Enzyme Catalyzed Formation of CoA Adducts of Fluorinated Hexanoic Acid Analogues using a Long-Chain acyl-CoA Synthetase from Gordonia sp. Strain NB4-1Y.

Per and polyfluoroalkyl substances (PFAS) are a large family of anthropogenic fluorinated chemicals of increasing environmental concern. Over recent years, numerous microbial communities have been found to be capable of metabolizing some polyfluoroalkyl substances, generating a range of low-molecular-weight PFAS metabolites. One proposed pathway for the microbial breakdown of fluorinated carboxylates includes ß-oxidation, this pathway is initiated by the formation of a CoA adduct. However, until recently no PFAS-CoA adducts had been reported. In a previous study, we were able to use a bacterial medium-chain acyl-CoA synthetase (mACS) to form CoA adducts of fluorinated adducts of propanoic acid and pentanoic acid but were not able to detect any products of fluorinated hexanoic acid analogues. Herein, we expressed and purified a long-chain acyl-CoA synthetase (lACS) and a A461K variant of mACS from the soil bacterium Gordonia sp. strain NB4-1Y and performed an analysis of substrate scope and enzyme kinetics using fluorinated and nonfluorinated carboxylates. We determined that lACS can catalyze the formation of CoA adducts of 1:5 fluorotelomer carboxylic acid (FTCA), 2:4 FTCA and 3:3 FTCA, albeit with generally low turnover rates (<0.02 s-1) compared with the nonfluorinated hexanoic acid (5.39 s-1). In addition, the A461K variant was found to have an 8-fold increase in selectivity toward hexanoic acid compared with wild-type mACS, suggesting that Ala-461 has a mechanistic role in selectivity toward substrate chain length. This provides further evidence to validate the proposed activation step involving the formation of CoA adducts in the enzymatic breakdown of PFAS.

Formation of CoA Adducts of Short-Chain Fluorinated Carboxylates Catalyzed by Acyl-CoA Synthetase from Gordonia sp. Strain NB4-1Y.

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) make up a group of anthropogenic chemicals with a myriad of applications. However, some PFAS have been shown to negatively impact human health and the environment, leading to increased regulation, with some countries making efforts to phase out their use. PFAS fate in the environment is driven by physical, chemical, and biological processes, with microbial communities in matrices such as soil and sewage sludge being known to generate a range of low-molecular-weight PFAS metabolites. Proposed metabolic intermediates for both mixed and pure microbial cultures include fluorinated carboxylates that may be activated by CoA prior to ß-oxidation and defluorination, although thus far, no PFAS-CoA adducts have been reported. Herein, we expressed and purified acyl-CoA synthetase (ACS) from the soil bacterium Gordonia sp. strain NB4-1Y and performed an analysis of substrate scope and enzyme kinetics using fluorinated and nonfluorinated carboxylates. We determined that ACS was able to catalyze the formation of CoA adducts of 3,3,3-trifluoropropionic acid, 5,5,5-trifluoropentanoic acid, 4,5,5-trifluoropent-4-enoic acid, and 4,4,5,5,5-pentafluoropentanoic acid. Kinetic analysis revealed a 90-98% decrease in kcat between nonfluorinated carboxylates and their fluorinated analogues. This provides evidence to validate proposed enzymatic pathways for microbial PFAS metabolism that proceed via an activation step involving the formation of CoA adducts.

S224 Presents a Catalytic Trade-off in PLP-Dependent l-Lanthionine Synthase from Fusobacterium nucleatum.

Lanthionine synthase from the oral bacterium Fusobacterium nucleatum is a fold type II pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the ß-replacement of l-cysteine by a second molecule of l-cysteine to form H2S and l-lanthionine. The meso-isomer of the latter product is incorporated into the F. nucleatum peptidoglycan layer. Herein, we investigated the catalytic role of S224, which engages in hydrogen-bond contact with the terminal carboxylate of l-lanthionine in the closed conformation of the enzyme. Unexpectedly, the S224A variant elicited a 7-fold increase in the turnover rate for H2S and lanthionine formation and a 70-fold faster rate constant for the formation of the α-aminoacrylate intermediate compared to the wild-type enzyme. Presteady state kinetic analysis further showed that the reaction between S224A and l-cysteine leads to the formation of the more reactive ketoenamine tautomer of the α-aminoacrylate. The α-aminoacrylate with the protonated Schiff base is not an observable intermediate in the analogous reaction with the wild type, which may account for its attenuated kinetic properties. However, the S224A substitution is detrimental to other aspects of the catalytic cycle; it facilitates the α,ß-elimination of l-lanthionine, and it weakens the enzyme's catalytic preference for the formation of l-lanthionine over that of l-cystathionine.