Genetic Adaptation of to Halogenated Aliphatic Compounds Dick B. Janssen, Jan R. van der Ploeg, and Frens Pries Department of Biochemistry, University of Groningen, The Netherlands The bacterial degradation and detoxification of chlorinated xenobiotic compounds requires the production of that are capable of recognizing and converting compounds which do not occur at significant concentrations in nature. We have studied the catabolic route of 1,2-dichloroethane as an example of a pathway for the conversion of such a synthetic compound. In strains of Xanthobacter and Ancylobacterthat have been isolated on 1,2-dichloroethane, the first catabolic step is catalyzed by a hydrolytic haloalkane dehalogenase. The converts 1,2-dichloroethane to 2- chloroethanol but is also active with many other environmentally important haloalkanes such as methylchloride, methylbromide, 1,2-dibromoethane, epichlorohydrin, and 1,3-dichloropropene. Further degradation of 2-chloroethanol proceeds by oxidation to the carboxylic acid and dehalogenation to glycolate. The aldehyde dehydrogenase prevents toxicity of the reactive chloroacetaldehyde that is formed as an intermediate and is necessary for establishing a functional 2-chloroethanol degradative pathway in a strain that is not capable of growth on this compound. - Environ Health Perspect 103(Suppl 5):29-32 (1995) Key words: chlorinated hydrocarbons, biodegradation, dehalogenase, evolution, adaptation

Introduction can cleave or labilize carbon-halogen verted by two different dehydrogenases to The observed environmental persistence bonds. A variety of bacterial enzymes that chloroacetate. This compound is cleaved and toxicity of a broad range of halo- have this capability are known (1). The by a hydrolytic dehalogenase to glycolate, genated aliphatic and aromatic compounds question then arises: how did such enzymes which supports growth of many bacteria. is due to the xenobiotic structural features evolve? They could be derived from en- Both dehalogenases are produced constitu- of these chemicals. Chlorine substitution zymes that convert natural halogenated tively, whereas the alcohol dehydrogenase causes natural compounds such as methane, substrates, many of which are known (2). and the aldehyde dehydrogenase are ethane, ethylene, propane, benzene, or Another possibility is that dehalogenases inducible (3-5). The haloalkane dehaloge- biphenyl to become recalcitrant to bio- have evolved from proteins that catalyze nase (dhIA) and the chloroacetalde- degradation by microorganisms. Recent the same reaction type on nonhalogenated hyde dehydrogenase are plasmid encoded studies on the microbial degradation of a compounds. Thus, a key question is (6). The haloalkane dehalogenase, the number of compounds with a low degree whether and how dehalogenases specifically aldehyde dehydrogenase, and the chloro- of chlorine substitution has led to the recognize the halogen function, and how acetate dehalogenase are only produced by identification of critical metabolic steps. mutations leading to recognition of syn- strains of Xanthobacter that utilize 1,2- Recalcitrance is usually caused by a lack of thetic compounds occur. dichloroethane (5,6). transformation at some point, which leads In this overview, we discuss the bacterial to partial transformation with formation of pathway of 1,2-dichloroethane conversion dead-end products, toxic effects, or even and the possible evolutionary changes that CH2CI-CH2CI dhlA complete inertness. If a productive meta- have led to organisms that can use it as a haloalkane bolic route is not present, i.e., in case of growth . Dichloroethane is, from dehalogenase dhlA KH0H20 35kDa co-metabolism the chemical does not stim- a production point of view, the largest HCI constitutive ulate growth, and adaptation at a popula- chlorinated chemical. It is used for various CH2CI-CH20H tion level does not occur. purposes and is a frequent groundwater alcohol r PQQ dehydrogenase An obvious critical step is the actual contaminant. A u1l mox ~Dnnu 2*(60+10) kDa dehalogenation reaction. Microbial KbJOp ruun2 inducible detoxification of chlorinated substrates Degradation of plasmid CH2CI-CHO aldehyde requires the production of enzymes that 1,2-Dichloroethane dehydrogenase ald NAD+H20 4*55 kDa The degradation of 1,2-dichloroethane, NADH2 inducible which is not known to occur naturally, was CH2CI-COOH investigated with cultures of Xanthobacter chromosome This paper was presented at the Conference on H20 dhIB d haloacetate Biodegradation: Its Role in Reducing Toxicity and autotrophicus and Ancylobacter aquaticus, dehydrogenase Exposure to Environmental Contaminants held which use it as a sole carbon source (3,4). HCI 28kDa 26-28 April 1993 in Research Triangle Park, North CH20H-COOH constitutive Carolina. Manuscript updated: fall 1994; manuscript This led to the establishment of the cata- received: January 23, 1995; manuscript accepted: bolic route shown in Figure 1. The path- February 13, 1995. way is composed of special enzymes and central metabolism Address correspondence to Dr. Dick B. Janssen, Department of Biochemistry, University of Groningen, more general enzymes. The first step is the 9747 AG, Groningen, The Netherlands. Telephone hydrolytic conversion of 1,2-dichloro- Figure 1. The catabolic pathway of 1,2-dichloroethane (31) 50-634209. Fax (31) 50-634165. ethane to 2-chloroethanol, which is con- in X. autotrophicus and A. aquaticus.

