Identification and characterization of the hydratase ThnL preparing for the second-ring cleavage in anaerobic naphthalene degradation.

Anaerobic degradation of polycyclic aromatic hydrocarbons (PAHs) proceeds through CoA-activated intermediates and β-oxidation-like reactions; however, the enzymes responsible for key hydration and hydrolytic steps remain insufficiently defined. In the sulfate-reducing enrichment culture N47, the thn operon encodes multiple putative hydratases and hydrolases proposed to participate in anaerobic naphthalene degradation. Here, we cloned, heterologously expressed, and purified six candidate enzymes (ThnA, ThnH, ThnI, ThnL, ThnM, and ThnU) to elucidate their substrate specificities. Among them, ThnL showed robust activity toward 2-carboxycyclohexylideneacetyl-CoA, catalyzing rapid hydration of the enoyl-CoA double bond to form the tertiary alcohol 1-(1-hydroxy-2-carboxycyclohexyl)acetyl-CoA. ThnL exhibited kinetic parameters of Kₘ = 0.11 mM and Vₘₐₓ = 350 µmol min⁻¹ mg⁻¹. Two additional hydratases, ThnU and ThnI, converted the substrate only weakly (0.26% and 0.08% of ThnL activity), indicating promiscuous rather than physiological roles. ThnL also catalyzed the hydrolytic C1-C2 ring cleavage of 2-oxocyclohexane-1-carbonyl-CoA to pimelyl-CoA, revealing a bifunctional hydratase/hydrolase activity, although this reaction is unlikely to be physiologically relevant based on growth assays. This study shows that the ThnL-catalyzed hydration of 2-carboxycyclohexylideneacetyl-CoA to the tertiary alcohol 1-(1-hydroxy-2-carboxycyclohexyl)acetyl-CoA completes the final preparatory step before the second-ring cleavage in anaerobic naphthalene degradation.IMPORTANCEPolycyclic aromatic hydrocarbons (PAHs), such as naphthalene, are widespread environmental pollutants that persist in oxygen-limited environments, such as marine sediments and contaminated aquifers. Although microorganisms are known to degrade these compounds anaerobically, the enzymatic mechanisms underlying key steps of these pathways remain poorly understood. In particular, the enzymes responsible for hydration reactions preceding ring cleavage in anaerobic naphthalene degradation have not been experimentally verified. In this study, we characterized several candidate enzymes encoded by the thn operon from a sulfate-reducing enrichment culture and identified ThnL as the enzyme catalyzing the hydration of 2-carboxycyclohexylideneacetyl-CoA. This finding clarifies a crucial preparatory step before the second-ring cleavage during anaerobic naphthalene metabolism and advances our understanding of microbial PAH degradation in anoxic environments.