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<p>The anaerobic degradation pathway of phenanthrene starts with activation through carboxylation to 2-phenanthroic acid, followed by thioesterification to produce 2-phenanthroyl-CoA, which is then reduced to hexahydro-2-phenanthroyl-CoA by two ATP-independent type III aryl-CoA reductases (AprB and AprC) to overcome the resonance energy of the aromatic rings. In this study, we elucidated the reduction of hexahydro-2-phenanthroyl-CoA to octahydro-2-phenanthroyl-CoA and its subsequent reduction to the fully dearomatized diene decahydro-2-phenanthroyl-CoA. The two-electron reduction steps were catalyzed by the heterologously produced and purified hexahydro-2-phenanthroyl-CoA reductase (AprD) and octahydro-2-phenanthroyl-CoA reductase (AprE). AprD and AprE have specific activities of 12.7 and 6.3 nmol min<sup>-1</sup> mg<sup>-1</sup> and <i>K</i><sub>M</sub> values of 15.1 and 63.9 nM, respectively. The ATP-independent and oxygen-sensitive reduction of hexahydro-2-phenanthroyl-CoA and octahydro-2-phenanthroyl-CoA catalyzed by AprD and AprE preferred dithionite-reduced methyl viologen as in vitro electron donor. AprD and AprE are monomeric enzymes with a molecular mass of ≈ 73 kDa and contain one FMN, one FAD, and one 4Fe-4S cluster, indicating that both reductases belong to the new type III aryl-CoA reductases of the old-yellow enzyme (OYE) family. The de-aromatization of 2-phenanthroyl-CoA aromatic rings through reduction was followed by water addition at the β-position of the cyclic diene, decahydro-2-phenanthroyl-CoA, producing β-hydroxydodecahydro-2-phenanthroyl-CoA. The hydration reaction was catalyzed by a homo-dimeric enoyl-CoA hydratase (ApcA) with a native molecular mass of ≈ 60 kDa and subunit molecular mass of ≈ 29 kDa. ApcA has a specific activity of 3.2 nmol min<sup>-1</sup> mg<sup>-1</sup> and a <i>K</i><sub>M</sub> value of 31.5 nM at pH value of 7.5. The catalytic activity of ApcA was oxygen-insensitive and the reaction did not need any cofactor or metal ion. These findings reveal a novel strategy in the anaerobic degradation of aromatic hydrocarbons where only ATP-independent type III aryl-CoA reductases are involved in breaking the aromaticity of the ring system before the metabolites enter subsequent beta-oxidation reactions.</p>

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Breaking aromaticity in the anaerobic degradation pathway of phenanthrene comprises only ATP-independent type III aryl-CoA reductases

  • 期刊论文

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  • 发表日期：

    2026年7月13日

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  • Nadia A. Samak,
  • Marvin Häßler,
  • Oliver J. Schmitz,
  • Khadija Adjir,
  • Rainer U. Meckenstock

摘要

The anaerobic degradation pathway of phenanthrene starts with activation through carboxylation to 2-phenanthroic acid, followed by thioesterification to produce 2-phenanthroyl-CoA, which is then reduced to hexahydro-2-phenanthroyl-CoA by two ATP-independent type III aryl-CoA reductases (AprB and AprC) to overcome the resonance energy of the aromatic rings. In this study, we elucidated the reduction of hexahydro-2-phenanthroyl-CoA to octahydro-2-phenanthroyl-CoA and its subsequent reduction to the fully dearomatized diene decahydro-2-phenanthroyl-CoA. The two-electron reduction steps were catalyzed by the heterologously produced and purified hexahydro-2-phenanthroyl-CoA reductase (AprD) and octahydro-2-phenanthroyl-CoA reductase (AprE). AprD and AprE have specific activities of 12.7 and 6.3 nmol min-1 mg-1 and KM values of 15.1 and 63.9 nM, respectively. The ATP-independent and oxygen-sensitive reduction of hexahydro-2-phenanthroyl-CoA and octahydro-2-phenanthroyl-CoA catalyzed by AprD and AprE preferred dithionite-reduced methyl viologen as in vitro electron donor. AprD and AprE are monomeric enzymes with a molecular mass of ≈ 73 kDa and contain one FMN, one FAD, and one 4Fe-4S cluster, indicating that both reductases belong to the new type III aryl-CoA reductases of the old-yellow enzyme (OYE) family. The de-aromatization of 2-phenanthroyl-CoA aromatic rings through reduction was followed by water addition at the β-position of the cyclic diene, decahydro-2-phenanthroyl-CoA, producing β-hydroxydodecahydro-2-phenanthroyl-CoA. The hydration reaction was catalyzed by a homo-dimeric enoyl-CoA hydratase (ApcA) with a native molecular mass of ≈ 60 kDa and subunit molecular mass of ≈ 29 kDa. ApcA has a specific activity of 3.2 nmol min-1 mg-1 and a KM value of 31.5 nM at pH value of 7.5. The catalytic activity of ApcA was oxygen-insensitive and the reaction did not need any cofactor or metal ion. These findings reveal a novel strategy in the anaerobic degradation of aromatic hydrocarbons where only ATP-independent type III aryl-CoA reductases are involved in breaking the aromaticity of the ring system before the metabolites enter subsequent beta-oxidation reactions.