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Strategies for the Biodegradation of Polyfluorinated Compounds

Strategies for the Biodegradation of Polyfluorinated Compounds Review Strategies for the Biodegradation of Polyfluorinated Compounds Lawrence P. Wackett Department of Biochemistry, Molecular Biology and Biophysics and BioTechnology Institute, University of Minnesota, Minneapolis, MN 55455, USA; [email protected] Abstract: Many cite the strength of C–F bonds for the poor microbial biodegradability of polyfluor- inated organic compounds (PFCs). However, commercial PFCs almost invariably contain more functionality than fluorine. The additional functionality provides a weak entry point for reactions that activate C–F bonds and lead to their eventual cleavage. This metabolic activation strategy is common in microbial biodegradation pathways and is observed with aromatic hydrocarbons, chlo- rinated compounds, phosphonates and many other compounds. Initial metabolic activation pre- cedes critical bond breakage and assimilation of nutrients. A similar strategy with commercial PFCs proceeds via initial attack at the non-fluorinated functionalities: sulfonates, carboxylates, chlorines, phenyl rings, or phosphonates. Metabolic transformation of these non-fluorinated groups can acti- vate the C–F bonds, allowing more facile cleavage than a direct attack on the C–F bonds. Given that virtually all compounds denoted as “PFAS” are not perfluorinated and are not alkanes, it is posited here that considering their individual chemical classes is more useful for both chemical and micro- biological considerations of their fate. Keywords: polyfluorinated; PFAS; biodegradation; bacteria; enzyme; metabolism; metabolic activation; weak bonds; defluorination Citation: Wackett, L.P. Strategies for the Biodegradation of Polyfluorinated Compounds. Microorganisms 2022, 10, 1664. https://doi.org/10.3390/ 1. Introduction microorganisms10081664 In 2022, a comprehensive search of all public sources identified more than nineteen Academic Editors: Maria De Fátima million fluorinated chemicals, almost all of which have been created by humans within Carvalho and Diogo Alexandrino the last century [1]. Commercial polyfluorinated organic compounds (PFCs) are used in more than two hundred different applications [2]. Between 1998 and 2020, more than half Received: 13 July 2022 of all International Organization for Standardization (ISO) approved agrichemicals were Accepted: 16 August 2022 fluorinated [3]. More than 20% of new pharmaceuticals are fluorinated, mostly aromatic Published: 17 August 2022 ring compounds [4,5]. While regulatory scrutiny is increasing, the vast majority of these Publisher’s Note: MDPI stays neu- fluorinated compounds remain unregulated and a knowledge of their biodegradation, tral with regard to jurisdictional and hence longevity in the environment, is widely lacking. claims in published maps and institu- In light of the general lack of information on PFC biodegradation, this review draws tional affiliations. on broad concepts of biodegradation to posit how PFCs might be transformed by natural and engineered microbes. As such, the review combines relevant literature on how mi- crobes activate difficult-to-cleave bonds with insights from the field of organic chemistry regarding carbon–fluorine bond cleavage mechanisms. Examples with relevant references Copyright: © 2022 by the author. will be provided throughout the review. The present compilation differs from previous Licensee MDPI, Basel, Switzerland. PFC biodegradation reviews that focus on a limited set of the >19 million fluorinated com- This article is an open access article pounds known. For example, previous reviews have covered fluoroalkyl acids and distributed under the terms and fluorotelomers, such as those used in firefighting foams [6–10]. However, there are mul- conditions of the Creative Commons Attribution (CC BY) license tiple classes of PFCs for which documented biodegradative metabolism is lacking. This (https://creativecommons.org/license contribution focuses on potential mechanisms by which these PFCs may be transformed s/by/4.0/). by microbes based on general principles of biodegradation and organofluorine chemistry. Microorganisms 2022, 10, 1664. https://doi.org/10.3390/microorganisms10081664 www.mdpi.com/journal/microorganisms Microorganisms 2022, 10, 1664 2 of 13 The vast majority, more than eighteen million, of anthropogenic fluorine compounds are aromatic [1]. While many of the commercial agrichemicals consist of multiply fluori- nated aromatic ring compounds, most biodegradation studies with fluoroaromatics were with monofluorinated compounds [11–13]. Much remains to be learned about the micro- bial mechanisms by which polyfluorinated aromatic, olefinic and alkyl compounds are biodegraded. In many of the metabolic pathways for PFCs that exist or may yet evolve, this author suggests that direct C–F bond cleavage is unlikely to occur. Instead, metabolic activation of adjacent atoms can activate the C–F bond to make biodegradation feasible. This concept is documented in literature from the chemical sciences on C–F bond activa- tion, and in literature on the activation and biodegradation of other very strong chemical bonds, such as the C–P bond of alkylphosphonic acids [14]. This author is unaware of other review articles on PFCs having this focus. Some of the steps for the metabolic activation of PFCs may take time to evolve given that no natural product PFCs are known to humans, and so may not have entered the biosphere until very recently. This contrasts with most functional groups found in com- mercial chemicals that also occur in natural products that have likely existed for eons [15]. Fluorinated natural products are rare (only several dozen have been identified) and they are all monofluorinated compounds [16,17]. There are no polyfluorinated natural prod- ucts known at this time. This contrasts with the synthetic and commercial production of fluorinated compounds, many of which are PFCs [1]. At one time, polychlorinated com- pounds were considered foreign to the biosphere, but over the past several decades, more than five thousand chlorinated natural products have been identified, many of which are polychlorinated [18]. Industry produced many chlorinated pesticides in the early to mid- 20th century to slow their biodegradation in the field [19], but has now largely shifted over to PFCs [4]. The sheer number of PFCs produced, their widespread use and their apparent foreignness to microbes presents a large challenge for biodegradation. It is in this context that all current knowledge of metabolic activation and C–F bond cleavage chemistry must be applied to this problem. 2. Definitions of PFAS and PFCs The term PFAS (per- and polyfluoroalkyl substances) is used to refer to many differ- ent classes of PFCs with a wide divergence in their chemical reactivity. The ambiguity of the term has set up an unfortunate fuzziness in thinking about their biodegradation. For example, some compounds that meet the current Organization for Economic Co-opera- tion and Development (OECD) definition of PFAS [20] are very biodegradable, while oth- ers are not. With few exceptions, the compounds referred to as per- and polyfluorinated alkyl substances (PFAS) contain aromatic rings, ether linkages and other functional groups that lead to a very large difference in biodegradability. An example of a perfluor- inated alkane is octadecafluorooctane (perfluorooctane) (Figure 1A, top). Perfluoroal- kanes have limited commercial uses compared to most compounds referred to as PFAS [21]. Virtually all of the regulated and targeted molecules for environmental remediation that are referred to as PFAS on PubChem [22] and other sites, contain functionalities in addition to their C–F bonds. Examples of some highly used and researched compounds are shown below the perfluoroalkane at the top of Figure 1A. If the term PFAS has become too pervasive to avoid it, users should minimally state upfront which definition of PFAS they are using, as discussed in Barnabas, et al. [1]. OECD, the organization representing mostly European countries, has defined PFAS as any compound with at least a -CF3 group, or a -CF2- group that does not have a H/Cl/Br/I atom associated with it [20]. A new United States Environmental Protection Agency defi- nition says that a PFAS is anything that contains (R1)(R2)(F)C–C(R3)F2 [1]. Barnabas and coworkers prefer the following PFAS definition: a compound that contains an (R1)(R2)(F)C–C(R3)F2 group where R groups are any atom except hydrogen and the bond between both aliphatic carbon atoms is a single bond. The Center for Disease Control (CDC) states, “PFAS are a group of chemicals used to make fluoropolymer coatings and Microorganisms 2022, 10, 1664 3 of 13 products that resist heat, oil, stains, grease and water” [23]. Others have also questioned the definitions of PFAS for the difficulties that they pose for regulatory decisions [24]. Moreover, it has been pointed out that the currently marketed fluorinated pharmaceuti- cals are classified as 1% being PFAS by one definition, and as 100% being PFAS by another definition [25]. Figure 1. Polyflourinated organic compounds (PFCs) and mechanisms for their defluorination. (A) A perfluorinated alkane (top) and commercial PFCs showing the types of functionalities typically combined with perfluorinated moieties. (B) Two types of mechanisms by which a functionality ad- jacent to a perfluorinated carbon can be attacked enzymatically leading to facile defluorination. Depending on which definition one might choose, biodegradation of a designated PFAS chemical might be easy or hard, or difficult to pin down if you are talking about the biodegradation of chemicals that “resist heat.” In this review, the term “polyfluorinated organic compounds” is used, abbreviated as PFCs, and defines specific chemical classes in the context of the nuances of their biodegradation. The classes discussed are: perfluoro- alkanes, substituted polyfluorinated alkanes, substituted polyfluorinated alkenes and substituted polyfluorinated aromatics. 3. Broad Principles for the Biodegradation of PFCs Perfluorooctane presents a significant challenge for biodegradation relative to other PFCs (Figure 1). With perfluorooctane, chemical hydrolysis is not observed, and reduction of a C–F bond requires electrochemical potentials below −2000 mV [26]. More recent stud- ies confirm these low potentials as being required [27,28]. The known lower limit for bio- logical reduction potential attained by an enzymatic cofactor is −622 mV [29], making it unlikely that biology will evolve a new cofactor to poise its potential at −2000 mV. With additional functionality attached to a highly fluorinated, even perfluorinated, chain or ring, many other chemical and metabolic options are available to bring about defluorina- tion. For example, the presence of an ether linkage with a C–O carbon containing a non- Microorganisms 2022, 10, 1664 4 of 13 fluorine substituent presents a potential soft spot for enzyme cleavage that could produce a gem-difluoro alcohol that would undergo spontaneous elimination and hydrolysis to release fluoride. Moreover, such a metabolic step potentially sets up the molecule for fur- ther degradation proceeding down the alkyl or alkenyl chain, or into a ring (Figure 1B, top). An ether cleavage that leads to spontaneous defluorination would be plausible, for example, with FRD-903 and F53-B. Similarly, fluorotelomers like the firefighting foam shown in Figure 1A could plausibly be attacked by a lyase catalyzing elimination of fluo- ride (Figure 1B, bottom). The resultant olefin is susceptible to water addition, oxygenation or other reactions to continue metabolism. Indeed, microbial transformations of fluorote- lomers have been described [30,31]. Bacteria carrying out these reactions include Gordonia sp. strain NB4-1Y, Pseudomonas fluorescens DSM 8341, Pseudomonas oleovorans and Myco- bacterium vaccae JOB5. Given that PFCs of the type shown in Figure 1 are very new to the biosphere, there has been little time on an evolutionary scale to put together consecutive reactions of the types shown to make dedicated metabolic pathways. This review discusses general prin- ciples as to how microbes may activate PFCs for defluorination by “attacking the weak points.” Since perfluorooctane and related perfluorinated alkanes are not the subject of any remediation activities this author is aware of, and virtually all molecules of interest have other functional groups, the dominant theme here is to attack non-fluorine function- alities and promote C–F bond cleavage indirectly. This review discusses the potential for this strategy in more detail, but first starts by describing the most well-understood exam- ple of direct enzymatic C–F bond cleavage. 