Enzymatic reactions involved in anaerobic naphthalene degradation

非取代芳烃的厌氧降解必须通过迄今为止表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基化为萘甲酸。这描述了一种新的羧化反应，这在生物学和化学中是前所未有的。在这里，我们要在粗细胞提取物中的萘羧化酶反应的特点。此外，我们将通过稳定同位素标记方法表征萘活化的机制。将测试可能的辅因子，如生物素或ATP，以及阳离子的需求等。应纯化酶活性，以鉴定酶复合物中涉及的亚基。厌氧萘降解中的第二个关键反应是萘酰辅酶A还原酶对双环系统的还原。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表征。该反应分两步进行，第一步还原为不依赖ATP的5，6，7，8-四氢-2-萘甲酸，第二步为未知产物。我们想要表征酶反应并鉴定第二步的产物。此外，我们希望纯化酶活性并阐明这两个反应步骤是由一种相同的酶复合物还是由两种不同的酶复合物进行的。蛋白质亚基将被测序，基因将在基因组上被识别。

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.