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J. Biol. Chem., Vol. 279, Issue 40, 41259-41262, October 1, 2004
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Minireview
Evolution of Enzymes for the Metabolism of New Chemical Inputs into the Environment*
From the Department of Biochemistry, Molecular Biology, and Biophysics and the Biotechnology Institute, University of Minnesota, St. Paul, Minnesota 55108
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INTRODUCTION |
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For all of the biological growth in the world, there is a corresponding amount of biodegradation. Together, biosynthesis and biodegradation underlie much of the global carbon cycle. The biological cycling of carbon is largely dependent on microbial enzymes because they are the most abundant and functionally divergent of biocatalysts (1). It is well documented that microbial catabolic enzymes biodegrade: (a) natural products, (b) compounds generated by humans, and (c) compounds derived from abiotic reactions. As an example of the latter, a structurally diverse set of aromatic hydrocarbons is generated abiotically in sediments by diagenesis or slow burning processes (2). Aromatic hydrocarbons have even been found in meteorites (3) and are proposed to exist in interstellar space (4). On top of these abiotic, naturally generated compounds, more than 65,000 chemical substances are in commerce. Most of these are thought to be biodegradable (5).
Globally, biodegradation by microorganisms protects the planet in a manner analogous to the mechanism by which the mammalian immune system protects the host organism (Fig. 1). In both examples, recognition of an antigen by IgG or a substrate by an enzyme leads to binding and catalysis, respectively. In both cases, successful events lead to cell proliferation and spread of the biological function. Immunological memory can protect an organism against infection for years. Similarly, environmental chemists have observed that soils exposed to a herbicide in one season will often biodegrade the herbicide more quickly in subsequent years. Studies indicate that the number of bacteria capable of biodegrading a herbicide usually declines after the herbicide is metabolized, but a low level population with the herbicide-degrading genotype is typically maintained for extended periods (6, 7).
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Another lesson being learned from biodegradation studies is that functionally significant enzyme evolution occurs on shorter time scales than previously appreciated: weeks, months, and years rather than eons. How do new enzymes and pathways arise in nature to handle new chemical inputs into the environment? This question will be addressed in the present review. First, the range of dehalogenation reactions will be discussed. Second, some ideas of enzyme promiscuity and evolution will be put forward. Third, two examples of recently evolved enzymes and metabolic pathways will be presented.
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Halogenated Compounds and Their Enzymatic Degradation |
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 TOPINTRODUCTION Halogenated Compounds and Their... Enzyme Origins, Promiscuity, and...Dichloromethane Dehalogenasetrans-3-Chloroacrylate...Rapid Evolution of...Global Distribution of...ConclusionsREFERENCES |
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Although some of the 3800 natural product organohalides (8), such as methyl chloride and methyl bromide, are thought to contribute significantly to the global atmospheric budget, organohalides from anthropogenic sources clearly contribute to the pollution of certain environments. Major chlorinated industrial solvents and pesticides, which sometimes migrate in contaminant plumes in groundwater, likely present new metabolic challenges for microbes in soil and water. However, microbes have adapted to these new inputs. For example, highly chlorinated polychlorinated biphenyls in the Hudson River and other environments were for decades thought to be poorly, if at all, metabolized (9). However, more recent studies show evidence of microbiologically mediated reductive dechlorination reactions (10), and certain bacteria have adapted to use polychlorinated biphenyls as their terminal electron acceptor (11).
It is instructive to consider which type of dehalogenase activities might have existed for eons and which type may have evolved in the modern industrial era. There is a broad range of dehalogenases, characterized by fundamentally different mechanisms: (a) reduction, (b) oxidation, (c) group transfer, (d) hydrolysis, or (e) elimination reactions. The current review selects several different enzyme examples, some of which have likely existed as dehalogenases over a long period and others that are likely of recent evolutionary origin.
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Enzyme Origins, Promiscuity, and Evolution |
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TOPINTRODUCTIONHalogenated Compounds and Their...Enzyme Origins, Promiscuity, and...Dichloromethane Dehalogenasetrans-3-Chloroacrylate...Rapid Evolution of...Global Distribution of...ConclusionsREFERENCES |
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How can one determine if the physiological function of an enzyme is to catalyze a dehalogenation reaction? The answer to this question is confounded by the well documented catalytic promiscuity of enzymes. Increasingly, known enzymes are being recognized to catalyze reactions other than those for which they were originally discovered to catalyze (12). In other cases, enzymes catalyzing a specific physiological reaction may also serve other roles (13). It has been pointed out that catalytic promiscuity may play an important role in evolution as cells may recruit a fortuitous enzymatic activity, and under selective pressure, that activity can improve and become fixed in populations.