4. Direct Enzymatic C–F Bond Cleavage Has Limitations Numerous bacteria have been isolated that defluorinate the widespread natural product fluoroacetate [32–37], largely mediated by fluoroacetate dehalogenase. This has become the mechanistic paradigm for direct SN2-type defluorination by a microbial en- zyme (Figure 2) [38,39]. There are X-ray structures for fluoroacetate dehalogenases [39,40], and computational chemistry has been applied to better understand the power and limi- tations of the reaction [41–43]. The enzyme uses a carboxylate anion nucleophile. The neg- ative charge on the substrate is shielded by the active site arginine and fluoride anion leaving is facilitated by a nearby triad of tyrosine, histidine and tryptophan residues. Wa- ter resolves the enzyme ester intermediately to produce the glycolic acid product. There is sufficient metabolic energy in glycolic acid to potentially support bacterial growth. Perfluorinated acetic and longer chain carboxylic acids are not known to react read- ily, if at all, with fluoroacetate dehalogenase. Moreover, studies with wastewater micro- organisms showed ready degradation of fluoroacetate, whereas di- and trifluoroacetate were recalcitrant [44]. Carbon–fluorine bond strength is known to increase with increasing fluorine substituents on an aliphatic carbon [45,46]. Moreover, unlike monofluoroacetate, di- and trifluoroacetate moieties are not known to be biosynthesized or found in natural products, hence microbial exposure to them has only occurred in recent history. Although fluoroacetate dehalogenase is a powerful enzyme, uniquely poised for direct defluorina- tion [47], its limitations highlight the need to pursue different mechanistic strategies for biodegrading polyfluorinated compounds. Given the observed hurdles in direct cleavage of unactivated C–F bonds, metabolic activation will likely be necessary to biodegrade many PFCs. This is not rare in biodegra- dative metabolism. As described in the next section, metabolic activation prior to degrad- ing stable molecules is the norm rather than the exception in biodegradation. Microorganisms 2022, 10, 1664 5 of 13 Figure 2. Fluoroacetate metabolism and enzyme from bacteria serves as the main, but rare, example of direct C–F bond cleavage. (A) Bacteria catalyzing defluorination of fluoroacetate. (B) Active site of the fluoroacetate dehalogenase from Rhodospeudomonas palustris CGA009 (3R3U). (C) Metabolic benefit. In addition to detoxification, some bacteria can use hydroxyacetic acid, or glycollate, as a sole source of carbon and energy to support growth. 5. Concept of Metabolic Activation in Biodegradation The cost of acquiring additional genes, and hence new metabolic functions, is often given as a reason why microbial genome size has largely stayed within 1–10 Mb confines [48]. Each added gene requires more resources and time to replicate. Microbes sometimes acquire, and evolve, new genes via plasmids, transposons and mutations that create new functions [49–51]. This strategy may be evolutionarily efficient by keeping genomes streamlined, gaining valuable functions and shedding genes if they fail to provide suffi- cient survival benefit. Given the extreme selective pressures against the unrestrained expansion of metab- olism, the common occurrence of metabolic activation attests to its importance. Metabolic activation refers to the initial steps in many biodegradative pathways in which energy or materials are not immediately gained. In many of those cases, the metabolic activation requires energy investment, which is later regained along with added energy and nutri- ents that support cell growth. In the present review, examples of metabolic activation are discussed that are most relevant to known and proposed microbial defluorination metab- olism. Hydrocarbons and dialkyl ethers are common industrial materials, and so biodeg- radation of such chemicals has been studied extensively. In aerobic environments, ali- phatic and aromatic hydrocarbons and ethers are often activated by oxygenases to pro- duce alcohols, aldehydes and carboxylic acids that can enter intermediary metabolic path- ways [52]. Enzymes that combine atmospheric oxygen with hydrocarbons must overcome a high energy barrier that requires activation of O2, typically with NAD(P)H (Figure 3A). Since there is often substantial energy gained from the complete aerobic oxidation of hy- drocarbons and dialkyl ethers to carbon dioxide, the energy investment is more than com- pensated for. An example of metabolic activation for dehalogenation has been observed with p- chlorobenzoate (Figure 3B) [53]. The carboxyl group is only mildly activating for the para position in nucleophilic aromatic substitution [54,55], causing some microorganisms to invest energy to make the para position more activated. This is achieved by transforming the carboxylate group into a coenzyme A-tethered thioether. The dechlorination can then proceed hydrolytically, and the ester is subsequently hydrolyzed to make the common growth substrate p-hydroxybenzoate. The thioesterification and its later hydrolytic cleav- age costs a cell two ATP-equivalents per metabolic cycle due to the CoA ligase enzyme Microorganisms 2022, 10, 1664 6 of 13 producing AMP and pyrophosphate. However, further of metabolism of p-hydroxyben- zoate more than recoups the energy investment. The cleavage of the C–P bond of alkyl phosphonates by C–P lyase provides an inter- esting parallel to plausible activation mechanisms for C–F bond cleavage (Figure 3C). The 2− overall reaction stoichiometry for methyl phosphonate is: CH3-PO3 + H2O → CH4 + 2− HOPO3 . However, this is not a direct addition of water to the C–P bond; the bond is cleaved only following a set of activation reactions requiring ATP and six proteins, with the key steps shown in the figure [56,57]. The extensive biological investment in this reac- tion reflects the extreme chemical stability of alkylphosphonates, that are known to be stable to refluxing conditions in an acid or base [58]. However, methylphosphonate is now known to be highly abundant in marine environments where phosphate is scarce [59]. This has presumably driven the evolution of this complex biochemical machinery to bio- degrade alkyl phosphonates for the release of the inorganic phosphate needed for cellular metabolism. The complexity of the physiology is underscored by the discovery that 14 gene products are encoded by the C–P lyase operon [60]. As shown in Figure 2C, the ulti- mate cleavage step requires ATP, is activated by a series of enzymatic steps and is ulti- mately driven by the stable formation of a cyclic phosphodiester with concomitant C–P bond breakage [56,57]. Figure 3. Examples of metabolic activation in microbial degradation pathways. (A) A major aerobic bacterial pathway for metabolizing benzenoid rings requires energy input to activate molecular ox- ygen for attack on the ring. The activated ring is then cleaved and fragments are assimilated in subsequent metabolism. (B) 4-Chlorobenzoate is activated for dichlorination via an initial energy- requiring thioesterification of the carboxylate moiety via coenzyme A. (C) The C–P bond of al- kylphosphonates is relatively unreactive and is activated using ATP to accomplish C–P bond cleav- age to release inorganic phosphate in phosphorus-poor environments. Other examples of metabolic activation to initiate biodegradation have been detailed in peer-reviewed literature [61,62]. Additionally, the present author perused the EAWAG- Biocatalysis/Biodegradation Database compilation of 219 biodegradation pathways [63] and found that ~2/3 showed an activation reaction(s) as initiating the pathway. Activation reactions are also apparent in the order of rules applied during biodegradation predic- tions [64] and in pesticide degradation records as captured on EnviPath [65]. Most com- pounds, for which biodegradation does not initiate with an activating reaction, are esters and amides that undergo simple hydrolysis reactions. For the majority of other metabo- lism requiring initial activations, oxygenase and CoA-ligase reactions are common. Other Microorganisms 2022, 10, 1664 7 of 13 activating reactions include azo and nitro group reduction, phosphorylation of phenol during anaerobic biodegradation, aryl and aliphatic carboxylation and anaerobic ethylbenzene dehydrogenation. The EAWAG-BBD lacks information on the biodegradation of PFCs by specific mi- crobes and enzymes, and it does not predict defluorination reactions [66]. By contrast, the database contains information on more than twenty dechlorinating enzymes, some of which have available X-ray structures. Polychlorinated natural products are abundant in several environments, indicating millions of years of exposure to the biosphere, and some microorganisms have evolved to use them for their carbon and energy requirements [67]. This sometimes necessitates metabolic activation of the type highlighted in Figure 2B. Metabolic activation, by definition, requires multiply coordinated enzyme reactions that, in turn, requires multiple genes to be expressed simultaneously. This can be accomplished via operonic gene organization, such as for the 4-chlorobenzoate pathway (Figure 2B), or at least constitutive gene expression. The latter was observed for the atrazine-degradation plasmid in Pseudomonas sp. ADP in which a dechlorinase, and two subsequent enzymes in the pathway are encoded by non-regulated, dispersed genes flanked by transposons that show evidence of evolutionarily recent insertion into the plasmid [68]. Similar to the metabolism of recently introduced herbicides like atrazine, the biodegradation of PFCs is likely at an early stage of evolution. However, the development of biodegradative metab- olism is rendered more difficult by the significantly greater strength of the C–F bond com- pared to the C–Cl bond, and so metabolic activation will more often be required to biode- grade PFCs. 6. Metabolic Activation of Trifluoromethyl Arenes for Indirect C–F Bond Cleavage Evolution is at an immature stage with respect to the biodegradation of polyfluori- nated compounds (PFCs), and in-depth mechanistic reports are lacking. This review draws on our limited knowledge of microbial PFC metabolism, and a more extensive knowledge of PFC chemistry. The first example given is for trifluoromethyl groups on benzenoid aromatic rings. Trifluoromethyl arenes are common moieties in many agrichemicals and pharma- ceuticals. One example, the antidepressant fluoxetine, more commonly known as Prozac, had already amassed more than $22 billion USD in sales by 2005 [69] and is increasingly detected in surface waters [70]. In 2010, 30% of blockbuster pharmaceuticals were fluori- nated, most of them aromatic [71], and thirteen new fluorinated drugs were approved in 2020 alone by the United States Food and Drug Administration [72]. Despite the signifi- cance and increasing emergence of these chemicals, studies on their mechanisms of bio- degradation are limited. In that context, this class serves as a major example of how bio- degradation is most likely to proceed via metabolic activation to labilize the C–F bond, rather than carrying out a direct C–F bond cleavage on an unaltered pesticide or pharma- ceutical. It is instructive here to compare chemical and microbiological transformations of (tri- fluoromethyl)benzene, also known as benzotrifluoride (Figure 4). For example, the reduc- tion potential for the direct C–F bond cleavage of benzotrifluoride, even with activating para-substituents, was below −1170 millivolts in all cases [73]. That potential is on the order of 500 millivolts below the lowest potential reported for a biological redox catalyst [29]. Yet, benzotrifluoride and their derivatives are reported to undergo defluorination by nu- merous microbes [74–77]. The mechanism is indirect, with metabolic activation occurring prior to C–F bond cleavage. The activation consists of the addition of oxygen to the ring that generates a low pKa phenolic hydroxyl group ortho to the trifluoromethyl group, with which it acts synergistically (Figure 4). Facile spontaneous deprotonation of the ortho-phe- nolic group leads to double bond rearrangement to displace a fluoride anion and generate an ortho-quinone methide. The quinone methide is highly reactive to nucleophilic attack at the exocyclic double bond that can react with water in the aqueous environment of a Microorganisms 2022, 10, 1664 8 of 13 bacterial cell to produce a gem-difluoro-benzyl alcohol. Further spontaneous defluorina- tion in water produces a non-fluorinated benzoic acid. Defluorination occurrence with microbially-produced trifluoromethyl catechols was first described in 1982 [74]. It has been described in cultures of Rhodococcus rubropertinctus N657 [75] and Pseudomonas putida F1 [77]. The defluorination in wild-type aromatic-de- grading bacteria was dependent upon the inability of a catechol dioxygenase enzyme to process the trifluoromethyl catechol, but a higher yield could be accomplished in a recom- binant Escherichia coli expressing toluene dioxygenase (TodABC1C2) and cis-dihydrodiol dehydrogenase (TodD) [77]. The defluorination is sufficiently facile to occur at neutral pH and 25 °C, suggestive that an evolved enzyme could catalyze highly efficient defluorina- tion by facilitating ortho-hydroxyl deprotonation and adding water to the quinone methide. Figure 4. Metabolic activation of benzotrifluoride leading to complete defluorination of the trifluo- romethyl group. (A) The direct cleavage of the C–F bond by a reductive mechanism is unlikely. (B) Dihydroxylation of the ring produces a phenolate anion at neutral pH that undergoes ring electron rearrangement that displaces one HF equivalent and produces an ortho-quinone methide. The latter may then undergo facile hydrolysis and complete defluorination. 