Cleavage of the Diketonitrile Derivative of the Herbicide Isoxaflutole by Extracellular Fungal Oxidases Christian Mougin,* Franc ¸ ois-Didier Boyer, Eliane Caminade, and Rachel Rama † INRA, Unite ´ de Phytopharmacie et Me ´diateurs Chimiques, Route de saint-Cyr, 78026 Versailles Cedex, France Isoxaflutole is a herbicide activated in soils and plants to its diketonitrile derivative, the active herbicide principle. The diketonitrile derivative undergoes cleavage to the inactive benzoic acid analogue. In this paper, it is established that an oxidative mechanism implicating two successive reactions in the presence of dimethyldioxirane can chemically initiate the cleavage of the diketonitrile. It is also shown that two white rot strains, Phanerochaete chrysosporium and Trametes versicolor, are able to convert the diketonitrile to the acid when cultured in liquid media. This main metabolite amounts to 24.6 and 15.1% of initial herbicide content after 12-15 days of culture. Another polar metabolite represents <3.7% of the parent compound amount during the same period. Oxidative enzymes produced by the fungi show a time course similar to that of diketonitrile degradation. Purified laccase (EC 1.10.3.2), in the presence of 2 mM 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) acting as a redox mediator at pH 3 supports the reaction with rates of 0.3-0.4 nmol h -1 unit -1 . Keywords: Herbicide; metabolism; dimethyldioxirane; white rot fungi; Phanerochaete chrysospo- rium; Trametes versicolor; laccases INTRODUCTION Isoxaflutole (IUPAC name: 5-cyclopropyl-1,2-oxazol- 4-yl R,R,R-trifluoro-2-mesyl-p-tolyl ketone, compound 1; Figure 1) is a recently developed herbicide for pre- and postemergence control of a wide range of important broadleaf and grass weeds in corn and sugarcane (Luscombe et al., 1995). After herbicide application, susceptible weed species show a bleaching symptomol- ogy of newly developed leaves, followed by growth suppression and necrosis prior to plant death, similar to that seen with herbicidal inhibitors of carotenoid biosynthesis. Furthermore, reduction of carotenoid and chlorophyll content is associated with an indirect inhibi- tion of the phytoene desaturase due to the depletion of the cofactor plastoquinone. That depletion is caused by the inhibition of the enzyme 4-hydroxy-phenylpyruvate dioxygenase (Pallett et al., 1998; Viviani et al., 1998). The dioxygenase catalyzes the oxidative decarboxylation of 4-hydroxyphenylpyruvate, forming homogentisate. In plants and soils, isoxaflutole is rapidly converted to a diketonitrile derivative (DKN, compound 2; Figure 1) by opening of the isoxazole ring (Viviani et al., 1998). DKN is the active herbicide principle and is a potent inhibitor of the dioxygenase (Pallett et al., 1998). DKN undergoes degradation to the inactive benzoic acid analogue (BZA, compound 3; Figure 1) in treated plants. The extent of this degradation is a basis for herbicidal selectivity, being more rapid in tolerant plants than in the susceptible species. Because of their implication in herbicide selectivity (agronomic impact) and breakdown (environmental impact), it is of great importance to identify enzymatic systems involved in the conversion of DKN to BZA. Enzymes responsible for the cleavage of diketone bonds have been poorly characterized to date. Sakai et al. (1986) reported a bacterial -diketone hydrolase (EC 3.7.1.7) involved in the degradation mechanism of poly- (vinyl alcohol). The enzyme was also active on aliphatic -diketones and on aromatic -diketones such as 1-phen- yl-1,3-butanedione, presenting a framework close to this of DKN. Nevertheless, such a hydrolytic mechanism producing a methyl ketone is not relevant to the formation of BZA. White rot fungi have been known for many years for their ability to transform various xenobiotics by using their rich enzymatic equipment (Barr and Aust, 1994). These organisms commonly live woody plants, but they can also found in soils. For that reason, their ability to cleave the diketone bond of the herbicide was investi- gated. We propose in this paper a hypothetical pathway for the oxidative transformation of DKN. Then we report herbicide transformation by two strains of white rot fungi cultured in liquid media and by purified oxidases. MATERIALS AND METHODS Chemicals and Reagents. The unlabeled DKN derivative of isoxaflutole, [ring-UL- 14 C]DKN (909 MBq/mmol, radio- chemical purity ) 96%), and standard of BZA were gifts from Rho ˆne-Poulenc Agro (Lyon, France). NAT 89 was a commercial phospholipid source supplied by Natterman Phospholipid GmbH (Cologne, Germany). All other chemicals and reagents were obtained from Sigma-Aldrich (St-Quentin Fallavier, France), and solvents came from Carlo Erba (Val de Reuil, France). Synthesis of Putative Transformation Products of DKN. NMR data ( 1 H, 300 MHz; 13 C, 75.5 MHz) were recorded on a Varian Gemini 300 instrument (Les Ulis, France). All NMR spectra were recorded in deuteriochloroform (CDCl3). Chemical shifts are reported in δ (parts per million) relative to CHCl3 (CDCl3) as internal reference: 7.27 ppm for 1 H (77.14 * Corresponding author (telephone +33-1-30-83-31-02; fax +33-1-30-83-31-19; e-mail mougin@versailles.inra.fr). † Present address: Aventis CropScience, 14-20 Rue P. Baizet, B.P. 9163, 69263 Lyon Cedex 09, France. 4529 J. Agric. Food Chem. 2000, 48, 4529-4534 10.1021/jf000397q CCC: $19.00 © 2000 American Chemical Society Published on Web 09/07/2000