In the anaerobic degradation pathway of naphthalene, initial activation of this unsubstituted bicyclic aromatic hydrocarbon is achieved via a naphthalene carboxylase (Mouttaki et al., 2012). Dearomatisation is then realised through aryl-CoA reductases (Eberlein et al., 2013a) which require a prior activation of the substrate by the formation of a CoA-thioester. The 2-naphthoate:CoA ligase identified in this work (chapter 2) therefore serves as a link for the previously described metabolic steps. The substrate specificity of this enzyme was characterised in cell free extracts of the sulphate-reducing naphthalene degraders N47 and NaphS2. In addition, the enzyme from NaphS2 could also be heterologously produced in E. coli. Further studies will focus on the characterisation of the pure ligase enzymes. An antecedent study showed that the intermediate 5,6,7,8-tetrahydro-2-naphthoyl-CoA (THNCoA) is further reduced to a hexahydro-2-naphthoyl-CoA by an ATP-dependent and oxygen-sensitive reductase with NADH as electron donor (Eberlein et al., 2013a). However, only a partial conversion of the substrate could be achieved in the referred study and the conformation of the product remained unclear. This work provides evidence that 2-oxoglutarate is the natural electron donor of the THNCoA reducing system and that most likely 4,4a,5,6,7,8-hexahydro-2-naphthoyl-CoA is the product of this reduction (chapter 4). This implies a downstream pathway analogous to the benzoyl-CoA pathway described for T aromatica (Harwood et al., 1999) with cleavage of the first ring occurring between C2 and C3. Through β-oxidation-like reactions, one side-chain of the cleavage-product is then shortened by one acetyl-CoA unit resulting in cis-2-(carboxymethy)cyclohexane-1-carboxyl-CoA. The free acid of this compound was also identified as a metabolite of the naphthalene-degrading enrichment culture N47 in prior metabolomic analyses (Annweiler et al., 2002). The enzymes responsible for these metabolic steps are encoded by the thn-operon which was identified in this work (chapter 3). This operon contains genes coding for the four subunits of the THNCoA reductase as well as for putative hydratases, hydrolases, dehydrogenases and thiolases. Most of these enzymes could be heterologously produced in E. coli and in some cases the postulated enzyme function could also be verified via enzyme tests with general substrate analogues. As elucidated in chapter 5, the further downstream pathway proceeds through cis-2-(2-carboxycyclohexyl)acetyl-CoA. Like the aforementioned cis-2-(carboxymethy)-cyclohexane-1-carboxyl-CoA, this compound is also a CoA-ester derivative of cis-2-(car-boxymethy)cyclohexane-1-carboxylic acid but with the CoA-thioester attached at the other carboxyl group. The putative CoA-transferase ThnP encoded by the thn-operon might catalyse the required intramolecular transfer of the CoA-thioester to form cis-2-(2-carboxycyclohexyl)acetyl-CoA. Subsequently, the latter is converted by an acyl-CoA dehydrogenase and an enoyl-CoA hydratase yielding cis-2-(1-hydroxy-2-carboxy-cyclohexyl)acetyl-CoA, which might be the substrate for a novel ring-opening lyase. This lyase reaction was deduced from sequence homologies of two enzymes encoded by the thn-operon (ThnQ and ThnS, presumably forming an enzyme complex ThnQS) to another proposed lyase. The lyase would yield 3-oxoazeloyl-CoA which implies a link to central metabolism via pimeloyl-CoA and glutaryl-CoA. The conversion of the latter two metabolites in cell free extracts of N47 and NaphS2 was demonstrated in this work. This is the first time that a complete metabolic route towards central metabolism was delineated for the anaerobic degradation of naphthalene. Regarding the involved enzymes, some discrepancies between the benzoyl-CoA and the 2-naphthoyl-CoA degradation pathway were observed: Although it was postulated that strict anaerobic benzoate degraders generally employ an ATP-independent benzoyl-CoA reductase (Loeffler et al., 2011), it could be shown that the reduction of the benzoyl-CoA analogue THNCoA in sulphate-reducing naphthalene degraders is catalysed by an ATP-dependant enzyme. Furthermore, no