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OnderzoekGBBBiotransformation and BiocatalysisResearch

Biodegradation of halogenated pollutants

D.B. Janssen
Biotransformation and Biocatalysis Group
Groningen Biomolecular Sciences and Biotechnology Institute
University of Groningen 

Synthetic chemicals, environmental pollution and biodegradation

Research on the biodegradation of environmental pollutants, mostly halogenated aliphatic and some aromatic compounds, has been a major research topic in the group for almost 20 years. The work was inspired by many cases of environmental pollution discovered in the early 80s, as well as the lack of scientific knowledge at that time on biodegradation pathways, evolution of new activities and enzyme mechanisms. In fact, the transformation of synthetic organic compounds by microorganisms and microbial enzymes is a scientifically intriguing process of considerable biotechnological importance. Many synthetic compounds have a xenobiotic molecular structure that did not occur on earth in biologically significant concentrations before the development of industrial organic synthesis some 70-100 years ago. Microbial degradation of such compounds therefore is expected to require the evolution of new catabolic pathways, either by acquisition of novel genes orby mutation of existing genes. This makes microorganisms that degrade xenobiotics very attractive for studying the evolution of enzyme specificity and the assembly of new metabolic routes. Furthermore, microorganisms that transform synthetic organic compounds often posses enzymes with interesting regioselectivities and stereoselectivities. Exploiting and engineering the catalytic potential of microbial enzymes can lead to the development of new biological processes for the production of valuable fine chemicals. 

Bacterial growth on halogenated chemicals

Many chlorinated and brominated chemicals that were originally expected to be recalcitrant to biodegradation now appear to be able to serve as a carbon source for some specialized microorganisms. Such organisms have developed a catabolic pathway that harvests energy and carbon from the halocarbon compound when degrading it. Examples of compounds for which we found organisms are listed in Table 1.

Other organohalogens can only be degraded under aerobic conditions by cometabolism or by anaerobic, reductive reactions. The former reactions are particularly well carried out by methanotrophic bacteria that produce a soluble type of methane monooxygenase. This enzyme has a very broad substrate range and can cooxidize various recalcitrant organohalogens, including trichloroethylene and dichloroethylenes. A drawback is that these cometabolic conversion often are accompanied by toxic effects, which are cause by the fact that various reactive transformation products can be formed.

In view of these effects, the isolation of organisms that can degrade and grow on organohalogens still is an important bottleneck towards the degradation of an efficient treatment process. How an organism that can degrade an organohalogen can be applied in bioremediation was nicely illustrated by Stucki and Thuer who developed a full scale process for groundwater treatment employing 1,2­dichloroethane degrading bacteria (Stucki G, and Thuer M. 1995. Experiences of a large-scale application of 1,2-dichloroethane degrading microorganisms for groundwater treatment. Environ. Sci. Technol. 29:2339-2345).

Isolated organisms that grow on organohalogens as sole carbon source.

Compound

Organism

Reference

dichloromethane

Hyphomicrobium GJ21

unpublished

1,2-dichloroethane

Xanthobacter autotrophicus GJ10, Ancylobacter aquaticus AD20

Janssen et al., 1985, van den Wijngaard et al., 1992

2-chloroethyl-vinylether

Ancylobacter aquaticus AD27

van den Wijngaard et al., 1992

2-chloroethanol

Pseudomonas GJ1

van der Ploeg et al., 1996

1,6-dichlorohexane

Rhodococcus GJ70

Janssen et al., 1987

epichlorohydrin

Agrobacterium radiobacter AD1

van den Wijngaard et al., 1989

1-chloro-2,3-propanediol

Arthrobacter AD2

van den Wijngaard et al., 1989

1,3-dichloropropene

Pseudomonas pavonaceae 170

Poelarends et al., 2000a

1,2-dibromoethane

Mycobacterium GP1

Poelarends et al., 2000b

chlorobenzene

Pseudomonas GJ31

Mars et al., 1997

1,2-dichlorobenzene

Pseudomonas GJ60

Oldenhuis et al., 1989

Dehalogenases cleave carbon-halogen bonds

Halogenated aliphatic compounds are widely used as solvents and intermediates in the production of agrochemicals and polymers. Studies on the biochemistry of catabolic pathways in organisms that use such compounds as a carbon source for growth (see Table 1) have led to the discovery of several new dehalogenating enzymes.

Four different classes of aliphatic dehalogenases are now well defined with X-ray structures solved and reliable mechanistic insight available (Table 1). These are: 1) haloalkane dehalogenases (e.g. DhlA from Xanthobacter autotrophicus and DhaA from Rhodococcus erythropolis), a group of proteins belonging to the α/β-hydrolase fold family; 2) halocarboxylic acid dehalogenases (e.g. DhlB of X. autotrophicus) of the HAD superfamily of proteins, which includes phosphatases; 3) halohydrin dehalogenases (e.g. HheC from Agrobacterium radiobacter), a class of proteins that shares similarity to the short chain dehydrogenase-reductase family of proteins; 4) chloroacrylic acid dehalogenases (e.g. CaaD from a 1,3-dichloropropene degrading Pseudomonas cichorii), enzymes that share similarity to the 4-oxalocrotonate tautomerase superfamily.

