Biochemistry of respiration in Dehalococcoides strain CBDB1

菌株CBDB 1将在几种不同的电子受体上生长。天然凝胶电泳方法结合质谱法将被应用于确定还原脱卤酶的生物体。将特别着重于区分用类似底物合成的酶组，如1，2，3-三氯苯与1，2，4-三氯苯或2，3，4-三氯联苯与2，4，5-三氯联苯。天然凝胶电泳（可能在用交联剂处理后结合消化肽的质谱法）将用于分析与呼吸链其他蛋白质的相互作用。生物化学试验（紫精活性试验、抑制研究）将更详细地表征已鉴定的脱卤酶的辅因子、底物光谱、膜取向以及B蛋白的结构和作用。在S。对于多噬菌体，B蛋白是一种小的跨膜蛋白，其总是在还原性脱卤酶操纵子中编码，并且被认为是膜锚。与子项目4合作，将研究菌株CBDB 1中的氢化酶和注释的甲酸脱氢酶，我们将尝试识别在氢化酶和还原性脱卤酶之间穿梭电子的膜结合电子介质。对于膜结合的氧化还原介质的识别，我们将使用GC-MS，电化学反应和预测推导出目前的比较基因组注释。

Strain CBDB1 will be grown on several different electron acceptors. A native gel electrophoresis approach in combination with mass spectrometry will be applied to identify reductive dehalogenases in the organism. Special emphasis will be focused on differentiation of the enzyme sets synthesized with comparable substrates such as 1,2,3- vs. 1,2,4-trichlorobenzene or 2,3,4-trichlorobiphenyl vs. 2,4,5-trichlorobiphenyl. Native gel electrophoresis, possibly after treatment with crosslinking agents in combination with mass spectrometry of digestion peptides, will be used to analyze interactions with other proteins of the respiratory chain. Biochemical tests (activity test with viologens, inhibition studies) will characterize in more detail identified dehalogenases with respect to cofactors, substrate spectra, orientation in the membrane and the structure and role of the B-protein. As in S. multivorans the B-protein is a small transmembrane protein that is always encoded in reductive dehalogenase operons and is thought to be a membrane anchor. In cooperation with subproject 4, which will study hydrogenases and the annotated formate dehydrogenase in strain CBDB1, we will try to identify membrane-bound electron mediators that shuttle electrons between hydrogenase(s) and the reductive dehalogenase(s). For the identification of membrane-bound redox mediators we will use GC-MS, electrochemical reactions and predictions deduced from current comparative genome annotations.