However, in some cases, the ability to catalyze a secondary reaction may not provide a benefit and represents a gratuitous reaction with a substrate that would only be encountered under laboratory conditions. For example, adenosine deaminase catalyzes the deamination of aminopurines as part of nucleotide cycling, but it will also catalyze the dehalogenation of chloropurines (14, 15). The latter is almost surely a chemical artifact. Adenosine deaminase is poised to catalyze a nucleophilic aromatic substitution reaction on purine rings, and the halogen substituent is an excellent leaving group that is displaced after attack on the ring by a metal-activated hydroxide anion (16).
In other cases, an enzyme may have evolved under selective pressure specifically to catalyze a dehalogenation reaction, and several diagnostic questions to address that issue are listed below. First, is there congruence between an enzyme's substrate specificity and the organism's growth substrate specificity? Second, does the dehalogenase gene cluster with other genes that might function coordinately with the dehalogenase? Third, is the substrate for the dehalogenase a natural product or does it closely resemble one? Fourth, is the dehalogenase gene present in different bacterial strains in which dehalogenation biochemistry might be important to the organism? Some specific examples in which these questions have been addressed experimentally are discussed below.
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Dichloromethane Dehalogenase |
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Dichloromethane dehalogenase initiates the metabolism of the widely used industrial solvent dichloromethane by methylotrophic bacteria obtained from dichloromethane-contaminated soils (17, 18). Methylotrophic bacteria metabolize naturally occurring single carbon compounds such as methanol, methylamine, and a large array of ubiquitous O-methyl ether compounds. The possession of a single gene encoding dichloromethane dehalogenase extends the growth substrate range of some methylotrophic bacteria to include dichloromethane. Dichloromethane dehalogenase requires glutathione (
-glutamylcysteinyl glycine) for activity, and formaldehyde is the product of the reaction (19). It was appreciated in initial studies that the enzyme likely catalyzes a direct attack of the glutathione thiol or thiolate on dichloromethane, generating an S-(chloromethyl)glutathione intermediate, which could undergo facile hydrolysis, either enzymatically or free in solution (19). This mechanism was first demonstrated directly using chlorofluoromethane as the substrate and observing the formation of the much more stable intermediate S-(fluoromethyl)glutathione by 19F NMR (20). Formation of an S-alkylated glutathione product is reminiscent of mammalian liver glutathione S-transferases that catalyze the reaction of the glutathione sulfur atom with a wide range of electrophilic substrates. In mammals, these reactions serve to protect against potentially toxic electrophilic compounds that are ingested or internalized through mucous membranes (21). Although there is no published x-ray structure available for dichloromethane dehalogenase, it is homologous to the mammalian glutathione S-transferases for which x-ray structures have been solved (22). Is dichloromethane dehalogenase a recently evolved enzymatic activity? Genome sequencing projects have revealed that glutathione S-transferase genes are widely distributed among prokaryotic species, although in many cases their physiological function is undefined (23). Various dichloromethane dehalogenases have been identified in dichloromethane-metabolizing bacteria, some of which show high sequence identity (>98%) in pairwise comparisons. In some of these bacteria, the dcm genes are found on large catabolic plasmids (24). Purified dichloromethane dehalogenase has a very narrow substrate specificity. It shows substantial activity with dihalomethanes, compounds that support growth of the bacterium harboring it, and a lower activity with monohaloethane substrates that form metabolic dead end products. Other substrates typically used to assay glutathione S-transferases are not substrates for dichloromethane dehalogenase (17). These observations suggest that enzymes acting specifically on dihalomethane substrates may have evolved and spread recently.