From the study of these dehalogenases, a number of important conclusions have emerged. First, it appears that enzymes that act on halogenated compounds really have evolved to carry out dehalogenation reactions. Thus, the capacity to cleave carbon-halogen bonds in xenobiotic structural elements is not just a side reaction of an enzyme that has a function in the conversion of some non-halogenated natural metabolite. Dehalogenases possess a specific halide-binding site, formed by groups that can donate hydrogen bonds to the halide ion, and that can bind both the halogenated substrate and the displaced halide.

Second, dehalogenases appear to be evolutionarily related to proteins that carry out conversions with natural compounds. For example, the halohydrin dehalogenases are related to the SDR (short-chain dehydrogenase-reductase) family of proteins, which is a diverse group of enzymes that oxidize alcohols and various other compounds (van Hylckama Vlieg et al., 2001).

Third, various open reading frames that show sequence similarity to known dehalogenases are present in many organisms, as shown by genome sequencing projects. The activity and function of the encoded proteins is not always clear, but they could be involved in the biodegradation of natural organohalogens, which may be formed by haloperoxidases or specific halogenating enzymes in a variety of environments. The abundance of these putative dehalogenase sequences varies a lot, some being rather common, such as genes that encode chlorocarboxylic acid dehalogenases, whereas others are very rare, e.g. the putative halohydrin dehalogenase gene sequences.

Our recent work has focused on halohydrin dehalogenases, which are involved in the biodegradation of vicinal chloro- or bromoalcohols. The enzymes possess unique biocatalytic potential and interesting evolutionary relations. 

Catabolic pathways for some haloaliphatics. 1, degradation of 1,2-dichloroethane by X. autotrophicus GJ10 (Janssen et al., 1985; Janssen, 2004); 2, degradation of 1,2-dibromoethane by Mycobacterium GP1. The route circumvents 2-bromoacetaldehyde, which is extremely toxic; 3, degradation of 1,3-dichloro-2-propanol via epichlorohydrin by Agrobacterium radiobacter AD1; 4, degradation of 1,3-dichloropropene by Pseudomonas pavonaceae 171.
Catabolic pathways for some haloaliphatics. 1, degradation of 1,2-dichloroethane by X. autotrophicus GJ10 (Janssen et al., 1985; Janssen, 2004); 2, degradation of 1,2-dibromoethane by Mycobacterium GP1. The route circumvents 2-bromoacetaldehyde, which is extremely toxic; 3, degradation of 1,3-dichloro-2-propanol via epichlorohydrin by Agrobacterium radiobacter AD1; 4, degradation of 1,3-dichloropropene by Pseudomonas pavonaceae 171.

Haloalkane dehalogenase

The best studied dehalogenases are undoubtedly the haloalkane dehalogenases, especially the enzyme from Xanthobacter autotrophicus GJ10, a 310 aa hydrolytic α/β-hydrolase fold enzyme, composed of a main domain with the general fold and a cap domain, which seems to be involved in determining the substrate specificity (Pries et al., 1994; Pikkemaat and Janssen, 2002). Its mechanism and kinetics have been investigated in detail.

A dehalogenase similar to the Xanthobacter enzyme has been discovered in a gram positive organism growing on 1-chlorobutane, a Rhodococcus (Janssen et al., 1987; Poelarends et al., 2000a, Janssen, 2004). This enzyme (DhaA) shares only about 28 % sequence identity with the haloalkane dehalogenase from Xanthobacter (DhlA). When we studied the genes involved in the initial dehalogenation reactions of 1,3-dichloropropylene in Pseudomonas pavonaceae 170 and Mycobacterium sp. GP1, it appeared that these organisms possessed haloalkane dehalogenase genes that were (almost) identical to the one found in Rhodococcus (Poelarends et al., 2000a, b). To understand the distribution of these genes, we have cloned and compared the sequences of the gene regions encoding the dehalogenases in these three organisms. The results indeed suggest that the dehalogenase gene clusters in bacteria growing on 1,2-dibromoethane and 1,3-dichloropropene originate from the more widespread 1­chlorobutane gene cluster of Rhodococcus erythropolis.

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Other dehalogenases

After the initial studies on haloalkane dehalogenases, several otherdehalogenases were mechanistically and structurally investigated. The Table gives a few examples.

