Reaction mechamism of oxidative rearrangement of flavanone in isoflavone biosynthesis

Abstract

Microsomes that were prepared from elicitor‐treated Pueraria lobata cell cultures catalyzed the conversion of liquiritigenin, a flavanone, into daidzein, an isoflavone. The reaction was resolved into two steps. 2, 7, 4'‐Trihydroxyisoflavanone was formed as a major product when liquiritigenin was incubated with carefully washed microsomes in the presence of NADPH. The structure of 2, 7, 4'‐trihydroxyisoflavanone was confirmed by mass and 1H NMR spectroscopies. The enzyme responsible for this rearrangement reaction is a cytochrome P‐450‐dependent monooxygenase. Upon treatment with a soluble enzyme fraction 2, 7, 4'‐trihydroxyisoflavone yielded daidzein quantitatively. The incorporation of 18O from 18O2 into the 2‐hydroxy group of 2, 7, 4'‐trihydroxyisoflavanone was demonstrated by the shift of molecular ion in its mass spectrum. Based on these observations a new reaction mechanism, hydroxylation associated with 1,2‐migration, is proposed for the oxidative rearrangement reaction catalyzed by the cytochrome P‐450 enzyme of Pueraria lobata.

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

“…In contrast with the previous prediction of 2R,3S configurations at C2 and C3 of the IFS product 2,7,49-trihydroxyisoflavanone (Hashim et al, 1990;Sawada et al, 2002;Akashi et al, 2003), x-ray crystallographic analysis revealed that 2,7,49-trihydroxyisoflavanone obtained from in vitro IFS reactions, HPLC purified under nonchiral resolving conditions, and then soaked into HI49OMT crystals possesses the 2S,3R stereochemical configuration ( Figure 8A). Moreover, in the HI49OMT structures solved with either pea 6a-hydroxymaackiain or pea pisatin bound, the conformations of the 6aR,11aR-stereoisomers of these pea compounds observed in the phenolic binding pocket of the barrel medic HI49OMT ( Figure 8B) are identical to the conformation of 2S,3R-2,7,49-trihydroxyisoflavanone described above.…”

“…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). The IFS-catalyzed facile suprafacial migration of the 2S aryl group was proposed to lead to a 3S aryl group configuration in the hydroxyisoflavanone product (Hashim et al, 1990;Hakamatsuka et al, 1998) (Figure 9). The 2R stereochemistry of the hydroxyl moiety at C2 was proposed based upon homology modeling of IFS with substrate docked into the active site and mechanistic comparisons with the known bacterial cytochrome P450 BM3 (Sawada et al, 2002).…”

“…Their biosynthesis diverges from the ubiquitous plant flavonoid pathway after the formation of committed flavanone intermediates, namely liquiritigenin or naringenin (Figure 1). The oxidative aryl ring migration and coupled introduction of a 2-OH moiety catalyzed by the cytochrome P450 isoflavone synthase (IFS) (Hashim et al, 1990) together with the subsequent 49-Omethylation of the IFS product, 2-hydroxyisoflavanone, comprise critical catalytic steps at the entry point into the formation of a structurally and functionally diverse group of isoflavonoid phytoalexins, including isoflavans, coumestans, and pterocarpans (Wong, 1975;Dewick, 1993;Dixon, 1999;Aoki et al, 2000).…”

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).…”

Biotransformation of Xenobiotics and Endogenous Substrates by Plant Cytochrome P450s

“…In leguminous cells, a P450 isoflavonc synthnse nlso acts on flavone to produce an isollavone via dehydration of 2-hydro:-..-yisollavonc (12,25). Severn!…”