7. Metabolic Activation of Chlorofluorocarbons for Indirect C–F Bond Cleavage The relative weakness of the C–Cl bond compared to the C–F bond provides a suita- ble weak entry point for metabolic activation, leading to defluorination. As an example, CF4 is highly inert but the refrigerant Freon-11, or trichlorofluoromethane was shown to undergo biotransformation via the heme enzyme cytochrome P450cam from the bacterium Pseudomonas putida G7 [78]. Driven by NADH, the enzyme catalyzed an overall two-elec- tron reduction to generate a dichlorofluoromethyl carbanion intermediate that rapidly and spontaneously eliminated a chloride anion (Figure 5A). The resultant fluorochloro- carbene is highly reactive with water and the expected defluorinated product, carbon monoxide, was demonstrated [78]. More recently, an anaerobic microbial-activated sludge community was tested with twelve chlorofluoro carboxylic acids from two to nine carbon atoms, and different ratios of chlorine to fluorine [79]. The reactivities leading to defluorination are illustrated with dichlorofluoroacetate (Figure 5B). Based on products detected, both reductive and hydro- lytic dechlorination were indicated and led to defluorination of the C2 carboxylic acid, producing end products that could conceivably serve as growth substrates for bacteria. Microorganisms 2022, 10, 1664 9 of 13 Figure 5. Metabolic activation in microbial defluorination. (A) Reductive dichlorination leading to ready defluorination catalyzed by the enzyme cytochrome P450cam from Pseudomonas putida G7. (B) Reductive and hydrolytic declorination catalyzed by a microbial community leading to defluorina- tion. (C) Fluoroolefin epoxidation leading to defluorination catalyzed by soluble methane monoox- ygenase from Methylosinus trichosporium OB3b. (D) Unusual displacement of a trifluoromethyl group from a polyfluorinated olefin by a microbial community, mechanism unknown. 8. Metabolic Activation of Fluoroolefins for Indirect C–F Bond Cleavage Electron-withdrawing fluorine substituents on carbon–carbon double bonds render the double bond more susceptible to nucleophilic attack [80]. For example, perfluoroiso- butene is so reactive with nucleophiles that it has been reported to react with solvents that are considered fairly inert, such as N,N-dimethylformamide [81]. Moreover, fluorine at- oms, being smaller than chlorine or methyl group substituents, offer less steric hindrance to reactions with oxidizing agents, although reactions with electrophilic reagents are typ- ically slowed. Tetrafluoroethylene is reported to react slowly but spontaneously with ox- ygen at low temperatures to give tetrafluoroethylene epoxide and uncharacterized poly- meric materials [82]. Therefore, it is not surprising that an enzyme generating a highly reactive oxygen species [83] could react with a fluorinated ethylene. Trifluoroethylene was observed to react with the soluble methane monooxygenase from Methylosinus tricho- sporium OB3b, albeit at only 8% the rate observed with ethylene [84] (Figure 5C); novel reactions can be expected. A recent report described trifluoromethyl group elimination from a highly fluorinated olefin by an aerobic activated sludge community (Figure 5D) [85]. While single elimination from such trifluoromethylated olefins are well-known in the organic literature [86], the trifluoromethyl group elimination appears novel. Moreo- ver, complete defluorination to small organic compounds was reported, a harbinger of further interesting microbial physiology. 9. Conclusions In discussing biodegradability and mechanisms of biodegradation, it is preferable to use the term polyfluorinated compounds (PFCs), rather than PFAS, and to describe the structure of the molecule beyond the C–F bond. This review discusses strategies for the biodegradation of substituted polyfluorinated alkanes, alkenes and arenes and specific substituent impacts on C–F bond reactivity. Considering biodegradation broadly, most environmental chemicals require initial metabolic activation prior to cleavage of the molecule into assimilatible fragments that provide benefit to the cell. Metabolic activation is particularly necessary as one considers plausible meachanisms for the biodegradation of PFCs. As the degree of fluorination in- creases, the direct reactivity of C–F bonds generally decreases. However, commercial Microorganisms 2022, 10, 1664 10 of 13 PFCs almost invariably contain functionalities other than C–F bonds. Those accessory functional groups provide the potential for metabolic activation to render C–F bonds weaker and facilitate their cleavage. Typical activating reactions may set up a carbanion, a carbon radical, or facilitate electron flow to a fluorinated carbon by shifting ring currents. These strategies are most likely to prove fruitful for the biodegradation of PFCs in natural and engineered environments. Funding: The author has been supported by the MnDRIVE program on Industry and the Environ- ment at the University of Minnesota. Data Availability Statement: Data discussed in the review are from, and available through, peer- reviewed published literature. Acknowledgments: The author thanks Serina Robinson for critical comments on the manuscript and Jack Richman for helpful discussions on organofluorine chemistry. Conflicts of Interest: The author declares no conflict of interest. References 1. Barnabas, S.J.; Böhme, T.; Boyer, S.K.; Irmer, M.; Ruttkies, C.; Wetherbee, I.; Kondić, T.; Schymanski, E.L.; Weber, L. Extraction of chemical structures from literature and patent documents using open access chemistry toolkits: A case study with PFAS. Digit. Discov. 2022, 1, 490–501. https://doi.org/10.26434/chemrxiv-2022-nmnnd-v2. 2. Evich, M.G.; Davis, M.J.B.; McCord, J.P.; Acrey, B.; Awkerman, J.A.; Knappe, D.R.U.; Lindstrom, A.B.;Speth, T.F.; Tebes-Stevens, C.; Strynar, M.J.; et al. Per- and polyfluoroalkyl substances in the environment. Science 2022, 375, eabg9065. https://doi.org/10.1126/science.abg9065. 3. Ogawa, Y.; Tokunaga, E.; Kobayashi, O.; Hirai, K.; Shibata, N. Current contributions of organofluorine compounds to the agrochemical industry. Iscience 2020, 23, 101467. https://doi.org/10.1016/j.isci.2020.101467. 4. Alexandrino, D.A.; Almeida, C.M.R.; Mucha, A.P.; Carvalho, M.F. Revisiting pesticide pollution: The case of fluorinated pesticides. Environ. Pollut. 2022, 292, 118315. https://doi.org/10.1016/j.envpol.2021.118315. 5. 5. Inoue, M.; Sumii, Y.; Shibata, N. Contribution of organofluorine compounds to pharmaceuticals. ACS Omega, 2020, 5, 10633– 10640. https://doi.org/10.1021/acsomega.0c00830. 6. Liu, J.; Avendaño, S.M. Microbial degradation of polyfluoroalkyl chemicals in the environment: A review. Environ. Int. 2013, 61, 98–114. https://doi.org/10.1016/j.envint.2013.08.022. 7. Ochoa-Herrera, V.; Field, J.A.;Luna-Valesco, A.; Sierra-Alvarez, R. Microbial toxicity and biodegradability of perfluorooctane sulfonate (PFOS) and shorter chain perfluoroalkyl and perfluoroalkyl substances (PFASs). Environ. Sci. Process Impacts., 2016, 18, 1236–1246. https://doi.org/10.1039/c6em00366d. 8. Zhang, W.; Pang, S.; Lin, Z.; Mishra, S.; Bhatt, P.; Chen, S. Biotransformation of perfluoroalkyl acid precursors from various environmental systems: Advances and perspectives. Environ. Pollut. 2021, 272, 115908. https://doi.org/10.1016/j.envpol.2020.115908. 9. Zhang, Z.; Sarkar, D.; Biswas, J.K.; Datta, R. Biodegradation of per- and polyfluoroalkyl substances (PFAS): A review. Bioresour Technol. 2022, 344 Pt B, 126223. https://doi.org/10.1016/j.biortech.2021.126223. 10. Weber, E.J.; Tebes-Steves, C.; Washington, J.W.; Gladstone, R. Development of a PFAS reaction library: Identifying plausible transformation pathways in environmental and biological systems. Environ. Sci. Process Impacts 2022, 25, 689–753. https://doi.org/10.1039/d1em00445j. 11. Carvalho, M.F.; Alves, C.C.; Ferreira, M.I.; De Marco, P.; Castro, P.M. Isolation and initial characterization of a bacterial consortium able to mineralize fluorobenzene. Appl. Environ. Microbiol. 2022, 68, 102–105. https://doi.org/10.1128/AEM.68.1.102- 105.2002. 12. Finkelstein, Z.I.; Baskunov, B.P.; Boerrsma, M.G.; Vervoot, J.; Golovlev, E.L.; van Berkel, W.J.; Govleva, L.A.; Riietjens, I.M. Identification of fluoropyrogallols as new intermediates in biotransformation of monofluorophenols in Rhodococcus opacus 1cp. Appl. Environ. Microbiol. 2000, 66, 2148–2153. https://doi.org/10.1128/AEM.66.5.2148-2153.2000. 13. Vargas, C.; Song, B.; Camps, M.; Häggblom, M.M. Anaerobic degradation of fluorinated aromatic compounds. Appl. Microbiol. Biotechnol. 2000, 53, 342–347. https://doi.org/10.1007/s002530050032. 14. Turner, A.M.; Abplanalp, M.J.; Bergantini, A.; Frigge, R.; Zhu, C.; Sun, B.J.; Hsiao, C.T.; Chang, A.H.; Meinert, C.; Kaiser, R.I. Sci. Adv. 2019, 5, eaaw4307. https://doi.org/10.1125/sciadv.aaw4307. 15. Wackett, L.P.; Robinson, S.L. The ever-expanding limits of enzyme catalysis and biodegradation: Polyaromatic, polychlorinated, polyfluorinated, and polymeric compounds. Biochem. J. 2020, 477, 2875–2891. https://doi.org/10.1042/BCJ20190720. 16. Harper, D.B.; O’Hagan, D.; Murphy, C.D. Fluorinated natural products: Occurrence and biosynthesis. Nat. Prod. Organohalogen Compd. 2003, 141–169. https://doi.org/10.1007/b10454. 17. Walker, M.C.; Chang, M.C. Natural and engineered biosynthesis of fluorinated natural products. Chem. Soc. Rev. 2014, 43, 6527– 6536. https://doi.org/10.1039/c4cs00027g. Microorganisms 2022, 10, 1664 11 of 13 18. Gribble, G.W. A recent survey of naturally occurring organohalogen compounds. Environ. Chem. 2015, 12, 396–405. https://doi.org/10.1071/EN15002. 19. Rosner, D.; Markowitz, G. Persistent pollutants: A brief history of the discovery of the widespread toxicity of chlorinated hydrocarbons. Environ. Res. 2013, 120, 126–133. https://doi.org/10.1016/j.envres.2012.08.011. 20. Wang, Z.; Buser, A.M.; Cousins, I.T.; Demattio, S.; Drost, W.; Johansson, O.; Ohno, K.; Patlewicz, G.; Richard, A.M.; Walker, G.W.; et al. A new OECD definition for per-and polyfluoroalkyl substances. Environ. Sci. Technol. 2021, 55, 15575–15578. https://doi.org/10.1021/acs.est.1c06896. 21. Freire, M.G.; Gomes, L.; Santos, L.M.; Marrucho, I.M.; Coutinho, J.A. Water solubility in linear fluoroalkanes used in blood substitute formulations. J. Phys. Chem. B 2006, 110, 22923–22929. https://doi.org/10.1021/jp0622942. 22. Kim, S.; Chen, J.; Cheng, T.; Gindulyte, A.; He, J.; He, S.; Li, Q.; Shoemaker, B.A.; Thiessem, P.A.; Yu, B.; et al. PubChem in 2021: New data content and improved web interfaces. Nucleic Acids Res. 2021, 49, D1388–D1395. https://doi.org/10.1093/nar/gkaa971. 23. CDC, Per- and Polyfluorinated Substances (PFAS) Factsheet. Available online: https://www.cdc.gov/biomonitoring/PFAS_FactSheet.html (accessed on 6 July 2022). 24. Wallington, T.J.; Andersen, M.S.; Nielsen, O.J. The case for a more precise definition of regulated PFAS. Env. Sci. Proc. Impacts 2021, 23, 1834–1838. https://doi.org/10.1039/d1em00296a. 25. Hammel, E.; Webster, T.F.; Gurney, R.; Heiger-Bernays, W. Implications of PFAS definitions using fluorinated pharmaceuticals. Iscience 2022, 25, 104020. https://doi.org/10.1016/j.isci.2022.104020. 26. Pud, A.A.; Shapoval, G.S.; Kukhar, V.P.; Mikulina, O.E.; Gervits, L.L. Electrochemical reduction of some saturated and unsaturated perfluorocarbons. Electrochim. Acta 1995, 40, 1157–1164. https://doi.org/10.1016/0013-4686(95)00030-I. 27. Combellas, C.; Kanoufi, F.; Thiebault, A. Reduction of polyfluorinated compounds. J. Phys. Chem. B 2003, 39, 10894–10905. https://doi.org/10.1021/jp034846b. 28. Houmam, A. Electron transfer initiated reactions: Bond formation and bond dissociation. Chem. Rev. 2008, 108, 2180–2237. https://doi.org/10.1021/cr068070x. 29. Huwiler, S.G.; Löffler, C.; Anselmann, S.E.; Stärk, H.J.; von Bergen, M.; Flechsler, J.; Rachel, R.; Boll, M. One-megadalton metalloenzyme complex in Geobacter metallireducens involved in benzene ring reduction beyond the biological redox window. Proc. Natl. Acad. Sci. USA 2019, 116, 2259–2264. https://doi.org/10.1073/pnas.1819636116. 30. Kim, M.H.; Wang, N.; Chu, K.H. 6:2 Fluorotelomer alcohol (6:2 FTOH) biodegradation by multiple microbial species under different physiological conditions. Appl. Microbiol. Biotechnol. 2014, 98, 1831–1840.. https://doi.org/10.1007/s00253-013-5131-3. 