Dehalogenation of halogenated benzoic acids in denitrifying bacteria

卤化芳烃是一类广泛分布的环境相关化合物，可在好氧和厌氧条件下进行生物降解。厌氧降解通常与膜结合的还原脱卤酶有关，这是厌氧有机卤化物呼吸的关键酶。相反，在反硝化、硫酸盐还原、铁（III）呼吸和厌氧光养细菌中，厌氧菌中卤苯甲酸盐的完全氧化降解已被报道。虽然初步证据表明，在古红假单胞菌中，3- cl -苯甲酸酯的降解途径是通过辅酶a酯进行的，包括还原性脱卤步骤，但迄今为止，脱卤反应尚未在体外得到证实。从卤苯甲酰辅酶a中还原去除卤素原子可以通过一类新的脱卤酶或通过脱芳化苯甲酰辅酶a还原酶来完成。在本项目中，我们将研究各种卤苯甲酸盐在反硝化模式生物绿生藻和埃文偶氮藻中的降解。主要目的是：(i)通过辅酶a酯代谢组分析和体外测定来确定脱卤反应的底物/产物；（ii）酶解脱卤过程的表征（动力学、机理、立体化学过程等）；（iii）分离/鉴定参与卤苯甲酸盐分解代谢的关键酶；（iv）测定在卤苯甲酸盐上生长的最低酶促先决条件。

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.