Products of enzymatic reduction of benzoyl‐CoA, a key reaction in anaerobic aromatic metabolism

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Abstract
Benzoyl‐coenzyme A is the most common central intermediate of anaerobic aromatic metabolism. Studies with whole cells of different bacteria and in vitro had shown that benzoyl‐CoA is reduced to alicyclic compounds, possibly via cyclohexadiene intermediates. This reaction is considered a ‘biological Birch reduction’. We have elucidated by NMR techniques the structures of six products of [ring‐13C6]benzoate reduction. The reaction is catalyzed by extracts from cells of a denitrifying Pseudomonas strain K172 anaerobically grown with benzoate and nitrate as sole carbon and energy sources. The assay mixture contained [ring‐13C6]benzoate plus traces of [U‐14C]benzoate, Mg2+, ATP, coenzyme A (CoA), and Ti(III) as reductant. The use of the multiply 13C‐labelled precursor increases the sensitivity of NMR detection and allows the analysis of crude product mixtures by two‐dimensional coherence transfer procedures such as total correlation 13C‐NMR spectroscopy and 13C‐filtered 1H‐NMR spectroscopy. The time course of product formation is consistent with the following order of events. Benzoyl‐CoA is formed from benzoate via benzoate‐CoA ligase. The first ring reduction product observed is cyclohex‐1,5‐diene‐1‐carboxyl‐CoA. The next intermediate is 6‐hydroxycyclohex‐1‐ene‐1‐carboxyl‐CoA which is derived from the diene by addition of water. Part of the diene seems to be reduced to cyclohex‐1‐ene‐1‐carboxyl‐CoA which becomes hydrated to trans‐2‐hydroxycyclohexane‐1‐carboxyl‐CoA; these two intermediates may be side products in vitro. The first non‐cyclic intermediate formed by β‐oxidation is 3‐hydroxypimelyl‐CoA. This aliphatic C7 dicarboxylic acid is proposed to be oxidized via glutaryl‐CoA and crotonyl‐CoA to three molecules of acetyl‐CoA and one molecule of CO2. A similar product pattern was observed in the benzoate‐degrading phototrophic bacterium Rhodopseudomonas palustris. This indicates that the enzymatic reduction of benzoyl‐CoA may be mechanistically similar in different anaerobes.