The 5,6,7,8-Tetrahydro-2-Naphthoyl-Coenzyme A Reductase Reaction in the Anaerobic Degradation of Naphthalene and Identification of Downstream Metabolites

Abstract

Anaerobic degradation of polycyclic aromatic hydrocarbons has been investigated mostly with naphthalene as a model compound. Naphthalene degradation by sulfate-reducing bacteria proceeds via carboxylation to 2-naphthoic acid, formation of a coenzyme A thioester, and subsequent reduction to 5,6,7,8-tetrahydro-2-naphthoyl-coenzyme A (THNCoA), which is further reduced to hexahydro-2-naphthoyl-CoA (HHNCoA) by tetrahydronaphthoyl-CoA reductase (THNCoA reductase), an enzyme similar to class I benzoyl-CoA reductases. When analyzing THNCoA reductase assays with crude cell extracts and NADH as electron donor via liquid chromatography-mass spectrometry (LC-MS), scanning for putative metabolites, we found that small amounts of the product of an HHNCoA hydratase were formed in the assays, but the downstream conversion by an NAD⁺-dependent β-hydroxyacyl-CoA dehydrogenase was prevented by the excess of NADH in those assays. Experiments with alternative electron donors indicated that 2-oxoglutarate can serve as an indirect electron donor for the THNCoA-reducing system via a 2-oxoglutarate:ferredoxin oxidoreductase. With 2-oxoglutarate as electron donor, THNCoA was completely converted and further metabolites resulting from subsequent β-oxidation-like reactions and hydrolytic ring cleavage were detected. These metabolites indicate a downstream pathway with water addition to HHNCoA and ring fission via a hydrolase acting on a β'-hydroxy-β-oxo-decahydro-2-naphthoyl-CoA intermediate. Formation of the downstream intermediate cis-2-carboxycyclohexylacetyl-CoA, which is the substrate for the previously described lower degradation pathway leading to the central metabolism, completes the anaerobic degradation pathway of naphthalene.IMPORTANCE Anaerobic degradation of polycyclic aromatic hydrocarbons is poorly investigated despite its significance in anoxic sediments. Using alternative electron donors for the 5,6,7,8-tetrahydro-2-naphthoyl-CoA reductase reaction, we observed intermediary metabolites of anaerobic naphthalene degradation via in vitro enzyme assays with cell extracts of anaerobic naphthalene degraders. The identified metabolites provide evidence that ring reduction terminates at the stage of hexahydro-2-naphthoyl-CoA and a sequence of β-oxidation-like degradation reactions starts with a hydratase acting on this intermediate. The final product of this reaction sequence was identified as cis-2-carboxycyclohexylacetyl-CoA, a compound for which a further downstream degradation pathway has recently been published (P. Weyrauch, A. V. Zaytsev, S. Stephan, L. Kocks, et al., Environ Microbiol 19:2819-2830, 2017, https://doi.org/10.1111/1462-2920.13806). Our study reveals the first ring-cleaving reaction in the anaerobic naphthalene degradation pathway. It closes the gap between the reduction of the first ring of 2-naphthoyl-CoA by 2-napthoyl-CoA reductase and the lower degradation pathway starting from cis-2-carboxycyclohexylacetyl-CoA, where the second ring cleavage takes place.

Keywords: THNCoA reductase; anaerobic catabolic pathways; naphthalene; polycyclic aromatic hydrocarbons.

FIG 1

First steps of the anaerobic naphthalene degradation pathway. Naphthalene (1) is initially carboxylated to 2-naphthoate (2) followed by the formation of 2-naphthoyl-CoA (3). The latter is stepwise reduced to 5,6-dihydro-2-naphthoyl-CoA (4) and 5,6,7,8-tetrahydro-2-naphthoyl-CoA (5) via ATP-independent and oxygen-insensitive class III aryl-CoA reductases. The subsequent reduction of 5,6,7,8-tetrahydro-2-naphthoyl-CoA via an ATP-dependent and oxygen-sensitive class II aryl-CoA reductase yields a hexahydro-2-naphthoyl-CoA (6) with the diene moiety being one of the possibilities shown.

FIG 2

LC-MS chromatograms of reductase assays with cell extracts of culture N47 and 5,6,7,8-tetrahydro-2-naphthoyl-CoA (THNCoA) as the substrate. Standard assays contained 50 μM THNCoA, 5 mM ATP, and 5 mM NADH. (A) 0 min incubation time. (B) 90 min incubation time. In a second experiment, 5 mM NAD⁺ was added to the standard assay after a preincubation time of 30 min. (C) Assay directly after addition of NAD⁺ (30 min). (D) 60 min after addition of NAD⁺. Note different y-axis scales in panels C and D. LC-MS chromatograms obtained in single-ion mode show accumulated ion counts of expected metabolites.