31. Shaw, D.M.; Munoz, G.; Bottos, E.M.; Duy, S.V.; Sauvé, S.; Liu, J.; Van Hamme, J.D. Degradation and defluorination of 6: 2 fluorotelomer sulfonamidoalkyl betaine and 6: 2 fluorotelomer sulfonate by Gordonia sp. strain NB4-1Y under sulfur-limiting conditions. Sci.Total Environ. 2019, 647, 690–698. https://doi.org/10.1016/j.scitotenv.2018.08.012. 32. Goldman, P. The enzymatic cleavage of the carbon-fluorine bond in fluoroacetate. J. Biol. Chem. 1965, 240, 3434–3438. https://doi.org/10.1016/S0021-9258(18)973236-4. 33. Liu, J.Q.; Kurihara, T.; Ichiyama, S.; Miyagi, M.; Tsunasawa, S.; Kawasaki, H.; Soda, K.; Esaki, N. Reaction mechanism of fluoroacetate dehalogenase from Moraxella sp. B. J. Biol. Chem. 1998, 273, 30897–30902. https://doi.org/10.1074/jbc.273.47.30897. 34. 34. Kurihara, T.; Yamauchi, T.; Ichiyama, S.; Takahata, H.; Esaki, N. Purification, characterization, and gene cloning of a novel fluoroacetate dehalogenase from Burkholderia sp. FA1. J. Mol. Catal. B Enzym. 2003, 23, 347–355. https://doi.org/10.1016/S1381- 1177(03)00098-5. 35. Donnelly, C.; Murphy, C.D. Purification and properties of fluoroacetate dehalogenase from Pseudomonas fluorescens DSM 8341. Biotechnol. Lett. 2009, 31, 245–250. https://doi.org/10.1007/s10529-008-9849-4. 36. 36. Davis, C.K.; Webb, R.I.; Sly, L.I.; Denman, S.E.; McSweeney, C.S. Isolation and survey of novel fluoroacetate-degrading bacteria belonging to the phylum Synergistetes. FEMS Microbiol. Ecol. 2012, 80, 671–684. https://doi.org/10.1111/j.1574- 6941.2012.01338.x. 37. Camboim, E.K.; Almeida, A.P.; Tadra-Sfeir, M.Z.; Junior, F.G.; Andrade, P.P.; McSweeney, C.S.; Melo, M.A.; Riet-Correa, F. Isolation and identification of sodium fluoroacetate degrading bacteria from caprine rumen in Brazil. Sci. World J. 2012, 2012, 178254. https://doi.org/10.1100/2012/178254. 38. Chan, W.Y.; Wong, M.; Guthrie, J.; Savchenko, A.V.; Yakunin, A.F.; Pai, E.F.; Edwards, E.A. Sequence-and activity-based screening of microbial genomes for novel dehalogenases. Microb. Biotechnol. 2010, 3, 107–120. https://doi.org/10.1111/j.1751- 7915.2009.00155.x. 39. Chan, P.W.; Yakunin, A.F.; Edwards, E.A.; Pai, E.F. Mapping the reaction coordinates of enzymatic defluorination. J. Am. Chem. Soc. 2011, 133, 7461–7468. https://doi.org/10.1021/ja200277d. 40. Jitsumori, K.; Omi, R.; Kurihara, T.; Kurata, A.; Mihara, H.; Miyahara, I.; Hirotsu, K.; Esaki, N. X-Ray crystallographic and mutational studies of fluoroacetate dehalogenase from Burkholderia sp. strain FA1. J. Bacteriol. 2009, 191, 2630–2637. https://doi.org/10.1128/JB.01654-08. 41. Kamachi, T.; Nakayama, T.; Shitamichi, O.; Jitsumori, K.; Kurihara, T.; Esaki, N.; Yoshizawa, K. The catalytic mechanism of fluoroacetate dehalogenase: A computational exploration of biological dehalogenation. Chemistry 2009, 15, 7394–7403.. https://doi.org/10.1002/chem.200801813. 42. Miranda-Rojas, S.; Fernández, I.; Kästner, J.; Toro-Labbé, A.; Mendizabal, F. Unraveling the nature of the catalytic power of fluoroacetate dehalogenase. ChemCatChem 2018, 10, 1052–1063. https://doi.org/10.1002/cctc.201701517. Microorganisms 2022, 10, 1664 12 of 13 43. Kang, H.; Zheng, M. Influence of the quantum mechanical region size in QM/MM modelling: A case study of fluoroacetate dehalogenase catalyzed CF bond cleavage. Comp. Theor. Chem. 2021, 1204, 113399. https://doi.org/10.1016/j.comptc.2021.113399. 44. Alexandrino, D.A.M.; Ribeiro, I.; Pinto, L.M.; Cambra, R.; Oliveira, R.S.; Pereira, F.; Carvalho, M.F. Biodegradation of mono-, di- and trifluoroacetate by microbial cultures with different origins. N. Biotechnol. 2018, 43, 23–29. https://doi.org/10.1016/j.nbt.2017.08.005. 45. Dolbier, W.R. Fluorine chemistry at the millenium. J. Fluor. Chem. 2005, 126, 157–163. https://doi.org/10.1016/j.jfluchem.2004.09.033. 46. O’Hagan, D. Understanding organofluorine chemistry. An introduction to the C-F bond. Chem. Soc. Rev. 2008, 37, 308–319. https://doi.org/10.1039/b711844a. 47. Chan, P.W.; Chakrabarti, N.; Ing, C.; Halgas, O.; To, T.K.; Wälti, M.; Petit, A.P.; Tran, C.; Savchenko, A.; Yakunin, A.F.; et al. Defluorination capability of l-2-haloacid dehalogenases in the HAD-like hydrolase superfamily correlates with active site compactness. ChemBioChem 2022, 23, e202100414. https://doi.org/10.1002/cbic.202100414. 48. Sela, I.; Wolf, Y.I.; Koonin, E.V. Theory of prokaryotic genome evolution. Proc. Natl. Acad. Sci. USA 2016, 113, 11399–11407. https://doi.org/10.1073/pnas.1614083113. 49. Wackett, L.P. Evolution of enzymes for the metabolism of new chemical inputs into the environment. J. Biol. Chem. 2004, 279, 41259–41262. https://doi.org/10.1074/jbc.R400014200. 50. Davidi, D.; Longo, L.M.; Jabłońska, J.; Milo, R.; Tawfik, D.S. A bird’s-eye view of enzyme evolution: Chemical, physicochemical, and physiological considerations. Chem. Rev. 2018, 118, 8786–8797. https://doi.org/10.1021/acs.chemrev.8b00039. 51. Copley, S.D. Setting the stage for evolution of a new enzyme. Curr. Opin. Struct. Biol. 2021, 69, 41–49. https://doi.org/10.1016/j.sbi.2021.03.001. 52. Gibson, D.T.; Parales, R.E. Aromatic hydrocarbon dioxygenases in environmental biotechnology. Curr. Opin. Biotechnol. 2000, 11, 236–243.. https://doi.org/10.1016/s0958-1669(00)00090-2. 53. Dunaway-Mariano, D.; Babbitt, P.C. On the origins and functions of the enzymes of the 4-chlorobenzoate to 4- hydroxybenzoate converting pathway. Biodegradation 1994, 5, 259–276. https://doi.org/10.1007/BF00696464. 54. Bunnett, J.F.; Morath, R.J.; Okamoto, T. The ortho:para ratio in activation of aromatic nucleophilic substitution by the carboxylate group. J. Am. Chem. Soc. 1955, 77, 5055–5057. https://doi.org/10.1021/ja01624a034. 55. Terrier, F. Modern Nucleophilic Aromatic Substitution; John Wiley & Sons: New York, NY, USA, 2013. 56. Kamat, S.S.; Raushel, F.M. The enzymatic conversion of phosphonates to phosphate by bacteria. Curr. Opin. Chem. Biol. 2013, 17, 589–596. https://doi.org/10.1016/j.cbpa.2013.06.006. 57. Yang, K.; Ren, Z.; Raushel, F.M.; Zhang, J. Structures of the carbon-phosphorus lyase complex reveal the binding mode of the nbd-like phnk. Structure 2016, 24, 37–42. https://doi.org/10.1016/j.str.2015.11.009. 58. Hoskin, F.C.G. Some observations concenring the biochemical inertness of methylphosphonic and isopropyl methylphosphonic acids. Can. J. Biochem. Physiol. 1955, 34, 743–746. https://doi.org/10.1139/o56-077. 59. Metcalf, W.W.; Griffin, B.M.; Cicchillo, R.M.; Gao, J.; Janga, S.C.; Cooke, H.A.; Circello, B.T.; Evans, B.S.; Martens-Habbena, W.; Stahl, D.A.; et al. Synthesis of methylphosphonic acid by marine microbes: A source for methane in the aerobic ocean. Science 2012, 337, 1104–1107. https://doi.org/10.1126/science.1219875. 60. Chen, C.M.; Ye, Q.Z.; Zhu, Z.M.; Wanner, B.L.; Walsh, C.T. Molecular biology of carbon-phosphorus bond cleavage. Cloning and sequencing of the phn (psiD) genes involved in alkylphosphonate uptake and C-P lyase activity in Escherichia coli B. J. Biol. Chem. 1990, 265, 4461–4471. 61. VanBriesen, J.M. Thermodynamic yield predictions for biodegradation through oxygenase activation reactions. Biodegradation 2001, 12, 265–281. https://doi.org/10.1023/a:1013179315518. 62. Coates, J.D.; Chakraborty, R.; McInerney, M.J. Anaerobic benzene biodegradation—a new era. Res. Microbiol. 2002, 153, 621–628. https://doi.org/10.1016/s0923-2508(02)01378-5. 63. Gao, J.; Ellis, L.B.; Wackett, L.P. The University of Minnesota biocatalysis/biodegradation database: Improving public access. Nucleic Acids Res. 2010, 38 (Suppl.1), D488–D491. https://doi.org/10.1093/nar/gkp771. 64. Fenner, K.; Gao, J.; Kramer, S.; Ellis, L.; Wackett, L.P. Data-driven extraction of relative reasoning rules to limit combinatorial explosion in biodegradation pathway prediction. Bioinformatics 2008, 24, 2079–2085. https://doi.org/10.1093/bioinformatics/btn378. 65. Latino, D.A.; Wicker, J.; Gütlein, M.; Schmid, E.; Kramer, S.; Fenner, K. Eawag-Soil in enviPath: A new resource for exploring regulatory pesticide soil biodegradation pathways and half-life data. Environ. Sci. Proc. Impacts 2017, 19, 449–464. https://doi.org/10.1039/c6em00697c. 66. Gao, J.; Ellis, L.B.; Wackett, L.P. The University of Minnesota Pathway Prediction System: Multi-level prediction and visualization. Nucleic Acids Res. 2011, 39, W406–W411. https://doi.org/10.1093/nar/gkr200. 67. Holliger, C.; Wohlfarth, G.; Diekert, G. Reductive dechlorination in the energy metabolism of anaerobic bacteria. FEMS Microbiol. Rev. 1998, 22, 383–398. https://doi.org/10.1111/j.1574-6976.1998.tb00377.x. 68. Martinez, B.; Tomkins, J.; Wackett, L.P.; Wing, R.; Sadowsky, M.J. Complete nucleotide sequence and organization of the atrazine catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP. J. Bacteriol. 2001, 183, 5684–5697. https://doi.org/10.1128/JB.183.19.5684-5697.2001. 69. Wenthur, C.J.; Bennett, M.R.; Lindsley, C.W. Classics in Chemical Neuroscience: Fluoxetine (Prozac). ACS Chem. Neurosci. 2013, 5, 14–23. https://doi.org/10.1021/cn400186j. Microorganisms 2022, 10, 1664 13 of 13 70. Martin, J.M.; Bertram, M.G.; Saaristo, M.; Fursdon, J.B.; Hannington, S.L.; Brooks, B.W.; Burket, S.R.; Mole, R.A.; Deal, N.D.; Wong, B.B. 2019. Antidepressants in surface waters: Fluoxetine influences mosquitofish anxiety-related behavior at environmentally relevant levels. Environ. Sci. Technol. 2019, 53, 6035–6043. https://doi.org/10.1021/acs.est.9b00944. 71. O’Hagan, D. Fluorine in health care: Organofluorine containing blockbuster drugs. J. Fluor. Chem. 2010, 131, 1071–1081. https://doi.org/10.1016/j.fluchem.2010.03.003. 72. Yu, Y.; Liu, A.; Dhawan, G.; Mei, H.; Zhang, W.; Izawa, K.; Soloshonok, V.A.; Han, J. Fluorine-containing pharmaceuticals approved by the FDA in 2020: Synthesis and biological activity. Chin. Chem. Lett. 2021, 32, 3342–335.. https://doi.org/10.1016/j.cclet.2021.05.042. 73. Combellas, C.; Kanoufi, F.; Thiébault, A. Reducibility of the carbon-fluorine bond in the trifluoromethyl group. J. Electroanal. Chem. 1996, 407, 195–202. https://doi.org/10.1016/0022-0728(95)04474-4. 74. Engesser, K.H. Der Einfluss der Trifluormethylgruppe auf die Biologische Abbaubarkeit von Aromaten. Doctoral Dissertation, University of Göttingen, Göttingen, Germany, 1982. 75. Engesser, K.H.; Cain, R.B.; Knackmuss, H.J. Bacterial metabolism of side chain fluorinated aromatics: Cometabolism of 3- trifluoromethyl(TFM)-benzoate by Pseudomonas putida (arvilla) mt-2 and Rhodococcus rubropertinctus N657. Arch. Microbiol. 1988, 149, 188–197. https://doi.org/10.1007/BF00422004. 76. Kiel, M.; Engesser, K.H. The biodegradation vs. biotransformation of fluorosubstituted aromatics. Appl. Microbiol. Biotechnol. 2015, 99, 7433–7464. https://doi.org/10.1007/s00253-015-6817-5. 77. Bygd, M.D.; Aukema, K.G.; Richman, J.E.; Wackett, L.P. Microwell fluoride screen for chemical, enzymatic, and cellular reactions reveals latent microbial defluorination capacity for -CF3 groups. Appl. Environ. Microbiol. 2022, 88, e00288-22. https://doi.org/10.1128/aem.00288-22. 78. Li, S.; Wackett, L.P. Reductive dehalogenation by cytochrome P450CAM: Substrate binding and catalysis. Biochemistry 1993, 32, 9355–9361. https://doi.org/10.1021/bi00087a014. 79. Jin, B.; Che, S.; Gao, J.; Yu, Y.; Liu, J.; Men, Y. Anaerobic defluorination of chlorine- substituted per- and polyfluorinated carboxylic acids triggered by microbial dechlorination. ChemRxiv 2022. https://doi.org/10.26434/chemrxiv-2022-44rx6. 80. Timperley, C.M.; Waters, M.J.; Greenall, J.A. Fluoroalkene chemistry: Part 3. Reactions of arylthiols with perfluoroisobutene, perfluoropropene and chlorotrifluoroethene. J. Fluor. Chem. 2006, 127, 249–256. https://doi.org/10.106/j.fluchem.2005.11.008. 81. Zeifman, Y.V.; Ter-Gabrielyan, E.G.; Gambaryan, N.P.; Knunyants, I.L. The chemistry of perfluoroisobutene. Russ. Chem Rev. 1984, 53, 256. 82. Gozzo, F.; Camaggi, G. Oxidation reactions of tetrafluoroethylene and their products—I: Auto-oxidation. Tetrahedron 1996, 22, 1765–1770. https://doi.org/10.1016/S0040-4020(01)82248-1. 83. Wallar, B.J.; Lipscomb, J.D. Dioxygen activation by enzymes containing binuclear non-heme iron clusters. Chem. Rev. 1996, 96, 2625–2658. https://doi.org/10.1021/cr9500489. 84. Fox, B.; Borneman, J.G.; Wackett, L.P.; Lipscomb, J.D. Haloalkene oxidation by the soluble methane monooxygenase from Methylosinus trichosporium OB3b: Mechanistic and environmental implications. Biochemistry 1990, 29, 6419–6427. https://doi.org/10.1021/bi00479a013. 85. Yu, Y.; Che, S.; Ren, C.; Bosen, J.; Zhenyu, T.; Liu, J.; Men, Y. Microbial defluorination of unsaturated per- and polyfluorinated carboxylic acids under anaerobic and aerobic conditions: A structure specificity study. Environ. Sci. Technol. 2022, 56, 4894–4904. https://doi.org/10.1021/acs.est.1c05509. 86. Liu, Y.; Zhou, Y.; Zhao, Y.; Qu, J. Synthesis of gem-difluoroallylboronates via FeCl2-catalyzed boration/β-fluorine elimination of trifluoromethyl alkenes. Org. Lett. 2017, 19, 946–949. https://doi.org/10.1021/acs.orglett.7b00168. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Microorganisms Multidisciplinary Digital Publishing Institute

Strategies for the Biodegradation of Polyfluorinated Compounds

Wackett, Lawrence P.
