Abstract
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.
Highlights
In the last decades fluorinated organic compounds have become relevant environmental contaminants formed by industrial, agricultural and pharmaceutical processes
The individual enzymatic steps can be summarized as follows (Figure 7): (i) activation of 2-F-benzoate to 2-F-benzoyl-coenzyme A (BzCoA) by promiscuous benzoate-CoA ligase (BCL); (ii) reduction of 2-F-BzCoA to a 1:2 mixture of 2-F-/6-F-1,5-dienoyl-CoA by promiscuous ATP-dependent class I BzCoA reductase (BCR); (iii) 1,4-hydration of 2-F-1,5-dienoyl-CoA isomer by either dienoyl-CoA hydratase (DCH) or oxo-1-enoylCoA hydrolase (OAH) to an instable 6-F-6-OH-1-enoyl-CoA that spontaneously decomposes to HF and 6-oxo-1-enoyl-CoA, and 1,4-hydration of the 6-F-1,5-dienoyl-CoA to stable 2-F6-OH-1-enoyl-CoA; (iv) conversion of 2-F-6OH-1-enoyl-CoA to the unstable 2-F-2,6-di-OH-cyclohexanecarboxyl-CoA via 1,2-hydration, catalyzed by OAH
Unlike 4-F-benzoate degradation, where class I BCR is directly involved in reductive defluorination, the downstream enzymes OAH and DCH are employed for C–F-bond cleavage during 2-F-benzoate degradation
Summary
In the last decades fluorinated organic compounds have become relevant environmental contaminants formed by industrial, agricultural and pharmaceutical processes. Fluorobenzoates (F-benzoates) are the best studied fluoroaromatic model compounds for microbial degradation (Murphy et al, 2009; Murphy, 2010; Kiel and Engesser, 2015; Tiedt et al, 2016). Some aerobically growing microorganisms of the genera Agrobacterium, Pseudomonas, Alcaligenes, Aureobacterium, Thauera and others use the 2-, 3- and 4-F-benzoate isomers as growth substrates (Kiel and Engesser, 2015). Mechanistic considerations for spontaneous or enzyme catalyzed fluorine release at this stage are on debate (Natarajan et al, 2005; Murphy, 2010; Kiel and Engesser, 2015)
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