Environmental Health Perspectives 29 JANSSEN ETAL.

Chloroacetate Dehalogenase composed of 60-kDa and 10-kDa sub- Table 1. Substrate range of haloalkane dehalogenase. The last step in 1,2-dichloroethane degra- units. The enzyme is induced by methanol dation is catalyzed by a 28-kDa haloacetate or 2-chloroethanol (5). There is no indica- Compound % Activitya Use, relevance dehalogenase that is active with chloroac- tion of adaptational mutations being 1,2-Dichloroethane 100 Vinyl chloride etate, dichloroacetates, bromoacetate, and needed for 2-chloroethanol oxidation by synthesis, solvent Chloromethane 28 Blowing agent, 2-chloropropionic acid. The enzyme is this enzyme. naturally produced produced constitutively, but higher levels Dichloromethane 9 Solvent are found in the stationary phase than in Haloalkane Dehalogenase Ethylchloride 24 Cooling agent the exponential phase of growth (7). The first step in 1,2-dichloroethane metab- 1-Chloropropane 48 - Sequence analysis and biochemical charac- olism is catalyzed by haloalkane dehaloge- 1,3-Dichloropropane 87 - terization (8) have shown that the nase, a protein that in 1,2-Dichloropropane 0.6 Chemical waste enzyme is identical different 1,3-Dichloropropene 96 Nematocide belongs to a class of L-chloropropionic acid isolates of X autotrophicus and A. aquaticus 2-Chloroethylvinylether 12 Waste dehalogenases that converts their substrates (4,12). Haloalkane dehalogenase is a solu- 2-Chloroethylmethylether 6 Waste product with inversion of configuration. Enzymes ble 35-kDa, 310-amino acid protein that Epichlorohydrin 8 Pharmaceutical of this class are not uncommon in various hydrolytically cleaves carbon-chlorine synthesis, resins haloacetate-degrading bacteria, and the bonds to produce alcohols. The substrate 2-Chloroethanol <1 Solvent natural occurrence of haloacetates makes it is 1,2-Dibromoethane 94 Nematocide, range very broad and includes a number gasoline probable that no specific adaptation was of environmentally important compounds Methylbromide 13 Nematocide needed for the acquisition of the last step (Table 1). The best substrate known is the Dibromomethane 112 Naturally produced of 1,2-dichloroethane conversion. nematocide 1,2-dibromoethane, a com- Ethylbromide 24 Aldehydes as Critical pound that has been used widely as a soil 2-Bromoethanol 24 fumigant and that is a frequent groundwa- aActivities are given at a substrate concentration of Intermediates ter contaminant. The Vma values with 1,2- 5 mM. Values are expressed as the percentage of A critical step in the degradation of halo- dichloroethane and 1,2-dibromoethane are activity found with 1,2-dichloroethane, which was 6 genated aliphatics is the oxidation of the both about 6 U/mg protein, but the K,, for pmole/min/mg of protein (turnover number kat 3.5 sub- aldehydes that are produced as inter- 1,2-dibromoethane is 0.008 mM, which is strate molecules/enzyme molecule per second). mediates. The 2-haloaldehydes are bifunc- 100-fold lower than for 1,2-dichloroethane. tionally reactive and can form covalent The evolutionary origin of the dehaloge- products with amines and nucleophilic nase is unknown, but the