Products of enzymatic reduction of benzoyl-CoA, a key reaction in anaerobic aromatic metabolism.
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.
Koch
J
J
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Federal Republic of Germany.
Eisenreich
W
W
Bacher
A
A
Fuchs
G
G
D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000693:Anaerobiosis; D001419:Bacteria / Q000378:metabolism*; D001565:Benzoates / Q000378:metabolism; D019817:Benzoic Acid; D003065:Coenzyme A / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000378:metabolism; D012241:Rhodopseudomonas / Q000378:metabolism; D012984:Software; D014025:Titanium / Q000378:metabolism
D000214:Acyl Coenzyme A; D001565:Benzoates; C037817:benzoyl-coenzyme A; D000255:Adenosine Triphosphate; D019817:Benzoic Acid; D014025:Titanium; D008274:Magnesium; D003065:Coenzyme A