Dehalogenation of halogenated benzoic acids in denitrifying bacteria

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Halogenated aromatics represent a widely distributed class of environmentally relevant compounds, which can be biologically degraded under both aerobic and anaerobic conditions. The anaerobic degradation has usually been associated with membrane-bound reductive dehalogenases, corrinoid-containing key enzymes of anaerobic organohalide respiration. In contrast, the complete oxidative degradation of halobenzoates in anaerobes has been reported in denitrifying, sulfate-reducing, Fe(III)-respiring and in anaerobic phototrophic bacteria. Although initial evidence has been obtained that in Rhodopseudomonas palustris the 3-Cl-benzoate degradation pathway proceeds via CoA-esters, including a reductive dehalogenation step, the dehalogenation reaction has thus far not been demonstrated in vitro. Reductive removal of the halogen atom from halobenzoyl-CoAs may be accomplished by a novel class of dehalogenases or by dearomatizing benzoyl-CoA reductases. In this project we will study the degradation of various halobenzoates in the denitrifying model organisms Thauera chlorobenzoica and Azoarcus evansii. The major objectives are: (i) determination of the substrate/product of the dehalogenation reactions by CoA-ester metabolome analysis and by the in vitro assays; (ii) characterization of the enzymatic dehalogenation process (kinetics, mechanism, stereochemical course, etc.); (iii) isolation/characterization of key enzymes involved in halobenzoate catabolism; and (iv) the determination of the minimal enzymatic prerequisites for growth on halobenzoates.