Abstract
Anthocyanidin synthase (ANS), flavonol synthase (FLS), and flavanone 3beta-hydroxylase (FHT) are involved in the biosynthesis of flavonoids in plants and are all members of the family of 2-oxoglutarate- and ferrous iron-dependent oxygenases. ANS, FLS, and FHT are closely related by sequence and catalyze oxidation of the flavonoid "C ring"; they have been shown to have overlapping substrate and product selectivities. In the initial steps of catalysis, 2-oxoglutarate and dioxygen are thought to react at the ferrous iron center producing succinate, carbon dioxide, and a reactive ferryl intermediate, the latter of which can then affect oxidation of the flavonoid substrate. Here we describe work on ANS, FLS, and FHT utilizing several different substrates carried out in 18O2/16OH2, 16O2/18OH2, and 18O2/18OH2 atmospheres. In the 18O2/16OH2 atmosphere close to complete incorporation of a single 18O label was observed in the dihydroflavonol products (e.g. (2R,3R)-trans-dihydrokaempferol) from incubations of flavanones (e.g. (2S)naringenin) with FHT, ANS, and FLS. This and other evidence supports the intermediacy of a reactive oxidizing species, the oxygen of which does not exchange with that of water. In the case of products formed by oxidation of flavonoid substrates with a C-3 hydroxyl group (e.g. (2R,3R)-trans-dihydroquercetin), the results imply that oxygen exchange can occur at a stage subsequent to initial oxidation of the C-ring, probably via an enzyme-bound C-3 ketone/3,3-gem-diol intermediate.
Highlights
Anthocyanidin synthase (ANS), flavonol synthase (FLS), and flavanone 3-hydroxylase (FHT) are involved in the biosynthesis of flavonoids in plants and are all members of the family of 2-oxoglutarate- and ferrous iron-dependent oxygenases
In the initial steps of catalysis, 2-oxoglutarate and dioxygen are thought to react at the ferrous iron center producing succinate, carbon dioxide, and a reactive ferryl intermediate, the latter of which can affect oxidation of the flavonoid substrate
In the case of products formed by oxidation of flavonoid substrates with a C-3 hydroxyl group (e.g. (2R,3R)-trans-dihydroquercetin), the results imply that oxygen exchange can occur at a stage subsequent to initial oxidation of the C-ring, probably via an enzymebound C-3 ketone/3,3-gem-diol intermediate
Summary
61.9 idin 12, being observed (Fig. 3B) [50]. Incubations of (2R,3S,4R)-trans-LCD 13 with ANS gave cis-DHQ 14, transDHQ 8/15, quercetin 9, and cyanidin 12 (Fig. 3C) [21, 49, 50]. Both ANS and FLS have been found to react with the unnatural substrate naringenin 4 (Fig. 3, D and E) [26, 28, 29, 49] These and other results led to the proposal that ANS mediates oxidation of the natural (2R,3S,4S)-cis-LCD 6 substrate via initial oxidation at C-3 followed by loss of water from the 3,3-gem-diol 16 to give a C-2,C-3 enol (a (4S)-flav-2-en-3,4-diol 17). In vitro, this may be (partially) retained in the active site to undergo a further round of oxidation to give quercetin 9. We report work that enhances mechanistic understanding of the non-heme dioxygenases of flavonoid biosynthesis by carrying out assays of ANS, FHT, and FLS in 18O-labeled dioxygen and water with a variety of substrates
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