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Metabolism of Dichloromethylcatechols as Central Intermediates in the Degradation of Dichlorotoluenes by Ralstonia sp. Strain PS12

Katrin Pollmann,¹ Stefan Kaschabek,^2, Victor Wray,³ Walter Reineke,² and Dietmar H. Pieper^1*

Department of Environmental Microbiology,¹ Department of Structure Research, GBF-German Research Center for Biotechnology, D-38124 Braunschweig,³ Chemical Microbiology, Bergische University, Wuppertal, Germany²

Received 17 April 2002/ Accepted 27 June 2002

Ralstonia sp. strain PS12 is able to use 2,4-, 2,5-, and 3,4-dichlorotoluene as growth substrates. Dichloromethylcatechols are central intermediates that are formed by TecA tetrachlorobenzene dioxygenase-mediated activation at two adjacent unsubstituted carbon atoms followed by TecB chlorobenzene dihydrodiol dehydrogenase-catalyzed rearomatization and then are channeled into a chlorocatechol ortho cleavage pathway involving a chlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, and dienelactone hydrolase. However, completely different metabolic routes were observed for the three dichloromethylcatechols analyzed. Whereas 3,4-dichloro-6-methylcatechol is quantitatively transformed into one dienelactone (5-chloro-2-methyldienelactone) and thus is degraded via a linear pathway, 3,5-dichloro-2-methylmuconate formed from 4,6-dichloro-3-methylcatechol is subject to both 1,4- and 3,6-cycloisomerization and thus is degraded via a branched metabolic route. 3,6-Dichloro-4-methylcatechol, on the first view, is transformed predominantly into one (2-chloro-3-methyl-trans-) dienelactone. In situ ¹H nuclear magnetic resonance analysis revealed the intermediate formation of 2,5-dichloro-4-methylmuconolactone, showing that both 1,4- and 3,6-cycloisomerization occur with this muconate and indicating a degradation of the muconolactone via a reversible cycloisomerization reaction and the dienelactone-forming branch of the pathway. Diastereomeric mixtures of two dichloromethylmuconolactones were prepared chemically to proof such a hypothesis. Chloromuconate cycloisomerase transformed 3,5-dichloro-2-methylmuconolactone into a mixture of 2-chloro-5-methyl-cis- and 3-chloro-2-methyldienelactone, affording evidence for a metabolic route of 3,5-dichloro-2-methylmuconolactone via 3,5-dichloro-2-methylmuconate into 2-chloro-5-methyl-cis-dienelactone. 2,5-Dichloro-3-methylmuconolactone was transformed nearly exclusively into 2-chloro-3-methyl-trans-dienelactone.

Present address: TU Bergakademie Freiberg, Interdisziplinäres Ökologisches Zentrum, D-09599 Freiberg, Germany.

This article has been cited by other articles:

• Pollmann, K., Wray, V., Pieper, D. H. (2005). Chloromethylmuconolactones as Critical Metabolites in the Degradation of Chloromethylcatechols: Recalcitrance of 2-Chlorotoluene. J. Bacteriol. 187: 2332-2340 [Abstract] [Full Text]  
• Pollmann, K., Wray, V., Hecht, H.-J., Pieper, D. H. (2003). Rational engineering of the regioselectivity of TecA tetrachlorobenzene dioxygenase for the transformation of chlorinated toluenes. Microbiology 149: 903-913 [Abstract] [Full Text]