Other observations indicate that DcmA1 has evolved a certain sophistication of function. First, dcmA is seen to be associated with a dcmR regulatory gene, which tightly controls expression of DcmA under conditions of dichloromethane exposure (25). Second, methylotrophs containing dcmA may have other adaptations that facilitate growth on dichloromethane (26, 27). Third, the catalytic cycle of DcmA may be tuned in a way to partially protect the cell against toxic intermediates (28). Initially, the relatively low kcat of 
0.5 s–1 for DcmA from a Hyphomicrobium sp. with dichloromethane (17) had been interpreted as suggesting its recent evolutionary origin. Moreover, the organism was found to contain 20% of its soluble protein as DcmA, indicating it was a catalytically inefficient enzyme. More recent data have shown that the processing of dichloromethane by DcmA leads to DNA alkylation, presumably via an intermediate S-(chloromethyl)glutathione species (27). Studies now indicate that the slow step in the DcmA catalytic cycle is likely the release of the S-(halomethyl)glutathione species off of the enzyme surface, at a rate that is competitive with the spontaneous hydrolysis of an S-(chloromethyl)thioether intermediate. In the context of evolutionary benefit, at least partial hydrolysis of an S-(chloromethyl)glutathione intermediate before full release into the cytoplasm could mitigate against DNA alkylation and thus be genetically selected (28). In vivo data are consistent with this hypothesis; DcmA-mediated dichloromethane turnover is less toxic to the cell than the same reaction catalyzed by mammalian liver glutathione S-transferase isozyme T1-1 (29).
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trans-3-Chloroacrylate Dehalogenase and 4-Oxalocrotonate Tautomerase |
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trans-Chloroacrylate dehalogenase activity was first detected in bacteria that grow on 1,3-dichloropropene, a compound widely applied to soils as a nematocide (30). The metabolic pathway proceeds via an initial allylic hydrolytic dechlorination reaction to yield 1-chloropropen-3-ol, two subsequent dehydrogenation reactions, and then hydrolytic dechlorination of 3-chloroacrylate to generate malonic acid semialdehyde (31). A recent evolutionary origin of the pathway is suggested by the observation that the enzyme catalyzing allylic dechlorination was acquired by apparent horizontal gene transfer (32). The allylic dehalogenase is homologous to haloalkane dehalogenase. In contrast, chloroacrylate dehalogenase is homologous to 4-oxalocrotonate tautomerase (33). 4-Oxalocrotonate tautomerase functions in the microbial metabolism of aromatic hydrocarbons, which are widespread naturally, and thus probably represents an enzyme activity that has existed over eons. This view is supported by widespread microbial genome sequencing projects that have demonstrated enzyme homologous to 4-oxalocrotonate tautomerase in a range of bacteria capable of degrading aromatic hydrocarbons and aromatic acids (34).
Mechanistic studies on the 4-oxalocrotonate family of enzymes has been revealing. Site directed mutagenesis of 3-chloroacrylate dehalogenase demonstrated the essentiality of the N-terminal proline residue in the 
-subunit of the enzyme, a residue previously shown to be critical for catalysis by 4-oxalocrotonate tautomerase (35). The mechanistic connection between the proteins was further strengthened by recent observations that 4-oxalocrotonate tautomerase and a homologous protein from Bacillus, YwhB, show 3-chloroacrylate dehalogenase activity (34). Although the activity was very low, measured on a time scale of days, the observation nonetheless suggests that a catalytically promiscuous tautomerase enzyme could have possessed sufficient dehalogenase activity to be recruited by bacteria that generated 3-chloroacrylate from metabolism of the nematocide 1,3-dichloropropene. Further evolutionary refinements of the enzyme could have produced a more efficient dehalogenase. The enzyme characterized by Poelarends et al. (33) was orders of magnitude more active than YwhB, showing a kcat/Km of 3.4 x 104M–1s–1. Note that the dehalogenase cannot be checked for low vestigial isomerase activity because the non-enzymatic rate of isomerization with the physiologically relevant substrate is too rapid (34).
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Rapid Evolution of Biodegradation Activity against New Chemical Inputs |
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Snapshots along a proposed recent evolutionary trajectory have been obtained with the enzymes tetrachlorohydroquinone (TCHQ) reductive dehalogenase and atrazine chlorohydrolase (AtzA). TCHQ dehalogenase catalyzes consecutive reductive dechlorination reactions starting with tetrachlorohydroquinone and yielding 2,6-dichlorohydroquinone. These reactions comprise part of a metabolic pathway for the biodegradation of the widely used wood preservative pentachlorophenol (36).
