) and a retrochalcone (echinatin) is rapidly induced upon elicitation. In this study, we obtained a full-length P450 cDNA, CYP Ge-8 (CYP93C2), from the cDNA library of elicited G. echinata cells. When the flavanones liquiritigenin and naringenin were incubated with the recombinant yeast microsome expressing CYP93C2, major products emerged and were readily converted to the isoflavones daidzein and genistein by acid treatment. The chemical structures of the products from liquiritigenin (2-hydroxyisoflavanone and isoflavone) were confirmed by mass spectrometry. CYP93C2 was thus shown to encode 2-hydroxyisoflavanone synthase, which catalyzes the hydroxylation associated with 1,2-aryl migration of flavanones. Northern-blot analysis revealed that transcripts of CYP93C2, in addition to those of other P450s involved in phenylpropanoid/flavonoid pathways, transiently accumulate upon elicitation.

Figure 1

Biosynthesis of isoflavonoids, retrochalcone, and flavones.

Figure 2

Amino acid sequence of CYP93C2 (CYP Ge-8; upper row) aligned with CYP93B1 (lower row). Gaps (−) are inserted to optimize alignment. Positions with identical and similar amino acid residues in both sequences are marked by asterisks and dots, respectively.

Figure 3

TLC autoradiograms of products from aerobic incubation of (2S)-[¹⁴C]liquiritigenin and NADPH with microsomal fractions of yeast cells expressing CYP93C2. A, Ethyl acetate extract of products from the incubation with recombinant yeast microsome (left lane) and with control yeast (transformed with the vector pYES2 without insert) microsome (right lane). B, Product after acid treatment of P1 scraped from the TLC plate: left lane, the material isolated from spot P1 in (A); right lane, acid treated sample. TLC, Cellulose; solvent, 15% (v/v) acetic acid. Liq, Liquiritigenin; Dai, daidzein; O, origin; F, solvent front.

Figure 4

HPLC analysis of products from (RS)-liquiritigenin in the IFS assay. A, Direct reaction products with control yeast microsome (top), microsome of the yeast transformed with CYP93C2 (middle), and products after acid treatment of the reaction mixture from IFS reaction (bottom). B, Substrate [(RS)-liquiritigenin] before the reaction (top) and recovered after 120 min of incubation (middle) on a chiral separation column. For reference, the chromatogram of (S)-liquiritigenin produced by the chalcone isomerase reaction is shown (bottom).

Figure 5

Electron impact mass spectra of P1 (2,7,4′-trihydroxy-isoflavanone) (A) and P3 (daidzein) (B).

Figure 6

TLC autoradiograms of the reaction products from (2S)-[¹⁴C]naringenin with microsome of yeast expressing CYP93C2 (A, left lane) or yeast harboring vector pYES2 without insert (A, right lane), and product (B, right lane) after HCl treatment of P4 (B, left lane) separated from the TLC plate (A). TLC, Cellulose; solvent, 15% (v/v) acetic acid. Nar, Naringenin; Gen, genistein.

Figure 7

TLC-chromatoscan data of reaction products from (2S)-[¹⁴C]liquiritigenin with microsomal fraction of elicitor-treated G. echinata cells (A) and products after HCl treatment (B). TLC conditions in A are the same as Figure 3; in B, TLC was performed on silica gel with the solvent toluene:ethyl acetate:methanol:light petroleum (6:4:1:3, v/v). Lico, Licodione; Liq, liquiritigenin; Dai, daidzein; 7,4′-D, 7,4′-dihydroxyflavone.

Figure 8

Northern-blot analysis of RNAs from cultured G. echinata cells after treatment with yeast extract (YE). For probes, P450 (IFS, CA4H, I2′H, and F2H) coding regions and the PCR fragment of the G. echinata actin gene (accession no. AB023637) labeled with alkaline phosphatase were used.

Figure 9

Comparison of partial stereostructures of (2S)-flavanone and (6aR)-pterocarpan.