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Phenanthrene is the only three-ring PAH where the anaerobic degradation has been partially elucidated. Phenanthrene is first activated via carboxylation producing 2-phenanthroate, which is further converted to 2-phenanthroyl-coenzyme A (CoA) via the enzyme 2-phenanthroate:CoA ligase. In this study, we elucidated the next degradation step, the reduction of 2-phenanthroyl-CoA to dihydro-2-phenanthroyl-CoA. We cloned the putative gene from the genome of culture TRIP_1 and heterologously expressed and purified the 2-phenanthroyl-CoA reductase enzyme from\n              <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n              . The identified monomeric flavo-enzyme belongs to the novel group of type III aryl-CoA reductases in the old-yellow enzyme family and has a molecular mass of 72 kDa. 2-Phenanthroyl-CoA reductase contains one FMN, one FAD, and one [4Fe-4S] iron-sulfur cluster as cofactors. The enzyme has a specific activity of 17.6 \u00b1 0.4 nmol\/min\/mg, a\n              <jats:italic toggle=\"yes\">K<\/jats:italic>\n              <jats:sub>m<\/jats:sub>\n              value of 1.8 \u00b5M, and a Vmax of 7.9 \u00b5mol\/min\/mg at pH 7.5, when reduced methyl viologen was used as electron donor. 2-Phenanthroyl-CoA reductase catalyzed a two-electron reduction step producing one of five possible isomers. Quantum mechanical calculations and nuclear magnetic resonance analysis of the reaction product suggested 9,10-dihydro-2-phenanthroyl-CoA as the most stable isomer. However, our experimental evidence suggests 7,8-dihydro-2-phenanthroyl-CoA (International Union of Pure and Applied Chemistry [IUPAC]: 1,2-dihydro-7-phenanthroyl-CoA) or 5,6-dihydro-2-phenanthroyl-CoA (IUPAC: 3,4-dihydro-7-phenanthroyl-CoA) as the most likely reduced product with a saturated bond in ring 3 of the substrate 2-phenanthroyl-CoA, before undergoing isomerization changes to reach the more stable structure of 9,10-dihydro-2-phenanthroyl-CoA.\n            <\/jats:p>\n            <jats:sec>\n              <jats:title>IMPORTANCE<\/jats:title>\n              <jats:p>PAHs are a group of highly toxic and persistent environmental pollutants. The anaerobic degradation of three-ring PAHs like phenanthrene is still poorly understood. Phenanthrene degradation starts with a carboxylation reaction to form 2-phenanthroic acid followed by a CoA-thioesterification reaction catalyzed by 2-phenanthroate:CoA ligase to produce 2-phenanthroyl-CoA. The next degradation step is the reduction of 2-phenanthroyl-CoA to dihydro-2-phenanthroyl-CoA to overcome the resonance energy of the aromatic ring system. Herein, we elucidated that the reduction reaction is catalyzed by the enzyme 2-phenanthroyl-CoA reductase. Furthermore, we provided biochemical and structural properties of the heterologously expressed and purified 2-phenanthroyl-CoA reductase, which confirmed that the enzyme belongs to the novel group of type III aryl-CoA reductases in the old-yellow enzyme family.<\/jats:p>\n            <\/jats:sec>\n          <\/jats:sec>","DOI":"10.1128\/aem.00166-25","type":"journal-article","created":{"date-parts":[[2025,4,17]],"date-time":"2025-04-17T13:01:22Z","timestamp":1744894882000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["Characterization of 2-phenanthroyl-CoA reductase, an ATP-independent type III aryl-CoA reductase involved in anaerobic phenanthrene degradation"],"prefix":"10.1128","volume":"91","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6575-2965","authenticated-orcid":true,"given":"Nadia A.","family":"Samak","sequence":"first","affiliation":[{"name":"Environmental Microbiology and Biotechnology 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