three-dimen- hydrolytic steps in the bacterial degrada- groups. A lack of aldehyde dehydrogenase sional structure that was recently solved tion of xenobiotic substrates (Figure 2). activity therefore causes toxicity of 2- (13) shows that the enzyme is composed of The sequence around the nucle- chloroethanol (5). Tardif et al. (6) have two domains. The main domain is similar ophilic residue is conserved. shown that X autotrophicus GJ10 produces to a number of hydrolytic enzymes that The mechanism of haloalkane dehaloge- a plasmid-encoded aldehyde dehydroge- can be classified as a/13-fold nase was recently investigated by X-ray nase for the conversion of chloroacetal- (14). This group of enzymes includes crystallography and involves nucleophilic dehyde. We have recently purified the other bacterial proteins catalyzing attack by Asp 124, followed by ester enzyme and found that it is a tetrameric protein with 55-kDa subunits. Genetic studies have indicated that chromosomally box ci ci encoded are also essential for chloro- * 4- ** CH2- CH2 acetaldehyde metabolism (9). Thus, the DhIA 1 ,2-Dichloroethane LVVQDWG G presence of these two activities is sufficient ci to allow growth on chloroethanol. DehHl Chloroacetate LVGHDR G G cH2 -cOOH The lack of a functional aldehyde dehy- DmpD 2-Hydroxymuconic semialdehyde LVGNS F G G R for seems to drogenase bromoacetaldehyde BphD 2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate LVGNSM G G be the reason that 1,2-dichloroethane ICOOH degraders do not use 1,2-dibromoethane XyIF 2-Hydroxy-6-methylmuconic semialdehyde IVGN S FG G OH for growth. In fact, both 1,2-dibro- TcbE Dichlorodienelactone LVGNSF G G moethane and 2-bromoethanol are very 0 4fdE Di- or trichlorodienelactone VVGYC L G G toxic for strains of X autotrophicus or A. COH aquaticus. This can be used to select ClcD Chlorodienelactone LVGYC L G G 0 mutants that lack the alcohol dehydroge- R2 nase (10) or the haloalkane dehalogenase (11). Figure 2. Sequence of a region around the active site nucleophile of several x/4-hydrolase fold enzymes involved in the bacterial degradation of xenobiotics. The enzymes are: DhIA, haloalkane dehalogenase from Xanthobacter Alcohol Dehydrogenase autotrophicus GJ10, from Janssen et al. (21); DehH1, haloacetate dehalogenase from Moraxella, from Kawasaki et The alcohol dehydrogenase for al. (17); DmpD, 2-hydroxymuconic semialdehyde hydrolase from Pseudomonas, from Nordlund and Shingler (22); responsible BphD, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase from Pseudomonas, from Kimbara et al. (23); XyIF, 2-chloroethanol conversion is the periplas- 2-hydroxymuconic semialdehyde hydrolase from Pseudomonas putida, from Horn et al. (24); TcbE, dienelactone mic quinoprotein alcohol dehydrogenase hydrolase from Pseudomonas P51, from van der Meer et al. (25); NfdE, dienelactone hydrolase from Alcaligenes that is commonly present in methylo- eutrophicus, from Perkins et al. (26); ClcD, dienelactone hydrolase from Pseudomonas B13, from Frantz et al. (27). trophic bacteria. The tetrameric enzyme is The -I indicates the position of the nucleophile: *, conserved residues; *, similar residues.

