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3-Acetyldeoxynivalenol is the major trichothecene produced by the fungus Fusarium culmorum. The first proven tricyclic intermediate in the biosynthesis of 3-acetyldeoxynivalenol has been shown by in vivo studies to be isotrichodermin, a natural metabolite of F. culmorum. Indeed, the feeding of ring-deuterated isotrichodermin resulted in ring-deuterated 3-acetyldeoxynivalenol as shown by NMR studies.In this work, we have shown that the 3-acetyl group of isotrichodermin is mostly lost in its metabolism to 3-acetyldeoxynivalenol. We have shown by two different approaches that the deacetylation occurs at an early step after the first oxygenation step at C-15. Derivatives of isotrichodermin lacking the 3-acetyl such as 3-deacetyl isotrichodermin or 3-oxo-12,13-epoxytrichothec-9-ene are not precursors to 3-acetyldeoxynivalenol. The role of this acetyl exchange mechanism is not clear presently.Fusarium culmorum has been shown to produce two main secondary metabolites ( Fig. 1): 3-acetyldeoxynivalenol (3-ADN) 1 and sambucinol (SOL) (1, 2). The biosynthesis of these two trichothecenes has been shown to differ at the tricyclic stage (3). Isotrichodermin (ITD), a natural metabolite (4), has been shown to be the major biosynthetic precursor of 3-ADN, whereas 12,13-epoxytrichothec-9-ene (EPT) is converted only to SOL (Fig. 1). There seems to be no metabolic interconversion between EPT and ITD (3). In addition, many of the oxidative metabolic products of isotrichodermin were elucidated (5). Preliminary data on cell-free experiments have been reported (6 -8).We have recently investigated the fate of the 3-acetyl group in isotrichodermin conversion to 3-acetyldeoxynivalenol. It has always been assumed without any proof that the 3-acetyl in 3-acetyldeoxynivalenol originates from the 3-acetyl in isotrichodermin. This was based on the seemingly textbook definition of a biosynthetic precursor: it was produced by F. culmorum cultures (4), it was 27% incorporated into 3-acetyldeoxynivalenol, and the incorporation site was rigorously determined by 2 H NMR of ring-deuterated isotrichodermin feedings (3). In order to ensure that we could use the easily synthesized [1Ј-14 C]acetyl isotrichodermin as a marker to isolate enzymes, we decided to confirm that indeed the 3-acetyl in isotrichodermin is retained in its conversion to 3-acetyldeoxynivalenol. We obtained surprising results, which will be discussed here. EXPERIMENTAL PROCEDURESInstrumentation-High performance liquid chromatography (HPLC) was performed on a Perkin-Elmer series 3B instrument coupled to a LC-75 variable wavelength detector (Perkin-Elmer) set at 204 nm and a Berthold LB 505 HPLC radioactivity monitor (Labserco, Oakville, Ontario). Thin layer chromatography separations using LHP-KF thin layer chromatography plates (Whatman) were analyzed with a Bioscan Imaging Scanner System 200 (Bioscan, Inc., Washington, D.C.). A Tracor Analytic Delta 300 instrument was used for liquid scintillation counting. Homogenization of F. culmorum cells was accomplished with a Bead-Beater (Bios...

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3-Acetyldeoxynivalenol is the major trichothecene produced by the fungus Fusarium culmorum. Studies in vivo with F. culmorum have established the following biosynthetic precursors of 3-acetyldeoxynivalenol: isotrichool → isotrichodiol → isotrichodermin → 15-deacetylcalonectrin, 7α-hydroxyisotrichodermin, 8α-hydroxyisotrichodermin → calonectrin → deoxynivalenol. In this paper, we describe in vitro investigations of one of these metabolic steps. The cell-free system of F. culmorum that converts isotrichodermin into 15-deacetylcalonectrin, 7α-hydroxyisotrichodermin, and 8α-hydroxyisotrichodermin is described here. This preparation requires NADPH but not NADH for activity and is not inhibited by carbon monoxide, cyanide, or known oxygenase inhibitors, such as SKF-525-A or ancymidol.Key words: trichothecene, Fusarium culmorum, cell-free system, isotrichodermin, 15-deacetylcalonectrin.

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