TCHQ dehalogenase is a member of the glutathione S-transferase superfamily (37). The reductive reaction it catalyzes is a variation of the nucleophilic displacement/addition reactions typical of the transferases. Overlaid on top of that is the participation of an active site cysteine residue that captures the intermediate thioether to generate a mixed enzyme-glutathione disulfide and an overall two-electron-reduced product (Fig. 2A). The resting enzyme is then regenerated via a thiol-disulfide interchange with a second molecule of glutathione to generate glutathione disulfide. One consequence of the catalytic mechanism is that the enzyme suffers from strong substrate inhibition by tetrachlorohydroquinone. Binding of TCHQ or trichlorohydroquinone to the mixed disulfide form of the enzyme is proposed to generate a dead end complex of the enzyme (38). This observation has been interpreted as evidence for a recent evolutionary origin of TCHQ dehalogenase. Moreover, the enzyme also shows substantial maleylacetate isomerase activity; this is considered to be a vestigial activity (39). The organism producing TCHQ dehalogenase contains another maleylacetate isomerase activity and so can readily grow on tyrosine in the absence of TCHQ dehalogenase (38). Additionally, TCHQ dehalogenase is constitutively expressed. Bacterial adaptation to growth on new compounds is often accompanied by the constitutive expression of relevant genes, thus uncoupling enzyme and regulatory adaptation and increasing metabolic versatility (40, 41).
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In contrast, AtzA does not have detectable vestigial deaminase activity; that might have remained from its heritage as a member of the amidohydrolase superfamily (42). AtzA is in the same enzyme superfamily as cytosine deaminase and adenosine deaminase (Fig. 2B) and, like those enzymes, contains a mononuclear metal center, which is essential for catalytic activity (43). Adenosine deaminase catalyzes the hydrolytic deamination of adenosine and is, thus, a relatively common bacterial enzyme functioning in intermediary metabolism. Many enzymes in the amidohydrolase superfamily catalyze displacement of an amino group from N-heterocyclic ring substrates (44). AtzA catalyzes only the hydrolytic removal of fluorine or chlorine substituents from s-triazine ring compounds; it will not displace amino, S-methyl, cyano, or azido substituents, and it will not act on chlorinated pyrimidines (45). Surprisingly, TriA, an enzyme with 98% amino acid sequence identity to AtzA, was shown to catalyze hydrolytic deamination of melamine or 2,4,6-triamino-1,3,5-triazine (46). TriA was not discernibly active with chlorinated s-triazine substrates. The change in catalytic activity, from deamination to dechlorination, was postulated to arise from a limited number of amino acid changes in the amidohydrolase active site leading to the differential stabilization of amino and chloride leaving groups, respectively (Fig. 2C). This hypothesis was supported by directed evolution experiments in which atzA and triA genes were shuffled to generate clonal variants with one or a few amino acid changes and with differential leaving group specificities in displacement from the s-triazine ring (47). It was proposed that a change in an aspartate (TriA) to an asparagine (AtzA) was important in its evolution into a dehalogenase (48).
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Global Distribution of Biodegradative Metabolism |
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The evolution of enzymes to attain new metabolic functions is just one facet of the global adaptation of microbes for the metabolism of new chemical inputs into the environment. The catabolism of most environmental pollutants requires the deployment of a series of enzymes. The spontaneous, independent evolution of several new enzymes in a single bacterium would be a highly improbable event and would be unlikely to explain observations of rapid microbial adaptation to new environmental chemicals. Rapid microbial adaptation is facilitated via the supplementation of chromosomal DNA with plasmid DNA. Catabolic plasmids carrying biodegradation genes were first described in the late 1960s (49). A few years later, a transmissible plasmid encoding camphor oxidation, including the cytochrome P450 monooxygenase that hydroxylates camphor, was discovered in Pseudomonas putida (50). Now it is known that many soil bacteria contain plasmids; for example, Pseudomonas sp. ADP contains at least 6 large plasmids ranging in size from 50 to 500 kilobases (51). Thus, plasmid DNA can represent a significant fraction of the total genomic DNA in soil bacteria.