Microorganisms , Volume 10 (8) – Aug 17, 2022
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Abstract

Review Strategies for the Biodegradation of Polyfluorinated Compounds Lawrence P. Wackett Department of Biochemistry, Molecular Biology and Biophysics and BioTechnology Institute, University of Minnesota, Minneapolis, MN 55455, USA; [email protected] Abstract: Many cite the strength of C–F bonds for the poor microbial biodegradability of polyfluor- inated organic compounds (PFCs). However, commercial PFCs almost invariably contain more functionality than fluorine. The additional functionality provides a weak entry point for reactions that activate C–F bonds and lead to their eventual cleavage. This metabolic activation strategy is common in microbial biodegradation pathways and is observed with aromatic hydrocarbons, chlo- rinated compounds, phosphonates and many other compounds. Initial metabolic activation pre- cedes critical bond breakage and assimilation of nutrients. A similar strategy with commercial PFCs proceeds via initial attack at the non-fluorinated functionalities: sulfonates, carboxylates, chlorines, phenyl rings, or phosphonates. Metabolic transformation of these non-fluorinated groups can acti- vate the C–F bonds, allowing more facile cleavage than a direct attack on the C–F bonds. Given that virtually all compounds denoted as “PFAS” are not perfluorinated and are not alkanes, it is posited here that considering their individual chemical classes is more useful for both chemical and micro- biological considerations of their fate. Keywords: polyfluorinated; PFAS; biodegradation; bacteria; enzyme; metabolism; metabolic activation; weak bonds; defluorination Citation: Wackett, L.P. Strategies for the Biodegradation of Polyfluorinated Compounds. Microorganisms 2022, 10, 1664. https://doi.org/10.3390/ 1. Introduction microorganisms10081664 In 2022, a comprehensive search of all public sources identified more than nineteen Academic Editors: Maria De Fátima million fluorinated chemicals, almost all of which have been created by humans within Carvalho and Diogo Alexandrino the last century [1]. Commercial polyfluorinated organic compounds (PFCs) are used in more than two hundred different applications [2]. Between 1998 and 2020, more than half Received: 13 July 2022 of all International Organization for Standardization (ISO) approved agrichemicals were Accepted: 16 August 2022 fluorinated [3]. More than 20% of new pharmaceuticals are fluorinated, mostly aromatic Published: 17 August 2022 ring compounds [4,5]. While regulatory scrutiny is increasing, the vast majority of these Publisher’s Note: MDPI stays neu- fluorinated compounds remain unregulated and a knowledge of their biodegradation, tral with regard to jurisdictional and hence longevity in the environment, is widely lacking. claims in published maps and institu- In light of the general lack of information on PFC biodegradation, this review draws tional affiliations. on broad concepts of biodegradation to posit how PFCs might be transformed by natural and engineered microbes. As such, the review combines relevant literature on how mi- crobes activate difficult-to-cleave bonds with insights from the field of organic chemistry regarding carbon–fluorine bond cleavage mechanisms. Examples with relevant references Copyright: © 2022 by the author. will be provided throughout the review. The present compilation differs from previous Licensee MDPI, Basel, Switzerland. PFC biodegradation reviews that focus on a limited set of the >19 million fluorinated com- This article is an open access article pounds known. For example, previous reviews have covered fluoroalkyl acids and distributed under the terms and fluorotelomers, such as those used in firefighting foams [6–10]. However, there are mul- conditions of the Creative Commons Attribution (CC BY) license tiple classes of PFCs for which documented biodegradative metabolism is lacking. This (https://creativecommons.org/license contribution focuses on potential mechanisms by which these PFCs may be transformed s/by/4.0/). by microbes based on general principles of biodegradation and organofluorine chemistry. Microorganisms 2022, 10, 1664. https://doi.org/10.3390/microorganisms10081664 www.mdpi.com/journal/microorganisms Microorganisms 2022, 10, 1664 2 of 13 The vast majority, more than eighteen million, of anthropogenic fluorine compounds are aromatic [1]. While many of the commercial agrichemicals consist of multiply fluori- nated aromatic ring compounds, most biodegradation studies with fluoroaromatics were with monofluorinated compounds [11–13]. Much remains to be learned about the micro- bial mechanisms by which polyfluorinated aromatic, olefinic and alkyl compounds are biodegraded. In many of the metabolic pathways for PFCs that exist or may yet evolve, this author suggests that direct C–F bond cleavage is unlikely to occur. Instead, metabolic activation of adjacent atoms can activate the C–F bond to make biodegradation feasible. This concept is documented in literature from the chemical sciences on C–F bond activa- tion, and in literature on the activation and biodegradation of other very strong chemical bonds, such as the C–P bond of alkylphosphonic acids [14]. This author is unaware of other review articles on PFCs having this focus. Some of the steps for the metabolic activation of PFCs may take time to evolve given that no natural product PFCs are known to humans, and so may not have entered the biosphere until very recently. This contrasts with most functional groups found in com- mercial chemicals that also occur in natural products that have likely existed for eons [15]. Fluorinated natural products are rare (only several dozen have been identified) and they are all monofluorinated compounds [16,17]. There are no polyfluorinated natural prod- ucts known at this time. This contrasts with the synthetic and commercial production of fluorinated compounds, many of which are PFCs [1]. At one time, polychlorinated com- pounds were considered foreign to the biosphere, but over the past several decades, more than five thousand chlorinated natural products have been identified, many of which are polychlorinated [18]. Industry produced many chlorinated pesticides in the early to mid- 20th century to slow their biodegradation in the field [19], but has now largely shifted over to PFCs [4]. The sheer number of PFCs produced, their widespread use and their apparent foreignness to microbes presents a large challenge for biodegradation. It is in this context that all current knowledge of metabolic activation and C–F bond cleavage chemistry must be applied to this problem. 2. Definitions of PFAS and PFCs The term PFAS (per- and polyfluoroalkyl substances) is used to refer to many differ- ent classes of PFCs with a wide divergence in their chemical reactivity. The ambiguity of the term has set up an unfortunate fuzziness in thinking about their biodegradation. For example, some compounds that meet the current Organization for Economic Co-opera- tion and Development (OECD) definition of PFAS [20] are very biodegradable, while oth- ers are not. With few exceptions, the compounds referred to as per- and polyfluorinated alkyl substances (PFAS) contain aromatic rings, ether linkages and other functional groups that lead to a very large difference in biodegradability. An example of a perfluor- inated alkane is octadecafluorooctane (perfluorooctane) (Figure 1A, top). Perfluoroal- kanes have limited commercial uses compared to most compounds referred to as PFAS [21]. Virtually all of the regulated and targeted molecules for environmental remediation that are referred to as PFAS on PubChem [22] and other sites, contain functionalities in addition to their C–F bonds. Examples of some highly used and researched compounds are shown below the perfluoroalkane at the top of Figure 1A. If the term PFAS has become too pervasive to avoid it, users should minimally state upfront which definition of PFAS they are using, as discussed in Barnabas, et al. [1]. OECD, the organization representing mostly European countries, has defined PFAS as any compound with at least a -CF3 group, or a -CF2- group that does not have a H/Cl/Br/I atom associated with it [20]. A new United States Environmental Protection Agency defi- nition says that a PFAS is anything that contains (R1)(R2)(F)C–C(R3)F2 [1]. Barnabas and coworkers prefer the following PFAS definition: a compound that contains an (R1)(R2)(F)C–C(R3)F2 group where R groups are any atom except hydrogen and the bond between both aliphatic carbon atoms is a single bond. The Center for Disease Control (CDC) states, “PFAS are a group of chemicals used to make fluoropolymer coatings and Microorganisms 2022, 10, 1664 3 of 13 products that resist heat, oil, stains, grease and water” [23]. Others have also questioned the definitions of PFAS for the difficulties that they pose for regulatory decisions [24]. Moreover, it has been pointed out that the currently marketed fluorinated pharmaceuti- cals are classified as 1% being PFAS by one definition, and as 100% being PFAS by another definition [25]. Figure 1. Polyflourinated organic compounds (PFCs) and mechanisms for their defluorination. (A) A perfluorinated alkane (top) and commercial PFCs showing the types of functionalities typically combined with perfluorinated moieties. (B) Two types of mechanisms by which a functionality ad- jacent to a perfluorinated carbon can be attacked enzymatically leading to facile defluorination. Depending on which definition one might choose, biodegradation of a designated PFAS chemical might be easy or hard, or difficult to pin down if you are talking about the biodegradation of chemicals that “resist heat.” In this review, the term “polyfluorinated organic compounds” is used, abbreviated as PFCs, and defines specific chemical classes in the context of the nuances of their biodegradation. The classes discussed are: perfluoro- alkanes, substituted polyfluorinated alkanes, substituted polyfluorinated alkenes and substituted polyfluorinated aromatics. 3. Broad Principles for the Biodegradation of PFCs Perfluorooctane presents a significant challenge for biodegradation relative to other PFCs (Figure 1). With perfluorooctane, chemical hydrolysis is not observed, and reduction of a C–F bond requires electrochemical potentials below −2000 mV [26]. More recent stud- ies confirm these low potentials as being required [27,28]. The known lower limit for bio- logical reduction potential attained by an enzymatic cofactor is −622 mV [29], making it unlikely that biology will evolve a new cofactor to poise its potential at −2000 mV. With additional functionality attached to a highly fluorinated, even perfluorinated, chain or ring, many other chemical and metabolic options are available to bring about defluorina- tion. For example, the presence of an ether linkage with a C–O carbon containing a non- Microorganisms 2022, 10, 1664 4 of 13 fluorine substituent presents a potential soft spot for enzyme cleavage that could produce a gem-difluoro alcohol that would undergo spontaneous elimination and hydrolysis to release fluoride. Moreover, such a metabolic step potentially sets up the molecule for fur- ther degradation proceeding down the alkyl or alkenyl chain, or into a ring (Figure 1B, top). An ether cleavage that leads to spontaneous defluorination would be plausible, for example, with FRD-903 and F53-B. Similarly, fluorotelomers like the firefighting foam shown in Figure 1A could plausibly be attacked by a lyase catalyzing elimination of fluo- ride (Figure 1B, bottom). The resultant olefin is susceptible to water addition, oxygenation or other reactions to continue metabolism. Indeed, microbial transformations of fluorote- lomers have been described [30,31]. Bacteria carrying out these reactions include Gordonia sp. strain NB4-1Y, Pseudomonas fluorescens DSM 8341, Pseudomonas oleovorans and Myco- bacterium vaccae JOB5. Given that PFCs of the type shown in Figure 1 are very new to the biosphere, there has been little time on an evolutionary scale to put together consecutive reactions of the types shown to make dedicated metabolic pathways. This review discusses general prin- ciples as to how microbes may activate PFCs for defluorination by “attacking the weak points.” Since perfluorooctane and related perfluorinated alkanes are not the subject of any remediation activities this author is aware of, and virtually all molecules of interest have other functional groups, the dominant theme here is to attack non-fluorine function- alities and promote C–F bond cleavage indirectly. This review discusses the potential for this strategy in more detail, but first starts by describing the most well-understood exam- ple of direct enzymatic C–F bond cleavage. 