30 Environmental Health Perspectives GENETIC ADAPTATION TO HALOALIPHATICS

general hydrolytic protein that fortuitously Evolution of a A Trppl75 also converts chlorinated substrates. 1,2-Dichloroethane Environmental Relevance of Degradative Pathway N Haloalkane Dehalogenase The evolution of an organism that can H CI The above data suggest that haloalkane grow on 1,2-dichloroethane seems to CH2 oX OH N'~~~~N H CH2 CI-'.HI 'g dehalogenase may have evolved from an require a number of steps. First, a host <----HN N N enzyme that converts related compounds organism that produces an alcohol dehy- o \- o H of natural origin. We have recently found drogenase which can be used for 2- used conversion may have Asp260 H N Trpl25 that 2-chloroethylvinylether may be chloroethanol His289 as a sole carbon source by strains of acquired a gene encoding a haloacetate Aspl24 0 A. aquaticus and that the haloalkane dehalogenase. Addition of a plasmid dehalogenases produced by these organisms encoding a chloroacetaldehyde dehydroge- B rpl75 are identical to the X autotrophicus GJ 10 nase and a haloalkane dehalogenase, enzyme (4,10). Little is known about the modified to become active with the syn- N bacterial utilization of most of the other thetic substrate 1,2-dichloroethane, allows H compounds listed in Table 1. It is con- utilization of this compound. Horizontal CH2 \ ceivable, however, that the enzymes transmission of the plasmid to other facul- C1 involved in the degradation of synthetic tative methylotrophs (Xanthobacter or H OH - H chloroaliphatics such as 1,2-dichloroethane Ancylobacter) that produce a haloacetate are related to enzymes involved in the dehalogenase may have led to a variety of Asp260 H N Trpl25 His289 / o degradation of some of the compounds 1,2-dichloroethane and 2-chloroethylvinyl- Aspl24 o listed in Table 1 or to enzymes that play a ether degrading organisms, as can now be role in the conversion of naturally pro- isolated from environmental samples. Figure 3. Proposed catalytic mechanism of haloalkane duced halogenated compounds. All 1,2-dichloroethane degraders isolated dehalogenase. (A) Cleavage of carbon-halogen chlo- The haloalkane dehalogenase is by far so far are facultative methylotrophs. An rine bond by nucleophilic substitution. (B) Hydrolysis of not a perfect enzyme. The kcat (turnover explanation for this could be that a quino- the covalent intermediate. number) for 1,2-dichloroethane is 3.5 protein alcohol dehydrogenase is needed sec , and the kcatlK is 5100 s-M-1. The for 2-chloroethanol conversion, but on the kinetics have consequences for the K other hand, not all organisms isolated on hydrolysis (15) (Figure 3). The enzyme is, (Monod constant) of the organism: a high 2-chloroethanol are methylotrophs. A dif- for several reasons, a specific dehalogenat- kcat and a low Km of the dehalogenase will ference between chloroethanol and 1,2- ing enzyme. First, there is a specific halide decrease the K (19), and this will lower dichloroethane utilization is that in the , which was identified by X-ray steady-state substrate concentrations in latter case 2-chloroethanol is generated in crystallography and fluorescence measure- mixed reactors. In both natural environ- the cell rather than taken up from the ments (16). It is formed by two tryp- ments and treatment systems, the produc- medium. It is conceivable that a periplasmic tophan residues. The site has a much tion of increased levels of dehalogenase or alcohol dehydrogenase helps to prevent higher affinity for bromide than for chlo- an enzyme with higher affinity would yield toxic cellular levels of chloroacetaldehyde ride, which can explain the lower Km value reduced concentrations of toxicants. that are produced as an intermediate. of 1,2-dibromoethane compared to that for It is conceivable that the dehalogenase The origin of the