Denitrifying Thauera Aromatica Research Articles

Promiscuous Defluorinating Enoyl-CoA Hydratases/Hydrolases Allow for Complete Anaerobic Degradation of 2-Fluorobenzoate.

Biodegradation of the environmentally hazardous fluoroaromatics has mainly been associated with oxygenase-dependent defluorination reactions. Only very recently a novel mode of oxygen-independent defluorination was identified for the complete degradation of para-substituted fluoroaromatics in the denitrifying Thauera aromatica: a promiscuous class I benzoyl-coenzyme A (BzCoA) reductase (BCR) catalyzed the ATP-dependent defluorination of 4-F-BzCoA to BzCoA. Here, we studied the unknown enzymatic defluorination during the complete degradation of 2-F-benzoate to CO2 and HF. We demonstrate that after activation of 2-F-benzoate by a promiscuous AMP-forming benzoate-CoA ligase, the 2-F-BzCoA formed is subsequently dearomatized by BCR to a mixture of 2-F- and 6-F-cyclohexa-1,5-diene-1-carboxyl-CoA (2-F-/6-F-1,5-dienoyl-CoA). This finding indicates that BCR is not involved in C–F-bond cleavage during growth with 2-F-benzoate. Instead, we identified defluorination of the two isomers by enoyl-CoA hydratases/hydrolases involved in down-stream reactions of the BzCoA degradation pathway. (i) The 1,5-dienoyl-CoA hydratase hydrated the F-1,5-dienoyl-CoA isomers to a mixture of the stable 2-F-6-OH-1-enoyl-CoA and the unstable α-fluorohydrin 6-F-6-OH-1-enoyl-CoA; the latter spontaneously decomposed to HF and 6-oxo-cyclohex-1-enoyl-CoA (6-oxo-1-enoyl-CoA), a common intermediate of the BzCoA degradation pathway. (ii) 6-Oxo-1-enoyl-CoA hydrolase/hydratase catalyzed the defluorination of 2-F-6-OH-1-enoyl-CoA to 2-oxo-6-OH-1-enoyl-CoA and HF again via water addition to an F-enoyl-CoA functionality. Based on these in vitro results, we demonstrate a previously overseen capability of 2-F-benzoate degradation for many but not all tested facultatively and obligately anaerobic bacteria that degrade aromatic compounds via the BzCoA degradation pathway. In conclusion, the newly identified enzymatic defluorination by enoyl-CoA hydratases via α-fluorohydrin formation represents an abundant, physiologically relevant principle of enzymatic defluorination.

ATP-Dependent C-F Bond Cleavage Allows the Complete Degradation of 4-Fluoroaromatics without Oxygen.

ABSTRACTComplete biodegradation of the abundant and persistent fluoroaromatics requires enzymatic cleavage of an arylic C–F bond, probably the most stable single bond of a biodegradable organic molecule. While in aerobic microorganisms defluorination of fluoroaromatics is initiated by oxygenases, arylic C–F bond cleavage has never been observed in the absence of oxygen. Here, an oxygen-independent enzymatic aryl fluoride bond cleavage is described during the complete degradation of 4-fluorobenzoate or 4-fluorotoluene to CO2 and HF in the denitrifying Thauera aromatica: the ATP-dependent defluorination of 4-fluorobenzoyl-coenzyme A (4-F-BzCoA) to benzoyl-coenzyme A (BzCoA) and HF, catalyzed by class I BzCoA reductase (BCR). Adaptation to growth with the fluoroaromatics was accomplished by the downregulation of a promiscuous benzoate-CoA ligase and the concomitant upregulation of 4-F-BzCoA-defluorinating/dearomatizing BCR on the transcriptional level. We propose an unprecedented mechanism for reductive arylic C–F bond cleavage via a Birch reduction-like mechanism resulting in a formal nucleophilic aromatic substitution. In the proposed anionic 4-fluorodienoyl-CoA transition state, fluoride elimination to BzCoA is favored over protonation to a fluorinated cyclic dienoyl-CoA.

Anaerobic toluene-catabolic pathway in denitrifying Thauera aromatica: activation and β-oxidation of the first intermediate, (R)-(+)-benzylsuccinate

Anaerobic catabolism of toluene is initiated by addition of the methyl group of toluene to the double bond of a fumarate cosubstrate to yield the first intermediate, benzylsuccinate. This reaction is catalysed by the glycyl-radical enzyme benzylsuccinate synthase, as shown for the denitrifying bacterium Thauera aromatica. Benzylsuccinate is further oxidized to benzoyl-CoA, the central intermediate of anaerobic degradation of aromatic compounds. The authors show here by experiments with cell extracts of toluene-grown T. aromatica that the pathway of benzylsuccinate oxidation requires activation of the free acid to a CoA-thioester, catalysed by a toluene-induced, reversible succinyl-CoA-dependent CoA-transferase. The product of the CoA-transferase reaction, benzylsuccinyl-CoA, is oxidized to benzoyl-CoA and succinyl-CoA in extracts of toluene-grown cells, adding proof to the proposed anaerobic toluene-catabolic pathway. The stereochemical preferences of the enzymes catalysing formation and activation of benzylsuccinate have been analysed. Benzylsuccinate synthase was found to produce exclusively (R)-(+)-benzylsuccinate, although the proposed reaction mechanism of this enzyme proceeds via radical intermediates. In accordance, the reaction of succinyl-CoA:benzylsuccinate CoA-transferase is also specific for (R)-(+)-benzylsuccinate and does not proceed with the (S)-(-)-enantiomer.