Enzymatic reactions involved in anaerobic naphthalene degradation

非取代芳烃的厌氧降解必须通过迄今表征不佳的活化反应进行。在我们的硫酸盐还原培养N47的初步工作中，我们可以证明萘直接羧化为萘酸。这描绘了一种新的羧基化反应，这在生物学和化学上是前所未有的。在这里，我们想表征粗细胞提取物中的萘羧化酶反应。此外，我们将通过稳定同位素标记方法表征萘活化的机制。可能的辅助因子如生物素或ATP将被测试，以及阳离子的需求等。酶活性必须纯化，以确定酶复合体中涉及的亚基。厌氧萘降解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。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-萘酸，第二步生成未知产物。我们想要表征酶的反应并鉴定第二步的产物。此外，我们想要纯化酶的活性，并阐明两个反应步骤是由一个相同的还是由两个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.