plasmid-encoded 1 ,2-dichloroethane. can evolve further to improve its kinetics haloalkane dehalogenase and chloroacetal- The nucleophile in haloalkane dehaloge- with 1,2-dichloroethane or other sub- dehyde dehydrogenase is unknown. It is nase and also that in haloacetate dehaloge- strates. This is also suggested by the finding likely that these enzymes are specifically nase (17) is an Asp rather than a Ser or that the same enzyme is present in organ- adapted to xenobiotic substrates, i.e., they Cys residue as in most a/n-fold hydrolases. isms isolated on 1,2-dichloroethane and 2- underwent mutations that were selected for Nucleophilic displacement of a halogen chloroethylvinylether; it seems unlikely as a result of the introduction in nature of from an alkylhalide by a Ser or Cys would that the same amino acid sequence yields a synthetic chlorinated compounds. It thus produce an ether intermediate, which is protein with optimal activity for two such remains to be determined from which pro- much more difficult to hydrolyze than an different substrates. teins the present specialized enzymes have ester. Thus, the presence of a carboxylate as Recently, we have experimentally shown evolved and which mutations took place a nucleophile is also directly related to the that the substrate range of the enzyme can during their adaptation to the degradation nature of the dehalogenase reaction (18). be modified to long-chain chloroalkanes by of toxic haloalkanes. The active site is located between the spontaneous mutations in the cap domain two domains of haloalkane dehalogenase. of the protein (20). The position and char- One of the tryptophan residues (Trp 125) acter of the mutations and the sequence of is located in the main domain and the the wild-type enzyme suggested that simi- REFERENCES other (Trp175) is located in the cap that lar mutations had occurred during the evo- lies on top of the main domain. Thus, both lution of the original enzyme. Thus, short 1. Janssen DB, Pries F, Van der Ploeg domains are specifically evolved to con- tandem sequence repeats that were JR. Genetics and biochemistry of tribute to the catalysis of carbon-halogen detected in the mutants were also present dehalogenating enzymes. Ann Rev bond cleavage. The enzyme clearly is not a in the wild type. Microbiol 48:163-191 (1994).

Volume 103, Supplement 5, June 1995 31 JANSSEN ETAL.

2. Gribble GW. The natural production of chlorinated com- 16. Verschueren KHG, Kingma J, Rozeboom HJ, Kalk KH, pounds. Environ Sci Technol 28:31OA-319A (1994). Janssen DB, Dijkstra BW. Crystallographic and fluorescence 3. Janssen DB, Scheper A, Dijkhuizen L, Witholt B. Degradation studies of the interaction of haloalkane dehalogenase with of halogenated aliphatic compounds by Xanthobacter autotroph- halide ions. Biochemistry 32:9031-9037 (1993). icus GJ10. Apple Environ Microbiol 49:673-677 (1985). 17. Kawasaki H, Tsuda K, Matsushita I, Tonomura K. Lack of 4. Van den Wijngaard AJ, Van der Kamp K, Van der Ploeg J, homology between two haloacetate dehalogenase genes encoded Kazemier B, Pries F, Janssen DB. Degradation of 1,2- on a plasmid from Moraxella sp. strain B. J Gen Microbiol dichloroethane by facultative methylotrophic bacteria. Apple 138:1317-1323 (1992). Environ Microbiol 58:976-983 (1992). 18. Pries F, Kingma J, Pentenga M, Van Pouderoyen G, 5. Janssen DB, Keuning S, Witholt B. Involvement of a quino- Jeronimus-Stratingh CM, Bruins AP, Janssen DB. Site-directed protein alcohol dehydrogenase and an NAD-dependent alde- mutagenesis and oxygen isotope incorporation studies of the hyde dehydrogenase in 2-chloroethanol metabolism in nucleophilic aspartate of haloalkane dehalogenase. Xanthobacter autotrophicus GJ1O. J Gen Microbiol 133:85-92 Biochemistry 33:1242-1247 (1994). (1987). 