4. Direct Enzymatic C–F Bond Cleavage Has Limitations Numerous bacteria have been isolated that defluorinate the widespread natural product fluoroacetate [32–37], largely mediated by fluoroacetate dehalogenase. This has become the mechanistic paradigm for direct SN2-type defluorination by a microbial en- zyme (Figure 2) [38,39]. There are X-ray structures for fluoroacetate dehalogenases [39,40], and computational chemistry has been applied to better understand the power and limi- tations of the reaction [41–43]. The enzyme uses a carboxylate anion nucleophile. The neg- ative charge on the substrate is shielded by the active site arginine and fluoride anion leaving is facilitated by a nearby triad of tyrosine, histidine and tryptophan residues. Wa- ter resolves the enzyme ester intermediately to produce the glycolic acid product. There is sufficient metabolic energy in glycolic acid to potentially support bacterial growth. Perfluorinated acetic and longer chain carboxylic acids are not known to react read- ily, if at all, with fluoroacetate dehalogenase. Moreover, studies with wastewater micro- organisms showed ready degradation of fluoroacetate, whereas di- and trifluoroacetate were recalcitrant [44]. Carbon–fluorine bond strength is known to increase with increasing fluorine substituents on an aliphatic carbon [45,46]. Moreover, unlike monofluoroacetate, di- and trifluoroacetate moieties are not known to be biosynthesized or found in natural products, hence microbial exposure to them has only occurred in recent history. Although fluoroacetate dehalogenase is a powerful enzyme, uniquely poised for direct defluorina- tion [47], its limitations highlight the need to pursue different mechanistic strategies for biodegrading polyfluorinated compounds. Given the observed hurdles in direct cleavage of unactivated C–F bonds, metabolic activation will likely be necessary to biodegrade many PFCs. This is not rare in biodegra- dative metabolism. As described in the next section, metabolic activation prior to degrad- ing stable molecules is the norm rather than the exception in biodegradation. Microorganisms 2022, 10, 1664 5 of 13 Figure 2. Fluoroacetate metabolism and enzyme from bacteria serves as the main, but rare, example of direct C–F bond cleavage. (A) Bacteria catalyzing defluorination of fluoroacetate. (B) Active site of the fluoroacetate dehalogenase from Rhodospeudomonas palustris CGA009 (3R3U). (C) Metabolic benefit. In addition to detoxification, some bacteria can use hydroxyacetic acid, or glycollate, as a sole source of carbon and energy to support growth. 5. Concept of Metabolic Activation in Biodegradation The cost of acquiring additional genes, and hence new metabolic functions, is often given as a reason why microbial genome size has largely stayed within 1–10 Mb confines [48]. Each added gene requires more resources and time to replicate. Microbes sometimes acquire, and evolve, new genes via plasmids, transposons and mutations that create new functions [49–51]. This strategy may be evolutionarily efficient by keeping genomes streamlined, gaining valuable functions and shedding genes if they fail to provide suffi- cient survival benefit. Given the extreme selective pressures against the unrestrained expansion of metab- olism, the common occurrence of metabolic activation attests to its importance. Metabolic activation refers to the initial steps in many biodegradative pathways in which energy or materials are not immediately gained. In many of those cases, the metabolic activation requires energy investment, which is later regained along with added energy and nutri- ents that support cell growth. In the present review, examples of metabolic activation are discussed that are most relevant to known and proposed microbial defluorination metab- olism. Hydrocarbons and dialkyl ethers are common industrial materials, and so biodeg- radation of such chemicals has been studied extensively. In aerobic environments, ali- phatic and aromatic hydrocarbons and ethers are often activated by oxygenases to pro- duce alcohols, aldehydes and carboxylic acids that can enter intermediary metabolic path- ways [52]. Enzymes that combine atmospheric oxygen with hydrocarbons must overcome a high energy barrier that requires activation of O2, typically with NAD(P)H (Figure 3A). Since there is often substantial energy gained from the complete aerobic oxidation of hy- drocarbons and dialkyl ethers to carbon dioxide, the energy investment is more than com- pensated for. An example of metabolic activation for dehalogenation has been observed with p- chlorobenzoate (Figure 3B) [53]. The carboxyl group is only mildly activating for the para position in nucleophilic aromatic substitution [54,55], causing some microorganisms to invest energy to make the para position more activated. This is achieved by transforming the carboxylate group into a coenzyme A-tethered thioether. The dechlorination can then proceed hydrolytically, and the ester is subsequently hydrolyzed to make the common growth substrate p-hydroxybenzoate. The thioesterification and its later hydrolytic cleav- age costs a cell two ATP-equivalents per metabolic cycle due to the CoA ligase enzyme Microorganisms 2022, 10, 1664 6 of 13 producing AMP and pyrophosphate. However, further of metabolism of p-hydroxyben- zoate more than recoups the energy investment. The cleavage of the C–P bond of alkyl phosphonates by C–P lyase provides an inter- esting parallel to plausible activation mechanisms for C–F bond cleavage (Figure 3C). The 2− overall reaction stoichiometry for methyl phosphonate is: CH3-PO3 + H2O → CH4 + 2− HOPO3 . However, this is not a direct addition of water to the C–P bond; the bond is cleaved only following a set of activation reactions requiring ATP and six proteins, with the key steps shown in the figure [56,57]. The extensive biological investment in this reac- tion reflects the extreme chemical stability of alkylphosphonates, that are known to be stable to refluxing conditions in an acid or base [58]. However, methylphosphonate is now known to be highly abundant in marine environments where phosphate is scarce [59]. This has presumably driven the evolution of this complex biochemical machinery to bio- degrade alkyl phosphonates for the release of the inorganic phosphate needed for cellular metabolism. The complexity of the physiology is underscored by the discovery that 14 gene products are encoded by the C–P lyase operon [60]. As shown in Figure 2C, the ulti- mate cleavage step requires ATP, is activated by a series of enzymatic steps and is ulti- mately driven by the stable formation of a cyclic phosphodiester with concomitant C–P bond breakage [56,57]. Figure 3. Examples of metabolic activation in microbial degradation pathways. (A) A major aerobic bacterial pathway for metabolizing benzenoid rings requires energy input to activate molecular ox- ygen for attack on the ring. The activated ring is then cleaved and fragments are assimilated in subsequent metabolism. (B) 4-Chlorobenzoate is activated for dichlorination via an initial energy- requiring thioesterification of the carboxylate moiety via coenzyme A. (C) The C–P bond of al- kylphosphonates is relatively unreactive and is activated using ATP to accomplish C–P bond cleav- age to release inorganic phosphate in phosphorus-poor environments. Other examples of metabolic activation to initiate biodegradation have been detailed in peer-reviewed literature [61,62]. Additionally, the present author perused the EAWAG- Biocatalysis/Biodegradation Database compilation of 219 biodegradation pathways [63] and found that ~2/3 showed an activation reaction(s) as initiating the pathway. Activation reactions are also apparent in the order of rules applied during biodegradation predic- tions [64] and in pesticide degradation records as captured on EnviPath [65]. Most com- pounds, for which biodegradation does not initiate with an activating reaction, are esters and amides that undergo simple hydrolysis reactions. For the majority of other metabo- lism requiring initial activations, oxygenase and CoA-ligase reactions are common. Other Microorganisms 2022, 10, 1664 7 of 13 activating reactions include azo and nitro group reduction, phosphorylation of phenol during anaerobic biodegradation, aryl and aliphatic carboxylation and anaerobic ethylbenzene dehydrogenation. The EAWAG-BBD lacks information on the biodegradation of PFCs by specific mi- crobes and enzymes, and it does not predict defluorination reactions [66]. By contrast, the database contains information on more than twenty dechlorinating enzymes, some of which have available X-ray structures. Polychlorinated natural products are abundant in several environments, indicating millions of years of exposure to the biosphere, and some microorganisms have evolved to use them for their carbon and energy requirements [67]. This sometimes necessitates metabolic activation of the type highlighted in Figure 2B. Metabolic activation, by definition, requires multiply coordinated enzyme reactions that, in turn, requires multiple genes to be expressed simultaneously. This can be accomplished via operonic gene organization, such as for the 4-chlorobenzoate pathway (Figure 2B), or at least constitutive gene expression. The latter was observed for the atrazine-degradation plasmid in Pseudomonas sp. ADP in which a dechlorinase, and two subsequent enzymes in the pathway are encoded by non-regulated, dispersed genes flanked by transposons that show evidence of evolutionarily recent insertion into the plasmid [68]. Similar to the metabolism of recently introduced herbicides like atrazine, the biodegradation of PFCs is likely at an early stage of evolution. However, the development of biodegradative metab- olism is rendered more difficult by the significantly greater strength of the C–F bond com- pared to the C–Cl bond, and so metabolic activation will more often be required to biode- grade PFCs. 6. Metabolic Activation of Trifluoromethyl Arenes for Indirect C–F Bond Cleavage Evolution is at an immature stage with respect to the biodegradation of polyfluori- nated compounds (PFCs), and in-depth mechanistic reports are lacking. This review draws on our limited knowledge of microbial PFC metabolism, and a more extensive knowledge of PFC chemistry. The first example given is for trifluoromethyl groups on benzenoid aromatic rings. Trifluoromethyl arenes are common moieties in many agrichemicals and pharma- ceuticals. One example, the antidepressant fluoxetine, more commonly known as Prozac, had already amassed more than $22 billion USD in sales by 2005 [69] and is increasingly detected in surface waters [70]. In 2010, 30% of blockbuster pharmaceuticals were fluori- nated, most of them aromatic [71], and thirteen new fluorinated drugs were approved in 2020 alone by the United States Food and Drug Administration [72]. Despite the signifi- cance and increasing emergence of these chemicals, studies on their mechanisms of bio- degradation are limited. In that context, this class serves as a major example of how bio- degradation is most likely to proceed via metabolic activation to labilize the C–F bond, rather than carrying out a direct C–F bond cleavage on an unaltered pesticide or pharma- ceutical. It is instructive here to compare chemical and microbiological transformations of (tri- fluoromethyl)benzene, also known as benzotrifluoride (Figure 4). For example, the reduc- tion potential for the direct C–F bond cleavage of benzotrifluoride, even with activating para-substituents, was below −1170 millivolts in all cases [73]. That potential is on the order of 500 millivolts below the lowest potential reported for a biological redox catalyst [29]. Yet, benzotrifluoride and their derivatives are reported to undergo defluorination by nu- merous microbes [74–77]. The mechanism is indirect, with metabolic activation occurring prior to C–F bond cleavage. The activation consists of the addition of oxygen to the ring that generates a low pKa phenolic hydroxyl group ortho to the trifluoromethyl group, with which it acts synergistically (Figure 4). Facile spontaneous deprotonation of the ortho-phe- nolic group leads to double bond rearrangement to displace a fluoride anion and generate an ortho-quinone methide. The quinone methide is highly reactive to nucleophilic attack at the exocyclic double bond that can react with water in the aqueous environment of a Microorganisms 2022, 10, 1664 8 of 13 bacterial cell to produce a gem-difluoro-benzyl alcohol. Further spontaneous defluorina- tion in water produces a non-fluorinated benzoic acid. Defluorination occurrence with microbially-produced trifluoromethyl catechols was first described in 1982 [74]. It has been described in cultures of Rhodococcus rubropertinctus N657 [75] and Pseudomonas putida F1 [77]. The defluorination in wild-type aromatic-de- grading bacteria was dependent upon the inability of a catechol dioxygenase enzyme to process the trifluoromethyl catechol, but a higher yield could be accomplished in a recom- binant Escherichia coli expressing toluene dioxygenase (TodABC1C2) and cis-dihydrodiol dehydrogenase (TodD) [77]. The defluorination is sufficiently facile to occur at neutral pH and 25 °C, suggestive that an evolved enzyme could catalyze highly efficient defluorina- tion by facilitating ortho-hydroxyl deprotonation and adding water to the quinone methide. Figure 4. Metabolic activation of benzotrifluoride leading to complete defluorination of the trifluo- romethyl group. (A) The direct cleavage of the C–F bond by a reductive mechanism is unlikely. (B) Dihydroxylation of the ring produces a phenolate anion at neutral pH that undergoes ring electron rearrangement that displaces one HF equivalent and produces an ortho-quinone methide. The latter may then undergo facile hydrolysis and complete defluorination. 