Polycyclic aromatic hydrocarbons (PAHs) threaten human lives since they are carcinogenic. They are widely distributed in the environment and oil. However, oxygen is rapidly depleted in water-saturated sediments containing PAHs, making anaerobic microorganisms responsible for biodegradation. So far, only the anaerobic degradation pathway of naphthalene as a model compound has been studied in more detail but the anaerobic degradation of larger PAHs such as phenanthrene is still unknown.
The anaerobic degradation of phenanthrene starts with the activation of the compound via carboxylation yielding 2-phenanthroic acid. An aryl-CoA ligase was confirmed to be responsible of the subsequent conversion of the produced 2-phenanthroic acid to 2-phenanthroyl-CoA.
Here, we studied putative following reduction reactions targeting the aromatic rings of phenanthrene to overcome the high resonance energy. We elucidated the function of four newly identified type III aryl-CoA reductases from the TRIP1 enrichment culture involved in anaerobic phenanthrene degradation. The corresponding genes were heterologously expressed in Escherichia coli, the enzymes purified, and their catalytic activity toward 2-phenanthroyl-CoA was confirmed by Liquid Chromatography–Mass Spectrometry and UV-vis spectroscopy. The oxygen-sensitive, ATP-independent enzymes reduced 2-phenanthroyl-CoA to decahydro-2-phenanthroyl-CoA with dithionite-reduced methyl viologen as electron donor. The reactions occurred in consecutive two-electron reduction steps each catalyzed by one enzyme. The four enzymes together could also reduce the last benzyl-ring of the former phenantroyl-CoA, which is thermodynamically very challenging. The four enzymes belong to the old yellow enzyme family, which contain the flavin cofactors flavin mononucleotide and flavin adenine dinucleotide. Iron content analysis and structural homology modeling confirmed that the four enzymes contain an iron-sulfur cluster which mediates electron transfer. In conclusion, we demonstrated that the ATP-independent NADH-Flavin oxidoreductases are responsible for phenanthrene ring reduction and belong to the recently discovered type III aryl-CoA reductases.