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Different types of microsomal enzymes catalyze ortho ‐ or para ‐hydroxylation in the biosynthesis of carnation phytoalexins
Author(s) -
Reinhard Klaus,
Matern Ulrich
Publication year - 1991
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(91)81345-9
Subject(s) - hydroxylation , cytochrome , microsome , monooxygenase , enzyme , biochemistry , chemistry , carnation , oxygenase , cytochrome p450 , stereochemistry , biology , botany
Cell suspension cultures of carnation ( Dianthus caryophyllus L.) accumulate, upon challenge with crude fungal elicitor, various dianthramide phytoalexins, all of which derive from N ‐benzoylanthranilate. In vitro, microsomes from the elicited carnation cells hydroxylated N ‐benzoylanthranilate in the 4‐ and/or 2′‐positions to yield the hydroxyanthranilate and/or salicyloyl derivatives. 2′‐Hydroxylation was shown to precede 4‐hydroxylation in the formation of N ‐salicyloyl‐4‐hydroxyanthranilate, and both these activities depended strictly on NADPH and molecular oxygen. 4‐Hydroxylation was shown to be catalyzed by cytochrome P ‐450‐dependent monooxygenase(s), whereas the 2′‐hydroxylating activity appeared to be due to a novel class of enzymes, also responding synergistically to NADH in combination with NADPH and showing apparent inhibition by cytochrome c but not by carbon monoxide. The difference in type of 4‐ and 2′‐hydroxylases was corroborated by the exclusive inhibition of either activity in imidazole vs. MOPS buffers as well as their differential heat sensitivities. In the course of these studies, low concentrations of N ‐salicyloylanthranilate turned out to inhibit the cytochrome P ‐450‐dependent 4‐hydroxylation more strongly than any of the commercial inhibitor chemicals tested, while neither the substrate, N ‐benzoylanthranilate, nor the final product, N ‐salicyloyl‐4‐hydroxyanthranilate, exhibited such significant inhibition. In addition, 2′‐hydroxylation activity was affected much less by N ‐benzoylanthranilate, N ‐salicyloylanthranilate or by inhibitor chemicals. The results demonstrate the requirement of two different classes of hydroxylase activities that appear to introduce the antimycotic quality to the dianthramides for phytoalexin defense.