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Appl. Environ. Microbiol., Jul 1997, 2560-2565, Vol 63, No. 7
Copyright © 1997, American Society for Microbiology

Structure of a Laccase-Mediated Product of Coupling of 2,4-Diamino-6-Nitrotoluene to Guaiacol, a Model for Coupling of 2,4,6-Trinitrotoluene Metabolites to a Humic Organic Soil Matrix

G Dawel, M Kastner, J Michels, W Poppitz, W Gunther and W Fritsche
Institute of Technical Microbiology, Institute of Inorganic and Analytic Chemistry, and Institute of Organic and Macromolecular Chemistry, Friedrich-Schiller-University, Jena, Germany

This work presents laccase-mediated model reactions for coupling of reduced 2,4,6-trinitrotoluene (TNT) metabolites to an organic soil matrix. The structure of an isolated coupling product of 2,4-diamino-6-nitrotoluene (2,4-DANT) to guaiacol as humic constituent was determined. Among several structures, the compound was identified conclusively to be the trinuclear coupling product 5-(2-amino-3-methyl-4-nitroanilino)-3,3(prm1)-dimethoxy-4,4(prm1)-diphenoqu inone. The compound has a weight of 409 g mol(sup-1) and may serve as a model reaction for the biogenic formation of bound residues in soil from TNT by coupling aminotoluenes (reduced TNT metabolites) to humic constituents. A linear correlation of the substrate consumption to the enzyme activity was detected. Based on this observation, the described reaction of 2,4-DANT coupling to guaiacol may be used for determination of laccase activity since the reaction was not inhibited by other compounds of culture supernatants. We propose a two-step mechanism for the coupling reaction because 2,4-DANT was not transformed by laccases in the absence of guaiacol and guaiacol oxidation was independent of the presence of 2,4-DANT. The first reaction step is a laccase-mediated dimerization of two guaiacol monomers with subsequent oxidation to a diphenoquinone. The second step is the nucleophilic addition of 2,4-DANT to the ortho position of the carbonyl group of the diphenoquinone structure.

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• Thiele, S., Fernandes, E., Bollag, J.-M. (2002). Enzymatic Transformation and Binding of Labeled 2,4,6-Trinitrotoluene to Humic Substances during an Anaerobic/Aerobic Incubation. J. Environ. Qual. 31: 437-444 [Abstract] [Full Text]  
• Esteve-Nunez, A., Caballero, A., Ramos, J. L. (2001). Biological Degradation of 2,4,6-Trinitrotoluene. Microbiol. Mol. Biol. Rev. 65: 335-352 [Abstract] [Full Text]