FIG 3

LC-MS chromatograms of a 5,6,7,8-tetrahydro-2-naphthoyl-CoA (THNCoA) reductase assay with cell extract of culture N47. The assay contained 50 μM THNCoA, 5 mM ATP, and 5 mM 2-oxoglutarate as electron donor. Samples were analyzed via LC-MS in single-ion mode scanning for expected metabolites. Chromatograms show accumulated ion counts for those metabolites.

FIG 4

LC-MS chromatograms (accumulated ion counts of single-ion mode scans) of 5,6,7,8-tetrahydro-2-naphthoyl-CoA (THNCoA) reductase assays in cell extract of culture N47. The assays contained 50 μM THNCoA, 5 mM ATP, and different ratios of NADH and NAD⁺ as follows: 5 mM NADH and 5 mM NAD⁺ (A); 5 mM NADH and 0.2 mM NAD⁺ (B); and 0.2 mM NADH and 5 mM NAD⁺ (C). Samples were taken after 90 min and analyzed via LC-MS in single-ion mode scanning for expected metabolites.

FIG 5

Rates of THNCoA consumption or HHNCoA accumulation in THNCoA reductase assays with cell extracts of culture N47 using NADH, 2-oxoglutarate, or citrate as electron donor (5 mM each). Error bars indicate standard deviations of triplicate incubations for each electron donor.

FIG 6

Comparison of the LC-MS chromatograms of the downstream metabolite with m/z = 962 observed in THNCoA reductase assays with 2-oxoglutarate (5 mM) as electron donor and chemically synthesized reference compounds (A1 and A2, both obtained as a mixture of cis-isomers with the CoA thioester formed on either carboxyl group). LC-MS analyses were conducted at pH 5.5 and pH 7.0 for each compound. Gray lines indicate the retention times of peaks of the unknown metabolite. The compounds with m/z = 962 were detected via the MS unit in single-ion mode.

FIG 7

Proposed degradation pathway of 5,6,7,8-tetrahydro-2-naphthoyl-CoA (THNCoA, compound 5, m/z = 926) assuming the product of the THNCoA reductase to be a hexahydro-2-naphthoyl-CoA (HHNCoA, m/z = 928) with double bonds in α,β- or β’,γ’-position (7). The subsequent β-oxidation-like pathway proceeds via β-hydroxyoctahydro-2-naphthoyl-CoA (8) (m/z = 946), β-oxooctahydro-2-naphthoyl-CoA (9) (m/z = 944), and β’-hydroxy-β-oxodecahydro-2-naphthoyl-CoA (10) (m/z = 962). Hydrolytic opening of ring I gives 4-(2-carboxycyclohexyl)-3-hydroxybutyryl-CoA (10a) or 3-(2-[carboxymethyl]cyclohexyl)-3-hydroxypropionyl-CoA (10b) (both m/z = 980). Another β-oxidation-like sequence then continues via 4-(2-carboxycyclohexyl)-3-oxobutyryl-CoA (11a) or 3-(2-[carboxymethyl]cyclohexyl)-3-oxopropionyl-CoA (11b) (both m/z = 978), and 2-(2-carboxycyclohexyl)acetyl-CoA (12a) or 2-(carboxymethyl)cyclohexane-1-carboxyl-CoA (12b) (both m/z = 936), from where a previously described downstream pathway starts.

FIG 8

Potential intermediates (as free acids) of anaerobic naphthalene degradation with m/z = 962 (as CoA thioester) correlating with the previously observed elemental composition C₁₁H₁₆O₄ (A and B) and their downstream metabolites (C). A1: 2-(3-carboxyallyl)cyclohexane-1-carboxylic acid; A2: 3-(2-[carboxymethyl]cyclohexyl)acrylic acid; B1: 3-hydroxy-1-oxodecahydro-2-naphthoic acid; B2: 1-hydroxy-3-oxodecahydro-2-naphthoic acid; C1: 4-(2-carboxycyclohexyl)-3-hydroxybutyric acid; C2: 3-(2-[carboxymethyl]cyclohexyl)-3-hydroxypropionic acid.

FIG 9

Synthesis of putative metabolite A1 with only one enantiomer shown. DIBAL-H, di-isobutylaluminum hydride; DCM, dichloromethane; THF, tetrahydrofuran; PCC, pyridinium chlorochromate.

FIG 10

Synthesis of putative metabolite A2 with only one enantiomer shown. Tf, CF₃SO₂.