Enzymic synthesis of isoflavones

Abstract

The NADPH and oxygen-dependent conversion of (2 S)-naringenin to genistein catalyzed by a microsomal preparation from elicitor-treated soybean cell suspension cultures has been resolved into two steps. In the first step (2s)-naringenin is converted to a product (P-2) which yields genistein in a second step. The chemical behaviour of P-2 and its ultraviolet and mass spectral data are consistent with a 2-hydroxyisoflavanone structure. The conversion of (2 S)-naringenin to P-2 requires NADPH, oxygen and cytochrome P-450. The participation of cytochrome P-450 was demonstrated by CO inhibition of the reaction and its partial reversal by light, and by inhibition with typical cytochrome P-450 inhibitors. On a Percoll gradient the membrane fraction which catalyzes P-2 formation coincides with marker enzymes for the endoplasmic reticulum and with the position of cytochrome P-450. Enzymatic activity for conversion of P-2 to genistein is mainly present in the supernatant of the 160000 x g fraction. This reaction, formally a dehydration, does not require NADPH or oxygen.Isoflavonoids comprise a large group of natural products. A recent survey by Ingham lists 510 naturally occurring isoflavonoids [l]. Isoflavonoids can have estrogenic, insecticidal, piscicidal and antimicrobial properties [2, 31. Antimicrobial isoflavonoids can act as phytoalexins in the defence of plants against potential pathogens [4].Detailed studies with radioactively labelled precursors have proved that isoflavones originate from a chalcone/ flavanone precursor by a 2,3-migration of the B-ring [5, 61, which has recently been shown to be an intramolecular rearrangement [28]. The finding that (-) (2 S)-5,7,4-trihydroxyflavanone [(2S)-naringenin] was stereospecifically incorporated into biochanin A in Cicer arietinum seedlings demonstrated that very probably the (2s)-flavanone and not the chalcone is the substrate for the rearrangement to isoflavone [7]. It was also shown that dihydrokaempferol is not a precursor for isoflavones in Cicer seedlings [8].The enzymology of the rearrangement of flavanone to isoflavone remained unknown until recently, when we were able to demonstrate the conversion of (2 S)-naringenin to genistein with a microsomal preparation from elicitorchallenged soybean cell cultures in the presence of NADPH and oxygen [9]. We now present a more detailed report on this enzyme system.

Structural Basis for Dual Functionality of Isoflavonoid O-Methyltransferases in the Evolution of Plant Defense Responses

“…IFS, which is used to prepare 2,7,49-trihydroxyisoflavanone, was previously shown to accept only 2S-flavanones as substrates (Hagmann and Grisebach, 1984;Kochs and Grisebach, 1986). Consistently, our studies here also demonstrated that the recombinant M. truncatula IFS only converted 2S-liquiritigenin (a 2S-flavanone) into 2,7,49-trihydroxyisoflavanone when incubated with a racemic mixture of flavanone substrates (data not shown).…”

“…To obtain the hydroxyisoflavanone substrate that is not commercially available and is chemically unstable, 2,7,49-trihydroxyisoflavanone was prepared biosynthetically using recombinant M. truncatula IFS incubated with a racemic mixture of 2R/2S-liquiritigenin (7,49-dihydroxyflavanone) substrates (Figure 1). In this reaction, only the 2S-isomer served as an IFS substrate (data not shown), consistent with previous reports of IFS substrate specificity (Hagmann and Grisebach, 1984;Kochs and Grisebach, 1986). However, the 2,7,49-trihydroxyisoflavanone product resulting from this IFS-catalyzed in vitro reaction existed as a pair of stereoisomers based upon rapid purification using chiral HPLC (data not shown).…”

Reaction mechamism of oxidative rearrangement of flavanone in isoflavone biosynthesis

“…Several hypotheses were proposed for the reaction mechanism of this oxidative 1,2-aryl migration reaction [6]. Kochs and Grisebach reported the isolation of 2-hydroxyisoflavanone as an intermediate in isoflavone synthesis by microsomes from elicitor challenged soybean cells, and proposed a mechanism which involves an enol epoxide of flavanone as a reaction intermediate [3,4]. Crombie et al suggested another mechanism in which epoxidation occurred on the side phenyl group of flavanone [7].…”

Inducible De Novo Biosynthesis of Isoflavonoids in Soybean Leaves by Spodoptera litura Derived Elicitors: Tracer Techniques Aided by High Resolution LCMS

“…Selective pathway activation was demonstrated by a comparative lack of [ 13 C 9 ]isotope incorporation into flavones and flavonol. This metabolic regulation is likely controlled by the activation of key enzymes in isoflavone biosynthesis, namely 2-hydroxyisoflavanone synthase (IFS) (Hashim et al 1990;Kochs and Grisebach 1986) and 2-hydroxyisoflavanone dehydrogenase (HID) (Hakamatsuka et al 1998) (Fig. 6).…”