7. Metabolic Activation of Chlorofluorocarbons for Indirect C–F Bond Cleavage The relative weakness of the C–Cl bond compared to the C–F bond provides a suita- ble weak entry point for metabolic activation, leading to defluorination. As an example, CF4 is highly inert but the refrigerant Freon-11, or trichlorofluoromethane was shown to undergo biotransformation via the heme enzyme cytochrome P450cam from the bacterium Pseudomonas putida G7 [78]. Driven by NADH, the enzyme catalyzed an overall two-elec- tron reduction to generate a dichlorofluoromethyl carbanion intermediate that rapidly and spontaneously eliminated a chloride anion (Figure 5A). The resultant fluorochloro- carbene is highly reactive with water and the expected defluorinated product, carbon monoxide, was demonstrated [78]. More recently, an anaerobic microbial-activated sludge community was tested with twelve chlorofluoro carboxylic acids from two to nine carbon atoms, and different ratios of chlorine to fluorine [79]. The reactivities leading to defluorination are illustrated with dichlorofluoroacetate (Figure 5B). Based on products detected, both reductive and hydro- lytic dechlorination were indicated and led to defluorination of the C2 carboxylic acid, producing end products that could conceivably serve as growth substrates for bacteria. Microorganisms 2022, 10, 1664 9 of 13 Figure 5. Metabolic activation in microbial defluorination. (A) Reductive dichlorination leading to ready defluorination catalyzed by the enzyme cytochrome P450cam from Pseudomonas putida G7. (B) Reductive and hydrolytic declorination catalyzed by a microbial community leading to defluorina- tion. (C) Fluoroolefin epoxidation leading to defluorination catalyzed by soluble methane monoox- ygenase from Methylosinus trichosporium OB3b. (D) Unusual displacement of a trifluoromethyl group from a polyfluorinated olefin by a microbial community, mechanism unknown. 8. Metabolic Activation of Fluoroolefins for Indirect C–F Bond Cleavage Electron-withdrawing fluorine substituents on carbon–carbon double bonds render the double bond more susceptible to nucleophilic attack [80]. For example, perfluoroiso- butene is so reactive with nucleophiles that it has been reported to react with solvents that are considered fairly inert, such as N,N-dimethylformamide [81]. Moreover, fluorine at- oms, being smaller than chlorine or methyl group substituents, offer less steric hindrance to reactions with oxidizing agents, although reactions with electrophilic reagents are typ- ically slowed. Tetrafluoroethylene is reported to react slowly but spontaneously with ox- ygen at low temperatures to give tetrafluoroethylene epoxide and uncharacterized poly- meric materials [82]. Therefore, it is not surprising that an enzyme generating a highly reactive oxygen species [83] could react with a fluorinated ethylene. Trifluoroethylene was observed to react with the soluble methane monooxygenase from Methylosinus tricho- sporium OB3b, albeit at only 8% the rate observed with ethylene [84] (Figure 5C); novel reactions can be expected. A recent report described trifluoromethyl group elimination from a highly fluorinated olefin by an aerobic activated sludge community (Figure 5D) [85]. While single elimination from such trifluoromethylated olefins are well-known in the organic literature [86], the trifluoromethyl group elimination appears novel. Moreo- ver, complete defluorination to small organic compounds was reported, a harbinger of further interesting microbial physiology. 9. Conclusions In discussing biodegradability and mechanisms of biodegradation, it is preferable to use the term polyfluorinated compounds (PFCs), rather than PFAS, and to describe the structure of the molecule beyond the C–F bond. This review discusses strategies for the biodegradation of substituted polyfluorinated alkanes, alkenes and arenes and specific substituent impacts on C–F bond reactivity. Considering biodegradation broadly, most environmental chemicals require initial metabolic activation prior to cleavage of the molecule into assimilatible fragments that provide benefit to the cell. Metabolic activation is particularly necessary as one considers plausible meachanisms for the biodegradation of PFCs. As the degree of fluorination in- creases, the direct reactivity of C–F bonds generally decreases. However, commercial Microorganisms 2022, 10, 1664 10 of 13 PFCs almost invariably contain functionalities other than C–F bonds. Those accessory functional groups provide the potential for metabolic activation to render C–F bonds weaker and facilitate their cleavage. Typical activating reactions may set up a carbanion, a carbon radical, or facilitate electron flow to a fluorinated carbon by shifting ring currents. These strategies are most likely to prove fruitful for the biodegradation of PFCs in natural and engineered environments. Funding: The author has been supported by the MnDRIVE program on Industry and the Environ- ment at the University of Minnesota. Data Availability Statement: Data discussed in the review are from, and available through, peer- reviewed published literature. Acknowledgments: The author thanks Serina Robinson for critical comments on the manuscript and Jack Richman for helpful discussions on organofluorine chemistry. Conflicts of Interest: The author declares no conflict of interest. References 1. Barnabas, S.J.; Böhme, T.; Boyer, S.K.; Irmer, M.; Ruttkies, C.; Wetherbee, I.; Kondić, T.; Schymanski, E.L.; Weber, L. Extraction of chemical structures from literature and patent documents using open access chemistry toolkits: A case study with PFAS. Digit. Discov. 2022, 1, 490–501. https://doi.org/10.26434/chemrxiv-2022-nmnnd-v2. 2. Evich, M.G.; Davis, M.J.B.; McCord, J.P.; Acrey, B.; Awkerman, J.A.; Knappe, D.R.U.; Lindstrom, A.B.;Speth, T.F.; Tebes-Stevens, C.; Strynar, M.J.; et al. Per- and polyfluoroalkyl substances in the environment. Science 2022, 375, eabg9065. https://doi.org/10.1126/science.abg9065. 3. Ogawa, Y.; Tokunaga, E.; Kobayashi, O.; Hirai, K.; Shibata, N. Current contributions of organofluorine compounds to the agrochemical industry. Iscience 2020, 23, 101467. https://doi.org/10.1016/j.isci.2020.101467. 4. Alexandrino, D.A.; Almeida, C.M.R.; Mucha, A.P.; Carvalho, M.F. Revisiting pesticide pollution: The case of fluorinated pesticides. Environ. Pollut. 2022, 292, 118315. https://doi.org/10.1016/j.envpol.2021.118315. 5. 5. Inoue, M.; Sumii, Y.; Shibata, N. Contribution of organofluorine compounds to pharmaceuticals. ACS Omega, 2020, 5, 10633– 10640. https://doi.org/10.1021/acsomega.0c00830. 6. Liu, J.; Avendaño, S.M. Microbial degradation of polyfluoroalkyl chemicals in the environment: A review. Environ. Int. 2013, 61, 98–114. https://doi.org/10.1016/j.envint.2013.08.022. 7. Ochoa-Herrera, V.; Field, J.A.;Luna-Valesco, A.; Sierra-Alvarez, R. Microbial toxicity and biodegradability of perfluorooctane sulfonate (PFOS) and shorter chain perfluoroalkyl and perfluoroalkyl substances (PFASs). Environ. Sci. Process Impacts., 2016, 18, 1236–1246. https://doi.org/10.1039/c6em00366d. 8. Zhang, W.; Pang, S.; Lin, Z.; Mishra, S.; Bhatt, P.; Chen, S. Biotransformation of perfluoroalkyl acid precursors from various environmental systems: Advances and perspectives. Environ. Pollut. 2021, 272, 115908. https://doi.org/10.1016/j.envpol.2020.115908. 9. Zhang, Z.; Sarkar, D.; Biswas, J.K.; Datta, R. Biodegradation of per- and polyfluoroalkyl substances (PFAS): A review. Bioresour Technol. 2022, 344 Pt B, 126223. https://doi.org/10.1016/j.biortech.2021.126223. 10. Weber, E.J.; Tebes-Steves, C.; Washington, J.W.; Gladstone, R. Development of a PFAS reaction library: Identifying plausible transformation pathways in environmental and biological systems. Environ. Sci. Process Impacts 2022, 25, 689–753. https://doi.org/10.1039/d1em00445j. 11. Carvalho, M.F.; Alves, C.C.; Ferreira, M.I.; De Marco, P.; Castro, P.M. Isolation and initial characterization of a bacterial consortium able to mineralize fluorobenzene. Appl. Environ. Microbiol. 2022, 68, 102–105. https://doi.org/10.1128/AEM.68.1.102- 105.2002. 12. Finkelstein, Z.I.; Baskunov, B.P.; Boerrsma, M.G.; Vervoot, J.; Golovlev, E.L.; van Berkel, W.J.; Govleva, L.A.; Riietjens, I.M. Identification of fluoropyrogallols as new intermediates in biotransformation of monofluorophenols in Rhodococcus opacus 1cp. Appl. Environ. Microbiol. 2000, 66, 2148–2153. https://doi.org/10.1128/AEM.66.5.2148-2153.2000. 13. Vargas, C.; Song, B.; Camps, M.; Häggblom, M.M. Anaerobic degradation of fluorinated aromatic compounds. Appl. Microbiol. Biotechnol. 2000, 53, 342–347. https://doi.org/10.1007/s002530050032. 14. Turner, A.M.; Abplanalp, M.J.; Bergantini, A.; Frigge, R.; Zhu, C.; Sun, B.J.; Hsiao, C.T.; Chang, A.H.; Meinert, C.; Kaiser, R.I. Sci. Adv. 2019, 5, eaaw4307. https://doi.org/10.1125/sciadv.aaw4307. 15. Wackett, L.P.; Robinson, S.L. The ever-expanding limits of enzyme catalysis and biodegradation: Polyaromatic, polychlorinated, polyfluorinated, and polymeric compounds. Biochem. J. 2020, 477, 2875–2891. https://doi.org/10.1042/BCJ20190720. 16. Harper, D.B.; O’Hagan, D.; Murphy, C.D. Fluorinated natural products: Occurrence and biosynthesis. Nat. Prod. Organohalogen Compd. 2003, 141–169. https://doi.org/10.1007/b10454. 17. Walker, M.C.; Chang, M.C. Natural and engineered biosynthesis of fluorinated natural products. Chem. Soc. Rev. 2014, 43, 6527– 6536. https://doi.org/10.1039/c4cs00027g. Microorganisms 2022, 10, 1664 11 of 13 18. Gribble, G.W. A recent survey of naturally occurring organohalogen compounds. Environ. Chem. 2015, 12, 396–405. https://doi.org/10.1071/EN15002. 19. Rosner, D.; Markowitz, G. Persistent pollutants: A brief history of the discovery of the widespread toxicity of chlorinated hydrocarbons. Environ. Res. 2013, 120, 126–133. https://doi.org/10.1016/j.envres.2012.08.011. 20. Wang, Z.; Buser, A.M.; Cousins, I.T.; Demattio, S.; Drost, W.; Johansson, O.; Ohno, K.; Patlewicz, G.; Richard, A.M.; Walker, G.W.; et al. A new OECD definition for per-and polyfluoroalkyl substances. Environ. Sci. Technol. 2021, 55, 15575–15578. https://doi.org/10.1021/acs.est.1c06896. 21. Freire, M.G.; Gomes, L.; Santos, L.M.; Marrucho, I.M.; Coutinho, J.A. Water solubility in linear fluoroalkanes used in blood substitute formulations. J. Phys. Chem. B 2006, 110, 22923–22929. https://doi.org/10.1021/jp0622942. 22. Kim, S.; Chen, J.; Cheng, T.; Gindulyte, A.; He, J.; He, S.; Li, Q.; Shoemaker, B.A.; Thiessem, P.A.; Yu, B.; et al. PubChem in 2021: New data content and improved web interfaces. Nucleic Acids Res. 2021, 49, D1388–D1395. https://doi.org/10.1093/nar/gkaa971. 23. CDC, Per- and Polyfluorinated Substances (PFAS) Factsheet. Available online: https://www.cdc.gov/biomonitoring/PFAS_FactSheet.html (accessed on 6 July 2022). 24. Wallington, T.J.; Andersen, M.S.; Nielsen, O.J. The case for a more precise definition of regulated PFAS. Env. Sci. Proc. Impacts 2021, 23, 1834–1838. https://doi.org/10.1039/d1em00296a. 25. Hammel, E.; Webster, T.F.; Gurney, R.; Heiger-Bernays, W. Implications of PFAS definitions using fluorinated pharmaceuticals. Iscience 2022, 25, 104020. https://doi.org/10.1016/j.isci.2022.104020. 26. Pud, A.A.; Shapoval, G.S.; Kukhar, V.P.; Mikulina, O.E.; Gervits, L.L. Electrochemical reduction of some saturated and unsaturated perfluorocarbons. Electrochim. Acta 1995, 40, 1157–1164. https://doi.org/10.1016/0013-4686(95)00030-I. 27. Combellas, C.; Kanoufi, F.; Thiebault, A. Reduction of polyfluorinated compounds. J. Phys. Chem. B 2003, 39, 10894–10905. https://doi.org/10.1021/jp034846b. 28. Houmam, A. Electron transfer initiated reactions: Bond formation and bond dissociation. Chem. Rev. 2008, 108, 2180–2237. https://doi.org/10.1021/cr068070x. 29. Huwiler, S.G.; Löffler, C.; Anselmann, S.E.; Stärk, H.J.; von Bergen, M.; Flechsler, J.; Rachel, R.; Boll, M. One-megadalton metalloenzyme complex in Geobacter metallireducens involved in benzene ring reduction beyond the biological redox window. Proc. Natl. Acad. Sci. USA 2019, 116, 2259–2264. https://doi.org/10.1073/pnas.1819636116. 30. Kim, M.H.; Wang, N.; Chu, K.H. 6:2 Fluorotelomer alcohol (6:2 FTOH) biodegradation by multiple microbial species under different physiological conditions. Appl. Microbiol. Biotechnol. 2014, 98, 1831–1840.. https://doi.org/10.1007/s00253-013-5131-3. 31. Shaw, D.M.; Munoz, G.; Bottos, E.M.; Duy, S.V.; Sauvé, S.; Liu, J.; Van Hamme, J.D. Degradation and defluorination of 6: 2 fluorotelomer sulfonamidoalkyl betaine and 6: 2 fluorotelomer sulfonate by Gordonia sp. strain NB4-1Y under sulfur-limiting conditions. Sci.Total Environ. 