Enzymatic detoxification of eutypine, a toxin from Eutypa lata, by Vitis vinifera cells: partial purification of an NADPH-dependent aldehyde reductase

Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzaldehyde, is a toxin produced by Eutypa lata (Pers.: Fr.) Tul., the causal agent of dying arm disease of Vitis vinifera L. (grapevine). Previously, we have shown that eutypine is involved in the development of disease symptoms. In the present study, the effects of V. vinifera cell-suspension cultures on the biological activity of the toxin were investigated. Eutypine was converted by grapevine tissues into a single compound, identified by mass spectrometry and nuclear magnetic resonance as 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzyl alcohol, designated eutypinol. This compound was found to be non-toxic for grapevine tissues. Unlike eutypine, eutypinol failed to affect the oxidation rate or membrane potential of isolated mitochondria. In grapevine cells, reduction of eutypine into the corresponding alcohol is an NADPH-dependent enzymatic reaction. An enzyme which reduced eutypine was partially purified, over 1000-fold, using a five-step purification procedure. By gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein was found to have a molecular mass of 54–56 kDa. The enzyme exhibited an apparent K _m for eutypine of 44 μM, and was active between pH 6.8 and 7.5 with a maximum at pH 7.0. The eutypine reductase activity was improved by Mn²⁺ and Mg²⁺ and inhibited by disulfiram and p-hydroxymercuribenzoate. The possible role of the eutypine-detoxification mechanism in the defense reactions of V. vinifera cells is discussed. Received: 20 April 1998 / Accepted: 22 September 1998