Biosynthetic origin of oxygen atoms in DIMBOA
--Glawischnig, E; Eisenreich, W; Bacher, A; Frey, M; Gierl, A
In maize, 2,4-dihydroxy-7-methoxy-1,4-benzoxazine-3-one (DIMBOA) is important for resistance against insects, fungi and bacteria. DIMBOA is synthesized in large quantities during the first days after germination and stored in the vacuole in a glucosylated form. Two reactions of the DIMBOA pathway, i. e. the C2-hydroxylation of the benzoxazinone and the N-hydroxylation of the 2-hydroxy-benzoxazinone (HBOA) have been studied in vitro (Bailey and Larson, Plant Physiol. (1991) 95:792-796; Kumar et al., Phytochemistry (1994) 36: 893-898). They both are characterized as cytochrome P450 monooxygenase catalyzed reactions. We have demonstrated that all four oxygen atoms bonded to carbon atoms of DIMBOA are introduced by monooxygenases (Glawischnig et al., Phytochemistry, in press).
Maize seeds were germinated in a gastight container filled with a mixture of 16O2 and 18O2 in a ratio of 1:2 (v/v). DIMBOA was isolated and NMR spectra were recorded in deuterated methanol. The presence of 18O2 induces an upfield isotope shift of the natural abundance 13C NMR signal of adjacent carbon atoms. The signals for carbon atoms 3, 7, 8a and the methyl group (Figure 1) show upfield satellites indicating the binding to 18O. The 13C signal for C2 appears with two upfield satellites indicating the presence of isotopomers with one and two 18O atoms bound at C2. This indicates that all four oxygen atoms bound to carbon atoms of DIMBOA acquired isotope labeling from molecular 18O. The relative intensities of the non-shifted signals (i. e. with adjacent 16O isotope) and the shifted signals (i. e. with adjacent 18O) are in the range of 1:2, thus reflecting faithfully the 16O2/18O2 composition in the growth chamber, indicating that O2 is the immediate source for the O-atoms. Therefore, it is likely that all oxygen atoms of DIMBOA are introduced via cytochrome P450 monooxygenases.
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