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

Abstract

Complete 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.

Liquid chromatography/isotope ratio mass spectrometry analysis of halogenated benzoates for characterization of the underlying degradation reaction in Thauera chlorobenzoica CB‐1T

“…It was shown that 3‐F‐benzoyl‐CoA is protonated yielding 3‐fluoro‐dienoyl‐CoA, which is not further converted, whereas 4‐F‐benzoyl‐CoA can be slowly dehalogenated. This results from the strength of the C–F bond . In contrast, as C–Cl and C–Br bonds are weaker, the reaction is more feasible but we also find differences in reaction rates according to the position of the substituent.…”

“…When considering the benzoyl‐CoA‐reduction and dehalogenation reaction, Tiedt et al showed differences in elimination reactions between meta ‐ and para ‐substitution of CoA‐halobenzoic acids. Whereas meta ‐substitution leads to spontaneous halogen abstraction by an E2‐elimination reaction, protonation of the anionic intermediate after hydride addition would lead to an unfavorable halo‐dienoyl‐CoA following an E1cB elimination of the para ‐substituent.…”

“…In contrast, as C–Cl and C–Br bonds are weaker, the reaction is more feasible but we also find differences in reaction rates according to the position of the substituent. Whereas 3‐Cl‐benzoic acid and 3‐Br‐benzoic acid were completely degraded after 46 h and 55 h, respectively, 4‐Cl‐benzoic acid was only completely degraded after 98 h. This points towards differences in the para ‐substituent removal reaction from meta‐ substituent removal confirming results by Tiedt et al…”

“…Thauera chlorobenzoica spp. and Thauera aromatica ; (ii) under sulfate‐reducing conditions; or (iii) by the phototrophic anaerobic microorganism Rhodopseudomonas palustris . Furthermore, T. chlorobenzoica 3CB‐1 T is capable of utilizing 3‐bromo‐, 2‐fluoro‐, 4‐fluorobenzoic acid and 4‐chlorobenzoic acid (O. Tiedt, University of Freiburg, personal communication) as substrates for growth.…”

“…The dehalogenation appears to occur enzymatically or spontaneously by release of hydrogen chloride (HCl) or hydrogen bromide (HBr) and re‐aromatization of the halobenzoyl‐CoA which follows the benzoyl‐CoA degradation pathway . Whereas 4‐fluorobenzoic acid can be dehalogenated completely, 3‐fluorobenzoic acid forms 3‐fluoro‐dienoyl‐CoA, which is not further converted …”