Enzymatic reactions involved in anaerobic naphthalene degradation

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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.