Pseudomonas sp. ADP metabolizes the herbicide atrazine to carbon dioxide and ammonia via the activities of 6 enzymes, all of which have been localized to plasmid pADP-1 (Fig. 3). Plasmid pADP-1 consists of 108,845 base pairs and was identified as belonging to a plasmid class known as the IncP-1 group (52). The plasmid backbone of pADP-1 shows 99% sequence identity to comparable regions of the following sequenced plasmids: (a) pR751 from Enterobacter aerogenes encoding trimethoprin resistance (53), (b) pUO1 from Delftia acidovorans encoding for the biodegradation of haloacetate (54), and (c) pB4 that was isolated from an environmental community of microorganisms and encoding resistance to chromate and various antibiotics (55).
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In the examples above, the basic plasmid backbone, which encodes plasmid maintenance and transfer, was highly conserved despite the wide divergence in taxonomic classes of the bacterial strains in which it is found. Consistent with this observation, IncP1-
plasmids are broad host range; that is, they have the ability to replicate, and thus be stably maintained, in a broad range of bacterial genera (56). The resistance and biodegradation genes were always found inserted into two regions of the IncP1- plasmid backbone near inverted repeat elements that probably serve as recognition sites for transposons (54). Thus, these plasmids may vary widely in size and the types of genes they carry according to the selective pressure imposed in given environmental pockets. In this context, Top and colleagues (56) have pointed out that IncP1- plasmids are often found in bacteria that have been isolated because of their ability to biodegrade synthetic industrial chemicals such as polychlorinated biphenyls, fumigants such as 1,3-dichloropropene, and herbicides such as bromoxynil. It is likely that the ease of DNA insertion and broad host range of the IncP1- plasmids is conducive to an environmental role in facilitating the evolution of metabolic pathways directed against chemicals that have been in the environment for only a short period of time on an evolutionary scale. Interestingly, bacteria isolated to biodegrade terpenoid compounds and hydrocarbons, classes of chemical compounds that occur naturally and thus have been in soil for millions of years, often contain the biodegradative genes on plasmids of the IncP2 or IncP9 class (56). A deeper understanding of how catabolic plasmids are organized and dispersed is emerging from ongoing work to determine their complete DNA sequences (54, 57–59). The DNA sequences obtained to date suggest that the catabolic plasmid genes have been assisted in their dissemination by insertion sequence (IS) elements. For example, the atzA, atzB, and atzC genes are seen to be flanked by IS1071 elements (Fig. 3). The movement of these atrazine genes, with rearrangement of plasmid pADP-1, has been observed during growth of Pseudomonas sp. ADP on laboratory media lacking atrazine and hence lacking any selective pressure to retain the genes. Other examples of catabolic transposons (60) carry genes to metabolize the following environmental pollutants: toluene (Tn4651/Tn4653), naphthalene (Tn4655), chlorobenzoate (Tn5271), chlorobenzene (Tn5280), and chlorobiphenyl (Tn4371).
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Conclusions |
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Bacteria are metabolic machines, their enzymatic activities being directly connected to acquiring carbon, nitrogen, phosphorus, and sulfur from their environments and fashioning that into a new cell. When those elements are contained within halogenated environmental pollutants, bacteria use dehalogenases to remove one or more halide ions and further metabolize the compounds to support growth of the cell. The dehalogenases are seen to derive from different enzyme superfamilies. There is evidence that catalytically efficient dehalogenases have arisen from precursor enzymes in the time since the introduction of pesticides or solvents into commercial use. The evolution of metabolic pathways for pollutants is facilitated by the horizontal transfer of genes on plasmids and insertion sequence elements. These processes ultimately contribute to cleansing the environment of new chemical compounds of anthropogenic origin.
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FOOTNOTES |
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* This minireview will be reprinted in the 2004 Minireview Compendium, which will be available in January, 2005.
To whom correspondence should be addressed: Dept. of Biochemistry, Molecular Biology, and Biophysics and the Biotechnology Institute, 140 Gortner Laboratory, University of Minnesota, St. Paul, MN 55108. Tel.: 612-625-3785; Fax: 612-625-5780; E-mail: wackett{at}biosci.cbs.umn.edu.
1 The abbreviations used are: DcmA, dichloromethane dehalogenase; AtzA, atrazine chlorohydrolase; TCHQ, tetrachlorohydroquinone; Tn, transposon; IS, insertion sequence.
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