{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T03:22:25Z","timestamp":1785813745259,"version":"3.56.0"},"reference-count":55,"publisher":"American Society for Microbiology","issue":"4","license":[{"start":{"date-parts":[[2026,4,22]],"date-time":"2026-04-22T00:00:00Z","timestamp":1776816000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,4,22]],"date-time":"2026-04-22T00:00:00Z","timestamp":1776816000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100005156","name":"Alexander von Humboldt-Stiftung","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100005156","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"published-print":{"date-parts":[[2026,4,22]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Polycyclic aromatic hydrocarbons (PAHs) are widespread pollutants and especially difficult to degrade under anoxic conditions. The non-substituted three-ring PAH phenanthrene is activated through carboxylation to 2-phenanthroic acid which is then ligated to Co-enzyme A to produce 2-phenanthroyl-CoA, followed by step-wise reduction of the aromatic rings. The first reduction reaction is catalyzed by 2-phenanthroyl-CoA reductase producing dihydro-2-phenanthroyl-CoA. In this study, we elucidated the second reductase enzyme in the pathway, catalyzing the reduction of dihydro-2-phenanthroyl-CoA to hexahydro-2-phenanthroyl-CoA. Dihydro-2-phenanthroyl-CoA reductase (AprC) was heterologously overproduced in\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      and purified and characterized. AprC has a specific activity of 15.8 \u00b1 0.3 nmol min\n                      <jats:sup>\u22121<\/jats:sup>\n                      mg\n                      <jats:sup>\u22121<\/jats:sup>\n                      and an apparent\n                      <jats:italic toggle=\"yes\">K<\/jats:italic>\n                      <jats:sub>\n                        <jats:italic toggle=\"yes\">m<\/jats:italic>\n                      <\/jats:sub>\n                      value of 59.9 \u00b1 3.8 nM at pH 7.5. Dithionite-reduced methyl viologen is the preferred\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      electron donor for the four electron-reduction reaction, but it also functions with NADH but not with NADPH. The intermediate appearance of tetrahydro-2-phenanthroyl-CoA during the reduction to hexahydro-2-phenanthroyl-CoA indicates that AprC catalyzes two consecutive two-electron reduction steps. AprC is a monomeric enzyme with a molecular mass of \u224873 kDa and contains one FMN, one FAD, and one 4Fe-4S cluster, indicating that the reductase belongs to the type III aryl-CoA reductases of the old-yellow enzyme family. The high amino acid sequence similarity between dihydro-2-phenanthroyl-CoA reductase and 2-naphthoyl-CoA reductase, as well as the similar cofactor coordination in both enzymes, suggested that dihydro-2-phenanthroyl-CoA reductase follows the same biochemical principle as naphthalene ring reduction by 2-naphthoyl-CoA reductase. Quantum chemical calculation indicated that 5,6,7,8,9,10-hexahydro-2-phenanthroyl-CoA (isomer\n                      <jats:bold>6a<\/jats:bold>\n                      ) is the most stable product of AprC out of eight possible isomers, although the real isomer is not known. The reductase also performed a two-electron reduction of chemically synthesized 9,10-dihydro-2-phenanthroyl-CoA to tetrahydro-2-phenanthroyl-CoA but at much lower reaction rates indicating that the 9,10-dihydro-2-phenanthroyl-CoA isomer is not the natural substrate of the enzyme and, thus, also not the real product of 2-phenanthroyl-CoA reduction by 2-phenanthroyl-CoA reductase AprB. Since 9,10-dihydro-2-phenanthroyl-CoA has two non-conjugated benzene rings, this also indicates that, surprisingly, type III aryl-CoA reductases can reduce benzene ring structures without additional ATP requirement.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>Polycyclic aromatic hydrocarbons (PAHs) are highly toxic, persistent pollutants found in the environment. The anaerobic degradation of larger three-ring PAHs like phenanthrene is still poorly understood. Here, we show that after activation to a CoA-ester, the resonance energy of the aromatic ring system of phenanthrene is overcome by consecutive two-electron reduction steps catalyzed by ATP-independent type III aryl-CoA reductases belonging to the old-yellow enzyme family. This finding contributes to our understanding of the anaerobic degradation of PAHs with three or more rings.<\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/aem.02248-25","type":"journal-article","created":{"date-parts":[[2026,3,13]],"date-time":"2026-03-13T13:00:16Z","timestamp":1773406816000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["The ATP-independent dihydro-2-phenanthroyl-CoA reductase AprC catalyzes two consecutive two-electron reduction steps of dihydro-2-phenanthroyl-CoA to hexahydro-2-phenanthroyl-CoA in anaerobic phenanthrene degradation"],"prefix":"10.1128","volume":"92","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6575-2965","authenticated-orcid":true,"given":"Nadia A.","family":"Samak","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04mz5ra38","id-type":"ROR","asserted-by":"publisher"}],"name":"Environmental 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