Publications

• Cyclohexa-1,5-diene-1-carbonyl-CoA hydratase, an enzyme involved in anaerobic metabolism of benzoyl-CoA in the denitrifying bacterium Thauera aromatica.

  Laempe D., Eisenreich W., Bacher A., Fuchs G.

  Many aromatic compounds can be metabolized by bacteria under anoxic conditions via benzoyl-CoA as the common intermediate. The central pathway of benzoyl-CoA metabolism is initiated by an ATP-driven reduction of the aromatic ring producing cyclohexa-1,5-diene-1-carbonyl-CoA. The 1,5-dienoyl-CoA in ... >> More

  Many aromatic compounds can be metabolized by bacteria under anoxic conditions via benzoyl-CoA as the common intermediate. The central pathway of benzoyl-CoA metabolism is initiated by an ATP-driven reduction of the aromatic ring producing cyclohexa-1,5-diene-1-carbonyl-CoA. The 1,5-dienoyl-CoA intermediate is thought to be transformed to 6-hydroxycyclohex-1-ene-1-carbonyl-CoA by a specific dienoyl-CoA hydratase catalyzing the formal addition of water to one of the double bonds. This dienoyl-CoA hydratase was detected in the denitrifying bacterium Thauera aromatica after anaerobic growth with benzoate. Substrate and product were confirmed and a convenient spectrophotometric assay was developed. The equilibrium concentrations of substrate and product were almost equal. Enzyme activity was induced after anoxic growth with benzoate, in contrast to acetate. The enzyme of 28 kDa was purified from T. aromatica and was found to be highly specific for the cyclic 1,5-dienoyl-CoA. A second 29-kDa enoyl-CoA hydratase acted on crotonyl-CoA; this highly active enoyl-CoA hydratase also acted slowly on cyclohex-1-ene-1-carbonyl-CoA. The regulation of expression of dienoyl-CoA hydratase activity, the kinetic constants, the substrate specificity, and the specific activity of the enzyme in cell extract provide evidence that dienoyl-CoA hydratase is the second enzyme of the central benzoyl-CoA pathway of anaerobic aromatic metabolism in T. aromatica. Extracts of Rhodopseudomonas palustris contained high activity of cyclohex-1-ene-1-carbonyl-CoA hydratase, but no 1,5-dienoyl-CoA hydratase activity. It appears that a variant of the benzoyl-CoA pathway is operating in R. palustris in which hydration of the 1,5-dienoyl-CoA does not take place. Rather, cyclohex-1-ene-1-carbonyl-CoA is hydrated to 2-hydroxycyclohexane-1-carbonyl-CoA [corrected]. << Less

  Eur. J. Biochem. 255:618-627(1998) [PubMed] [EuropePMC]

• Cyclohexa-1,5-diene-1-carbonyl-coenzyme A (CoA) hydratases of Geobacter metallireducens and Syntrophus aciditrophicus: Evidence for a common benzoyl-CoA degradation pathway in facultative and strict anaerobes.

  Peters F., Shinoda Y., McInerney M.J., Boll M.

  In the denitrifying bacterium Thauera aromatica, the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyclohexa-1,5-diene-1-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series o ... >> More

  In the denitrifying bacterium Thauera aromatica, the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyclohexa-1,5-diene-1-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series of beta-oxidation-like reactions of the so-called benzoyl-CoA degradation pathway resulting in ring cleavage. Recently, evidence was obtained that obligately anaerobic bacteria that use aromatic growth substrates do not contain an ATP-dependent benzoyl-CoA reductase. In these bacteria, the reactions involved in dearomatization and cleavage of the aromatic ring have not been shown, so far. In this work, a characteristic enzymatic step of the benzoyl-CoA pathway in obligate anaerobes was demonstrated and characterized. Dienoyl-CoA hydratase activities were determined in extracts of Geobacter metallireducens (iron reducing), Syntrophus aciditrophicus (fermenting), and Desulfococcus multivorans (sulfate reducing) cells grown with benzoate. The benzoate-induced genes putatively coding for the dienoyl-CoA hydratases in the benzoate degraders G. metallireducens and S. aciditrophicus were heterologously expressed and characterized. Both gene products specifically catalyzed the reversible hydration of dienoyl-CoA to 6-hydroxycyclohexenoyl-CoA (Km, 80 and 35 microM; Vmax, 350 and 550 micromol min(-1) mg(-1), respectively). Neither enzyme had significant activity with cyclohex-1-ene-1-carbonyl-CoA or crotonyl-CoA. The results suggest that benzoyl-CoA degradation proceeds via dienoyl-CoA and 6-hydroxycyclohexanoyl-CoA in strictly anaerobic bacteria. The steps involved in dienoyl-CoA metabolism appear identical in all nonphotosynthetic anaerobic bacteria, although totally different benzene ring-dearomatizing enzymes are present in facultative and obligate anaerobes. << Less

  J Bacteriol 189:1055-1060(2007) [PubMed] [EuropePMC]

• Products of enzymatic reduction of benzoyl-CoA, a key reaction in anaerobic aromatic metabolism.

  Koch J., Eisenreich W., Bacher A., Fuchs G.

  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 'biologica ... >> More

  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 beta-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. << Less

  Eur J Biochem 211:649-661(1993) [PubMed] [EuropePMC]

• Shedding light on anaerobic benzene ring degradation: a process unique to prokaryotes?

  Harwood C.S., Gibson J.

  J Bacteriol 179:301-309(1997) [PubMed] [EuropePMC]

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