19. Van den Wijngaard AJ, Wind R, Janssen DB. Kinetics of bac- 6. Tardif G, Greer CW, Labbe D, Lau PCK. Involvement of a terial growth on chlorinated aliphatic compounds. Apple large plasmid in the degradation of 1,2-dichloroethane by Environ Microbiol 59:2041-2048 (1993). Xanthobacter autotrophicus GJ1O. Apple Environ Microbiol 20. Pries F, Van den Wijngaard AJ, Bos R, Pentenga M, Janssen 57:1853-1857 (1991). DB. The role of spontaneous cap domain mutations in 7. Greer CW, Baumier D, Samson R. Application of on-line sen- haloalkane dehalogenase specificity and evolution. J Biol Chem sors during growth of the dichloroethane degrading bacterium 269:17490-17494 (1994). Xanthobacter autotrophicus. J Biotechnol 12:261-274 (1989). 21. Janssen DB, van der Ploeg JR, Pries F. Genetics and biochem- 8. Van der Ploeg JR, Van Hall G, Janssen DB. Characterization istry of 1,2-dichloroethane degradation. Biodegradation of the halocid dehalogenase from Xanthobacter autotrophicus 48:163-191 (1994). GJ1O and sequencing of the dhIB gene. J Bacteriol 22. Nordlund I, Shingler V. Nucleotide sequence of the meta- 173:7925-7933 (1991). cleavage pathway enzymes 2-hydroxymuconic semialdehyde 9. Van der Ploeg JR, Smidt MP, Landa AS, Janssen DB. dehydrogenase and 2-hydroxymuconic semialdehyde hydrolase Identification of a chloroacetaldehyde dehydrogenase involved from Pseudomonas CF600. Biochim Biophys Acta in 1,2-dichloroethane degradation. Apple Environ Microbiol 1049:227-230 (1990). 60:1599-1605 (1994). 23. Kimbara K, Hashimoto T, Fukuda M, Koana T, Takagi M, 10. Van den Wijngaard AJ, Prins J, Smal AJAC, Janssen DB. Oishi M, Yano K. Cloning and sequencing of two tandem Degradation of 2-chloroethylvinylether by Ancylobacter aquati- genes involved in degradation of 2,3-dihydroxybiphenyl to cus AD25 and AD27. Appl Environ Microbiol 59:2777-2783 benzoic acid in the polychlorinated biphenyl-degrading soil (1993). bacterium Pseudomonas sp. strain KKS 102. J Bacteriol 11. Janssen DB, Pries F, Van der Ploeg J, Kazemier B, Terpstra P, 171:2740-2747 (1989). Witholt B. Cloning of 1,2-dichloroethane degradation genes 24. Horn JM, Harayama S, Timmis KN. DNA sequence determi- of Xanthobacter autotrophicus GJ 10, and expression and nation of the TOL plasmid (pWWO) xylGFJ genes of sequencing of the dAIA gene. J Bacteriol 171:6791-6799 Pseudomonas putida: implications for the evolution of aromatic (1989). catabolism. Mol Microbiol 5:2459-2474 (1991). 12. Keuning S, Janssen DB, Witholt B. Purification and characteri- 25. Van der Meer JR, Eggen RI, Zehnder AJ, de Vos WM. zation ofhydrolytic haloalkane dehalogenase from Xanthobacter Sequence analysis of the Pseudomonas sp. strain P51 tcb gene autotrophicus GJ1O. J Bacteriol 163:635-639 (1985). cluster, which encodes metabolism of chlorinated catechols: 13. Franken SM, Rozeboom HJ, Kalk KH, Dijkstra BW. Crystal evidence for specialization of catechol 1,2-dioxygenases for structure of haloalkane dehalogenase: an enzyme to detoxify chlorinated substrates. J Bacteriol 173:2425-2434 (1991). halogenated alkanes. EMBO J 10: 1297-1302 (1991). 26. Perkins EJ, Gordon MP, Caceres 0, Lurquin PF. Organization 14. Ollis DL, Cheah E, Cygler M, Dijkstra BW, Frolow F, and sequence analysis of the 2,4-dichlorophenol hydroxylase Franken S, Hare M, Memington J, Silman I, Schrag J, Sussman and dichlorocatechol oxidative operons of plasmid pJP4. J J, Goldman A. The cx/p hydrolase fold. Protein Eng 5:197-211 Bacteriol 172:2351-2359 (1992). (1992). 27. Frantz B, Ngai KL, Chatterjee DK, Ornston LN, Chakrabarty 15. Verschueren KHG, Selj&e F, Rozeboom HJ, Kalk KH, Dijkstra AM. Nucleotide sequence and expression of clcD, a plasmid- BW. Crystallographic analysis of the catalytic mechanism of borne dienelactone hydrolase gene from Pseudomonas sp. B 13. haloalkane dehalogenase. Nature 363:693-698 (1993). J Bacteriol 169:704-709 (1987).

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