Enzymatic reactions involved in anaerobic naphthalene degradation

非取代芳香烃的厌氧降解必须通过迄今特征不佳的活化反应进行。在我们的硫酸盐还原培养物N47的初步工作中，我们可以证明萘直接羧化为萘酸。这描述了一种在生物和化学上前所未有的新型羧化反应。在这里，我们想要表征粗细胞提取物中的萘羧酸酶反应。此外，我们还将通过稳定的同位素标记方法来表征萘的活化机理。可能的辅因子，如生物素或三磷酸腺苷，以及阳离子的需求等将进行测试。酶活性应进行纯化，以确定酶复合体中涉及的亚基。厌氧降解过程中的第二个关键反应是双环体系被萘甲酰辅酶A还原。在初步工作中，我们可以测量酶反应，现在将对其进行更详细的描述。该反应分两步进行，第一步还原为不依赖于ATP的5，6，7，8-四氢-2-萘甲酸，第二步生成未知产物。我们想要对酶反应进行表征，并鉴定第二步的产物。此外，我们想要纯化酶的活性，并阐明这两个反应步骤是由一个相同的还是由两个不同的酶复合体执行的。蛋白质亚单位将被测序，基因将在基因组上识别。

Anaerobic degradation of non-substituted aromatic hydrocarbons must proceed via so far poorly characterized activation reactions. In preliminary work with our sulphate-reducing culture N47, we could show that naphthalene is directly carboxylated to naphthoic acid. This depicts a novel carboxylation reaction which is unprecedented in biology and chemistry. Here, we want to characterize the naphthalene carboxylase reaction in crude cell extracts. Furthermore, we will characterize the mechanism of naphthalene activation by stable isotope labelling approaches. Possible cofactors such as biotin or ATP will be tested as well as the demand of cations etc. The enzyme activity shall be purified in order to identify the subunits involved in the enzyme complex. A second key reaction in anaerobic naphthalene degradation is the reduction of the bicyclic ring system by naphthoyl-CoA reductase. In preliminary work we could measure the enzyme reaction which shall now be characterized in more detail. The reaction occurs in two steps, a first reduction to 5,6,7,8-tetrahydro-2-naphthoic acid which is not ATP-dependent and a second step with an unknown product. We want to characterize the enzyme reaction and identify the product of the second step. Furthermore, we want to purify the enzyme activity and elucidate if the two reaction steps are performed by one identical or by two different enzyme complexes. The protein subunits will be sequenced and the genes will be identified on the genome.