Halocarboxylic acid dehalogenases of the HAD family are enantioselective with 2-chloropropionic acid and related to a broad family of proteins that share a conserved topology and positioning of the catalytic residues. The family includes several phosphatases (Janssen et al., 2005).

Halohydrin dehalogenases belong to the SDR family of proteins (van Hylckama Vlieg et al., 2001). They do not catalyze a hydrolytic reaction, but instead convert vicinal halohydrins to epoxides.

Chloroacrylic acid dehalogenases convert 3-chloroacrylic acid to malonic semialdehyde. These proteins belong to 4-OT family of tautomerases-isomerases (Poelarends et al., 2001). 

Further reading

Janssen DB, Scheper A, Dijkhuizen L, & Witholt B. 1985. Degradation of halogenated aliphatic compounds by Xanthobacter autotrophicus GJ10 Appl Environ Microbiol 49: 673-677.

Janssen DB, Jager D, Witholt B. 1987. Degradation of n-haloalkanes and alpha, omega-dihaloalkanes by wild-type and mutants of Acinetobacter sp. strain GJ70. Appl Environ Microbiol 53:561-6. Note: the organism described here was later classified as a Rhodococcus (Poelarends et al., 2000a).

Janssen DB, Dinkla IJ, Poelarends GJ, Terpstra P. 2005. Bacterial degradation of xenobiotic compounds: evolution and distribution of novel enzyme activities. Environ Microbiol 7:1868-82.

Janssen DB, Oppentocht JE & Poelarends GJ. 2001. Microbial dehalogenation. Current Opinion Biotechnol 12, 254-258

Janssen DB. 2004. Evolving haloalkane dehalogenases. Curr Opin Chem Biol 8:150-9.

Mars AE, Kasberg T, Kaschabek SR, van Agteren MH, Janssen DB, Reineke W. 1997. Microbial degradation of chloroaromatics: use of the meta-cleavage pathway for mineralization of chlorobenzene. J Bacteriol 179:4530-7.

Oldenhuis R, Kuijk L, Lammers A, Janssen DB, & Witholt B. 1989. Degradation of chlorinated and non-chlorinated aromatic solvents in soil suspensions by pure bacterial cultures Appl Microbiol Biotechnol 30: 211-217.

Pikkemaat MG, Janssen DB. 2002. Generating segmental mutations in haloalkane dehalogenase: a novel part in the directed evolution toolbox. Nucleic Acids Res. 30:E35-5.

Poelarends GJ, Kulakov LA, Larkin MJ, van Hylckama Vlieg JET, Janssen DB. 2000a. Roles of horizontal gene transfer and gene integration in evolution of 1,3 dichloropropene- and 1,2 dibromoethane-degradative pathways. J Bacteriol 182: 2191-9.

Poelarends GJ, Zandstra M, Bosma T, Kulakov A, Larkin MJ, Marchesi JR, Weightman AJ, Janssen, DB. 2000b. Haloalkane-utilizing Rhodococcus strains isolated from geographically distinct locations possess a highly conserved gene cluster encoding haloalkane catabolism. J Bacteriol 182: 2725-31.

Poelarends GJ, Saunier R & Janssen DB. 2001. The trans-3-chloroacrylic acid dehalogenase from Pseudomonas pavonaceae shares structural and mechanistic similarities with 4-oxalocrotonate tautomerase. J Bacteriol 183: 4269-77.

Pries F, van den Wijngaard AJ, Bos R, Pentenga M, Janssen DB. 1994. The role of spontaneous cap domain mutations in haloalkane dehalogenase specificity and evolution. J Biol Chem 269:17490-4.

Van den Wijngaard, AJ, Janssen, DB & Witholt, B. 1989. Degradation of epichlorohydrin and halohydrins by bacterial cultures isolated from freshwater sediment Journal of General Microbiology 135: 2199-2208.

Van den Wijngaard, AJ., Van der Kamp, KWHJ., Van der Ploeg, JR, Pries, F, Kazemier, B & Janssen, DB. 1992. Degradation of 1,2-dichloroethane by Ancylobacter aquaticus and other facultative methylotrophs Appl Environ Microb 58: 976-983.

Van der Ploeg JR, Kingma J, De Vries EJ, Van der Ven JG, Janssen DB. 1996. Adaptation of Pseudomonas sp. GJ1 to 2-bromoethanol caused by overexpression of an NAD-dependent aldehyde dehydrogenase with low affinity for halogenated aldehydes. Arch Microbiol 165:258-64.

Van Hylckama Vlieg JET, Tang L, Lutje Spelberg JH, Smilda T, Poelarends GJ, Bosma T, van Merode AE, Fraaije MW, Janssen DB. 2001. Halohydrin dehalogenases are structurally and mechanistically related to short-chain dehydrogenases/reductases. J Bacteriol 183: 5058-66.

Last modified:03 October 2012 09.40 a.m.