enzyme similar to the crotonase-like 6-oxo-cyclohex-1-ene-1-carboxyl-CoA hydratase/hydrolase that usually occurs in the benzoyl-CoA pathway proceeding via the cyclic diene intermediate cyclohexadienecarboxyl-CoA (Kuntze et al., 2008) is present in N47 or NaphS2. The cyclic diene metabolite β’-hydroxy-β-oxodecahydro-2-naphthoyl-CoA identified in the downstream pathway of anaerobic naphthalene degradation (see chapter 4) might be converted by the concerted action of two separate enzymes, a hydratase and hydrolase, leading to the cleavage of the first ring. In general, the phylogeny of the crotonase-like enzymes encoded by the thn-operon indicates reactions on intermediates derived from the cyclic monoene octahydro-2-naphthoyl-CoA, but the metabolites detected downstream of the THNCoA reductase product clearly prove that the β-oxidation-like sequence starts from hexahydro-2-naphthoyl-CoA. Therefore, this sequence seems to be catalysed by unusual enzymes that might be specific for anaerobic of PAH degraders. Furthermore, the putative ring-opening lyase ThnQS, which is assumed to mediate cleavage of the second ring, would represent a completely novel enzyme once its function is proven. The respective genes (thnQ and thnS) as well as the ones coding for the aforementioned unusual crotonase-like enzymes might therefore be suitable as gene markers for the detection of anaerobic bacteria capable of degrading naphthalene or other PAHs. Comparable lyase reactions might be a common principle of degradation pathways of polycyclic compounds since they offer a convenient solution for cleaving ring systems with more than one side-chain or for the linearisation of branched intermediates. Either of these types of metabolites inevitably occurs during the degradation of polycyclic compounds. Thus it will be interesting to see if enzymes similar to ThnQS can also be identified in other PAH-degrading cultures in the future. In this work, most of the metabolic steps in the downstream pathway of anaerobic naphthalene degradation starting from THNCoA were measured in cell free extracts of sulphate-reducing naphthalene degraders and furthermore the thn-operon coding for the enzymes involved in the downstream pathway was identified. The objective of further studies should be the linkage of the observed reactions in cell free extracts to the catalytic function of specific proteins. Up to now, this attempt was hampered by the fact that the conformation of the hexahydro-2-naphthoyl-CoA isomer produced by the THNCoA reductase could not be identified and the conformations of downstream metabolites were likewise unknown. Therefore, the heterologously produced enzymes could not be assayed towards their native substrates yet. The results obtained in this work revealed that the reductase product is most likely 4,4a,5,6,7,8-hexahydro-2-naphthoyl-CoA, albeit this needs to be verified by NMR-analysis. This identification enables a targeted chemical synthesis of the naturally occurring isomer. However, the appropriate activity of the 2-naphthoate:CoA ligase from N47 towards its substrate analogue 5,6,7,8-tetrahydro-2-naphthoate (see chapter 2) might enable a more convenient enzymatic synthesis via a coupled system consisting of a CoA-ligase reaction on 5,6,7,8-tetrahydro-2-naphthoate and a subsequent THNCoA reductase reaction in cell free extracts. When running the reductase assay with 2-oxo-glutarate as electron donor, a complete conversion of THNCoA can be achieved and downstream processes can by inhibited by amending the assay mixture with an excess of NADH (see chapter 4). This strategy has the advantage that it should yield only the naturally occurring isomer of hexahydro-2-naphthoyl-CoA. Starting from this hexahydro-2-naphthoyl-CoA isomer, a stepwise conversion through candidate enzymes encoded by the thn-operon should be possible, whereas the product of one conversion would serve as substrate for the subsequent enzyme. Finally, these coupled enzymatic conversions should result in a reproduction of the metabolic steps observed in assays with cell free extracts and to an assignment of a metabolic function to all enzymes encoded by the thn-operon.