Aromatizing cyclohexa-1,5-diene-1-carbonyl-coenzyme A oxidase. Characterization and its role in anaerobic aromatic metabolism.
Benzoyl-CoA reductases (BCRs) are key enzymes of anaerobic aromatic metabolism in facultatively anaerobic bacteria. The highly oxygen-sensitive enzymes catalyze the ATP-dependent reductive de-aromatization of the substrate, yielding cyclohexa-1,5-diene-1-carbonyl-CoA (1,5-dienoyl-CoA). In extracts from anaerobically grown denitrifying Thauera aromatica, we detected a benzoate-induced, benzoyl-CoA-forming, 1,5-dienoyl-CoA:acceptor oxidoreductase activity. This activity co-purified with BCR but could be partially separated from it by hydroxyapatite chromatography. After activity staining on native gels, a monomeric protein with a subunit molecular weight of M(r) 76,000 was identified. Mass spectrometric analysis of tryptic digests identified peptides from NADH oxidases/2,4-dienoyl-CoA reductases/"old yellow" enzymes. The UV-visible spectrum of the enriched enzyme suggested the presence of flavin and Fe/S-cofactors, and it was bleached upon the addition of 1,5-dienoyl-CoA. The enzyme had a high affinity for dioxygen as electron acceptor (K(m) = 10 microm) and therefore is referred to as 1,5-dienoyl-CoA oxidase (DCO). The likely product formed from dioxygen reduction was H(2)O. DCO was highly specific for 1,5-dienoyl-CoA (K(m) = 27 microm). The initial rate of DCO followed a Nernst curve with half-maximal activity at +10 mV. We propose that DCO provides protection for the extremely oxygen-sensitive BCR enzyme when the bacterium degrades aromatic compounds at the edge of steep oxygen gradients. The redox-dependent switch in DCO guarantees that DCO is only active during oxidative stress and circumvents futile de-aromatization/re-aromatization reactions catalyzed by BCR and DCO.
Thiele
Bärbel
B
Institute of Biochemistry, University of Leipzig, Brüderstrasse 34, Leipzig, Germany.
Rieder
Oliver
O
Jehmlich
Nico
N
von Bergen
Martin
M
Müller
Michael
M
Boll
Matthias
M
D000255:Adenosine Triphosphate / Q000737:chemistry; D002384:Catalysis; D004952:Esters; D006836:Hydro-Lyases / Q000737:chemistry*; D062385:Hydroxybenzoates / Q000737:chemistry; D000860:Hypoxia; D007700:Kinetics; D008956:Models, Chemical; D009243:NAD / Q000737:chemistry; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013379:Substrate Specificity; D014466:Ultraviolet Rays
D004952:Esters; D062385:Hydroxybenzoates; D010455:Peptides; D009243:NAD; D000255:Adenosine Triphosphate; D006836:Hydro-Lyases; C114397:cyclohexa-1,5-diene-1-carboxyl-CoA hydratase; D010100:Oxygen