2019, 647, 690–698. https://doi.org/10.1016/j.scitotenv.2018.08.012. 32. Goldman, P. The enzymatic cleavage of the carbon-fluorine bond in fluoroacetate. J. Biol. Chem. 1965, 240, 3434–3438. https://doi.org/10.1016/S0021-9258(18)973236-4. 33. Liu, J.Q.; Kurihara, T.; Ichiyama, S.; Miyagi, M.; Tsunasawa, S.; Kawasaki, H.; Soda, K.; Esaki, N. Reaction mechanism of fluoroacetate dehalogenase from Moraxella sp. B. J. Biol. Chem. 1998, 273, 30897–30902. https://doi.org/10.1074/jbc.273.47.30897. 34. 34. Kurihara, T.; Yamauchi, T.; Ichiyama, S.; Takahata, H.; Esaki, N. Purification, characterization, and gene cloning of a novel fluoroacetate dehalogenase from Burkholderia sp. FA1. J. Mol. Catal. B Enzym. 2003, 23, 347–355. https://doi.org/10.1016/S1381- 1177(03)00098-5. 35. Donnelly, C.; Murphy, C.D. Purification and properties of fluoroacetate dehalogenase from Pseudomonas fluorescens DSM 8341. Biotechnol. Lett. 2009, 31, 245–250. https://doi.org/10.1007/s10529-008-9849-4. 36. 36. Davis, C.K.; Webb, R.I.; Sly, L.I.; Denman, S.E.; McSweeney, C.S. Isolation and survey of novel fluoroacetate-degrading bacteria belonging to the phylum Synergistetes. FEMS Microbiol. Ecol. 2012, 80, 671–684. https://doi.org/10.1111/j.1574- 6941.2012.01338.x. 37. Camboim, E.K.; Almeida, A.P.; Tadra-Sfeir, M.Z.; Junior, F.G.; Andrade, P.P.; McSweeney, C.S.; Melo, M.A.; Riet-Correa, F. Isolation and identification of sodium fluoroacetate degrading bacteria from caprine rumen in Brazil. Sci. World J. 2012, 2012, 178254. https://doi.org/10.1100/2012/178254. 38. Chan, W.Y.; Wong, M.; Guthrie, J.; Savchenko, A.V.; Yakunin, A.F.; Pai, E.F.; Edwards, E.A. Sequence-and activity-based screening of microbial genomes for novel dehalogenases. Microb. Biotechnol. 2010, 3, 107–120. https://doi.org/10.1111/j.1751- 7915.2009.00155.x. 39. Chan, P.W.; Yakunin, A.F.; Edwards, E.A.; Pai, E.F. Mapping the reaction coordinates of enzymatic defluorination. J. Am. Chem. Soc. 2011, 133, 7461–7468. https://doi.org/10.1021/ja200277d. 40. Jitsumori, K.; Omi, R.; Kurihara, T.; Kurata, A.; Mihara, H.; Miyahara, I.; Hirotsu, K.; Esaki, N. X-Ray crystallographic and mutational studies of fluoroacetate dehalogenase from Burkholderia sp. strain FA1. J. Bacteriol. 2009, 191, 2630–2637. https://doi.org/10.1128/JB.01654-08. 41. Kamachi, T.; Nakayama, T.; Shitamichi, O.; Jitsumori, K.; Kurihara, T.; Esaki, N.; Yoshizawa, K. The catalytic mechanism of fluoroacetate dehalogenase: A computational exploration of biological dehalogenation. Chemistry 2009, 15, 7394–7403.. https://doi.org/10.1002/chem.200801813. 42. Miranda-Rojas, S.; Fernández, I.; Kästner, J.; Toro-Labbé, A.; Mendizabal, F. Unraveling the nature of the catalytic power of fluoroacetate dehalogenase. ChemCatChem 2018, 10, 1052–1063. https://doi.org/10.1002/cctc.201701517. Microorganisms 2022, 10, 1664 12 of 13 43. Kang, H.; Zheng, M. Influence of the quantum mechanical region size in QM/MM modelling: A case study of fluoroacetate dehalogenase catalyzed CF bond cleavage. Comp. Theor. Chem. 2021, 1204, 113399. https://doi.org/10.1016/j.comptc.2021.113399. 44. Alexandrino, D.A.M.; Ribeiro, I.; Pinto, L.M.; Cambra, R.; Oliveira, R.S.; Pereira, F.; Carvalho, M.F. Biodegradation of mono-, di- and trifluoroacetate by microbial cultures with different origins. N. Biotechnol. 2018, 43, 23–29. https://doi.org/10.1016/j.nbt.2017.08.005. 45. Dolbier, W.R. Fluorine chemistry at the millenium. J. Fluor. Chem. 2005, 126, 157–163. https://doi.org/10.1016/j.jfluchem.2004.09.033. 46. O’Hagan, D. Understanding organofluorine chemistry. An introduction to the C-F bond. Chem. Soc. Rev. 2008, 37, 308–319. https://doi.org/10.1039/b711844a. 47. Chan, P.W.; Chakrabarti, N.; Ing, C.; Halgas, O.; To, T.K.; Wälti, M.; Petit, A.P.; Tran, C.; Savchenko, A.; Yakunin, A.F.; et al. Defluorination capability of l-2-haloacid dehalogenases in the HAD-like hydrolase superfamily correlates with active site compactness. ChemBioChem 2022, 23, e202100414. https://doi.org/10.1002/cbic.202100414. 48. Sela, I.; Wolf, Y.I.; Koonin, E.V. Theory of prokaryotic genome evolution. Proc. Natl. Acad. Sci. USA 2016, 113, 11399–11407. https://doi.org/10.1073/pnas.1614083113. 49. Wackett, L.P. Evolution of enzymes for the metabolism of new chemical inputs into the environment. J. Biol. Chem. 2004, 279, 41259–41262. https://doi.org/10.1074/jbc.R400014200. 50. Davidi, D.; Longo, L.M.; Jabłońska, J.; Milo, R.; Tawfik, D.S. A bird’s-eye view of enzyme evolution: Chemical, physicochemical, and physiological considerations. Chem. Rev. 2018, 118, 8786–8797. https://doi.org/10.1021/acs.chemrev.8b00039. 51. Copley, S.D. Setting the stage for evolution of a new enzyme. Curr. Opin. Struct. Biol. 2021, 69, 41–49. https://doi.org/10.1016/j.sbi.2021.03.001. 52. Gibson, D.T.; Parales, R.E. Aromatic hydrocarbon dioxygenases in environmental biotechnology. Curr. Opin. Biotechnol. 2000, 11, 236–243.. https://doi.org/10.1016/s0958-1669(00)00090-2. 53. Dunaway-Mariano, D.; Babbitt, P.C. On the origins and functions of the enzymes of the 4-chlorobenzoate to 4- hydroxybenzoate converting pathway. Biodegradation 1994, 5, 259–276. https://doi.org/10.1007/BF00696464. 54. Bunnett, J.F.; Morath, R.J.; Okamoto, T. The ortho:para ratio in activation of aromatic nucleophilic substitution by the carboxylate group. J. Am. Chem. Soc. 1955, 77, 5055–5057. https://doi.org/10.1021/ja01624a034. 55. Terrier, F. Modern Nucleophilic Aromatic Substitution; John Wiley & Sons: New York, NY, USA, 2013. 56. Kamat, S.S.; Raushel, F.M. The enzymatic conversion of phosphonates to phosphate by bacteria. Curr. Opin. Chem. Biol. 2013, 17, 589–596. https://doi.org/10.1016/j.cbpa.2013.06.006. 57. Yang, K.; Ren, Z.; Raushel, F.M.; Zhang, J. Structures of the carbon-phosphorus lyase complex reveal the binding mode of the nbd-like phnk. Structure 2016, 24, 37–42. https://doi.org/10.1016/j.str.2015.11.009. 58. Hoskin, F.C.G. Some observations concenring the biochemical inertness of methylphosphonic and isopropyl methylphosphonic acids. Can. J. Biochem. Physiol. 1955, 34, 743–746. https://doi.org/10.1139/o56-077. 59. Metcalf, W.W.; Griffin, B.M.; Cicchillo, R.M.; Gao, J.; Janga, S.C.; Cooke, H.A.; Circello, B.T.; Evans, B.S.; Martens-Habbena, W.; Stahl, D.A.; et al. Synthesis of methylphosphonic acid by marine microbes: A source for methane in the aerobic ocean. Science 2012, 337, 1104–1107. https://doi.org/10.1126/science.1219875. 60. Chen, C.M.; Ye, Q.Z.; Zhu, Z.M.; Wanner, B.L.; Walsh, C.T. Molecular biology of carbon-phosphorus bond cleavage. Cloning and sequencing of the phn (psiD) genes involved in alkylphosphonate uptake and C-P lyase activity in Escherichia coli B. J. Biol. Chem. 1990, 265, 4461–4471. 61. VanBriesen, J.M. Thermodynamic yield predictions for biodegradation through oxygenase activation reactions. Biodegradation 2001, 12, 265–281. https://doi.org/10.1023/a:1013179315518. 62. Coates, J.D.; Chakraborty, R.; McInerney, M.J. Anaerobic benzene biodegradation—a new era. Res. Microbiol. 2002, 153, 621–628. https://doi.org/10.1016/s0923-2508(02)01378-5. 63. Gao, J.; Ellis, L.B.; Wackett, L.P. The University of Minnesota biocatalysis/biodegradation database: Improving public access. Nucleic Acids Res. 2010, 38 (Suppl.1), D488–D491. https://doi.org/10.1093/nar/gkp771. 64. Fenner, K.; Gao, J.; Kramer, S.; Ellis, L.; Wackett, L.P. Data-driven extraction of relative reasoning rules to limit combinatorial explosion in biodegradation pathway prediction. Bioinformatics 2008, 24, 2079–2085. https://doi.org/10.1093/bioinformatics/btn378. 65. Latino, D.A.; Wicker, J.; Gütlein, M.; Schmid, E.; Kramer, S.; Fenner, K. Eawag-Soil in enviPath: A new resource for exploring regulatory pesticide soil biodegradation pathways and half-life data. Environ. Sci. Proc. Impacts 2017, 19, 449–464. https://doi.org/10.1039/c6em00697c. 66. Gao, J.; Ellis, L.B.; Wackett, L.P. The University of Minnesota Pathway Prediction System: Multi-level prediction and visualization. Nucleic Acids Res. 2011, 39, W406–W411. https://doi.org/10.1093/nar/gkr200. 67. Holliger, C.; Wohlfarth, G.; Diekert, G. Reductive dechlorination in the energy metabolism of anaerobic bacteria. FEMS Microbiol. Rev. 1998, 22, 383–398. https://doi.org/10.1111/j.1574-6976.1998.tb00377.x. 68. Martinez, B.; Tomkins, J.; Wackett, L.P.; Wing, R.; Sadowsky, M.J. Complete nucleotide sequence and organization of the atrazine catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP. J. Bacteriol. 2001, 183, 5684–5697. https://doi.org/10.1128/JB.183.19.5684-5697.2001. 69. Wenthur, C.J.; Bennett, M.R.; Lindsley, C.W. Classics in Chemical Neuroscience: Fluoxetine (Prozac). ACS Chem. Neurosci. 2013, 5, 14–23. https://doi.org/10.1021/cn400186j. Microorganisms 2022, 10, 1664 13 of 13 70. Martin, J.M.; Bertram, M.G.; Saaristo, M.; Fursdon, J.B.; Hannington, S.L.; Brooks, B.W.; Burket, S.R.; Mole, R.A.; Deal, N.D.; Wong, B.B. 2019. Antidepressants in surface waters: Fluoxetine influences mosquitofish anxiety-related behavior at environmentally relevant levels. Environ. Sci. Technol. 2019, 53, 6035–6043. https://doi.org/10.1021/acs.est.9b00944. 71. O’Hagan, D. Fluorine in health care: Organofluorine containing blockbuster drugs. J. Fluor. Chem. 2010, 131, 1071–1081. https://doi.org/10.1016/j.fluchem.2010.03.003. 72. Yu, Y.; Liu, A.; Dhawan, G.; Mei, H.; Zhang, W.; Izawa, K.; Soloshonok, V.A.; Han, J. Fluorine-containing pharmaceuticals approved by the FDA in 2020: Synthesis and biological activity. Chin. Chem. Lett. 2021, 32, 3342–335.. https://doi.org/10.1016/j.cclet.2021.05.042. 73. Combellas, C.; Kanoufi, F.; Thiébault, A. Reducibility of the carbon-fluorine bond in the trifluoromethyl group. J. Electroanal. Chem. 1996, 407, 195–202. https://doi.org/10.1016/0022-0728(95)04474-4. 74. Engesser, K.H. Der Einfluss der Trifluormethylgruppe auf die Biologische Abbaubarkeit von Aromaten. Doctoral Dissertation, University of Göttingen, Göttingen, Germany, 1982. 75. Engesser, K.H.; Cain, R.B.; Knackmuss, H.J. Bacterial metabolism of side chain fluorinated aromatics: Cometabolism of 3- trifluoromethyl(TFM)-benzoate by Pseudomonas putida (arvilla) mt-2 and Rhodococcus rubropertinctus N657. Arch. Microbiol. 1988, 149, 188–197. https://doi.org/10.1007/BF00422004. 76. Kiel, M.; Engesser, K.H. The biodegradation vs. biotransformation of fluorosubstituted aromatics. Appl. Microbiol. Biotechnol. 2015, 99, 7433–7464. https://doi.org/10.1007/s00253-015-6817-5. 77. Bygd, M.D.; Aukema, K.G.; Richman, J.E.; Wackett, L.P. Microwell fluoride screen for chemical, enzymatic, and cellular reactions reveals latent microbial defluorination capacity for -CF3 groups. Appl. Environ. Microbiol. 2022, 88, e00288-22. https://doi.org/10.1128/aem.00288-22. 78. Li, S.; Wackett, L.P. Reductive dehalogenation by cytochrome P450CAM: Substrate binding and catalysis. Biochemistry 1993, 32, 9355–9361. https://doi.org/10.1021/bi00087a014. 79. Jin, B.; Che, S.; Gao, J.; Yu, Y.; Liu, J.; Men, Y. Anaerobic defluorination of chlorine- substituted per- and polyfluorinated carboxylic acids triggered by microbial dechlorination. ChemRxiv 2022. https://doi.org/10.26434/chemrxiv-2022-44rx6. 80. Timperley, C.M.; Waters, M.J.; Greenall, J.A. Fluoroalkene chemistry: Part 3. Reactions of arylthiols with perfluoroisobutene, perfluoropropene and chlorotrifluoroethene. J. Fluor. Chem. 2006, 127, 249–256. https://doi.org/10.106/j.fluchem.2005.11.008. 81. Zeifman, Y.V.; Ter-Gabrielyan, E.G.; Gambaryan, N.P.; Knunyants, I.L. The chemistry of perfluoroisobutene. Russ. Chem Rev. 1984, 53, 256. 82. Gozzo, F.; Camaggi, G. Oxidation reactions of tetrafluoroethylene and their products—I: Auto-oxidation. Tetrahedron 1996, 22, 1765–1770. https://doi.org/10.1016/S0040-4020(01)82248-1. 83. Wallar, B.J.; Lipscomb, J.D. Dioxygen activation by enzymes containing binuclear non-heme iron clusters. Chem. Rev. 1996, 96, 2625–2658. https://doi.org/10.1021/cr9500489. 84. Fox, B.; Borneman, J.G.; Wackett, L.P.; Lipscomb, J.D. Haloalkene oxidation by the soluble methane monooxygenase from Methylosinus trichosporium OB3b: Mechanistic and environmental implications. Biochemistry 1990, 29, 6419–6427. https://doi.org/10.1021/bi00479a013. 85. Yu, Y.; Che, S.; Ren, C.; Bosen, J.; Zhenyu, T.; Liu, J.; Men, Y. Microbial defluorination of unsaturated per- and polyfluorinated carboxylic acids under anaerobic and aerobic conditions: A structure specificity study. Environ. Sci. Technol. 2022, 56, 4894–4904. https://doi.org/10.1021/acs.est.1c05509. 86. Liu, Y.; Zhou, Y.; Zhao, Y.; Qu, J. Synthesis of gem-difluoroallylboronates via FeCl2-catalyzed boration/β-fluorine elimination of trifluoromethyl alkenes. Org. Lett. 2017, 19, 946–949. https://doi.org/10.1021/acs.orglett.7b00168.

Journal

Microorganisms – Multidisciplinary Digital Publishing Institute

Published: Aug 17, 2022

Keywords: polyfluorinated; PFAS; biodegradation; bacteria; enzyme; metabolism; metabolic activation; weak bonds; defluorination

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