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Bacterial degradation of xenobiotic compounds: evolution and distribution of novel enzyme
activities
Janssen, D.B.; Dinkla, I.J.T.; Poelarends, G.J.; Terpstra, P
Published in:Environmental Microbiology
DOI:
10.1111/j.1462-2920.2005.00966.x
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Janssen, D. B., Dinkla, I. J. T., Poelarends, G. J., & Terpstra, P. (2005). Bacterial degradation of xenobiotic
compounds: evolution and distribution of novel enzyme activities: evolution and distribution of novel
enzyme activities. Environmental Microbiology, 7(12), 1868 - 1882. https://doi.org/10.1111/j.14622920.2005.00966.x

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Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2912© 2005 The Authors; Journal compilation © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd71218681882Review ArticleBacterial degradation of xenobiotic compoundsD. B. Janssen, I. J. T. Dinkla, G. J. Poelarends and P. Terpstra

Environmental Microbiology (2005) 7(12), 1868–1882

doi:10.1111/j.1462-2920.2005.00966.x

Minireview
Bacterial degradation of xenobiotic compounds:
evolution and distribution of novel enzyme activities
Dick B. Janssen,1* Inez J. T. Dinkla,1Gerrit J. Poelarends1 and Peter Terpstra2
1
Biochemical Laboratory, Groningen Biomolecular
Sciences and Biotechnology Institute, University ofGroningen, Nijenborgh 4, 9747 AG Groningen, theNetherlands.
2
Section Medical Biology, University Medical Centre
Groningen, University of Groningen, A. Deusinglaan 1,
9713 AV Groningen, the Netherlands.
Summary
Bacterial dehalogenases catalyse the cleavage ofcarbon-halogen bonds, which is a key step in aerobic
mineralization pathways of many halogenated compounds that occur as environmental pollutants. Thereis a broad range of dehalogenases, which can beclassified in different protein superfamilies and havefundamentally different catalytic mechanisms. Identical dehalogenases have repeatedly been detected inorganisms that were isolated at different geographicallocations, indicating that only a restricted number ofsequences are used for a certain dehalogenation
reaction in organohalogen-utilizing organisms. At thesame time, massive random sequencing of environmental DNA, and microbial genome sequencing
projects have shown that there is a large diversity ofdehalogenase sequences that is not employed by
known catabolic pathways. The corresponding proteins may have novel functions and selectivities that
could be valuable for biotransformations in the future.
Apparently, traditional enrichment and metagenome
approaches explore different segments of sequencespace. This is also observed with alkane hydroxylases, a category of proteins that can be detected on
basis of conserved sequence motifs and for which alarge number of sequences has been found in isolated bacterial cultures and genomic databases. It is
likely that ongoing genetic adaptation, with the

Received 25 May, 2005; accepted 19 October, 2005. *For correspondence. E-mail d.b.janssen@rug.nl; Tel. (+31) 503 634 209; Fax(+31) 503 634 165.

recruitment of silent sequences into functional catabolic routes and evolution of substrate range by mutations in structural genes, will further enhance thecatabolic potential of bacteria toward synthetic organohalogens and ultimately contribute to cleansing
the environment of these toxic and recalcitrant
chemicals.
Introduction: degradable and recalcitrant compounds
The recalcitrance of many synthetic chemicals to biodegradation is mainly due to a lack of enzymes that can carryout critical steps in a catabolic pathway. This especiallyholds for low-molecular weight halogenated compounds.
These xenobiotic chemicals are relatively water soluble
and bioavailable, and theoretically could be converted byshort metabolic routes to intermediates that support cellular growth under aerobic conditions. Yet, no organisms
have been found that oxidatively degrade and use as acarbon source important environmental chemicals suchas chloroform, trichloroethylene, 1,1,1-trichloroethane,
1,2-dichloropropane and 1,2,3-trichloropropane (Fig. 1).
Attempts to obtain enrichments or pure cultures that aerobically grow on these chemicals have met no success.
However, some other halogenated chemicals are easily
biodegradable, and cultures that utilize chloroacetate,2-chloropropionate and 1-chlorobutane can be readilyenriched from almost any soil sample (Leisinger, 1996;
van Agteren et al., 1998). For still other compounds, degradative organisms can be isolated, but only after prolonged adaptation or if a suitable inoculum is used inwhich the catabolic activity that is searched for apparently
is already enriched due to pre-exposure to halogenatedchemicals in the environment. Compounds of this class ofintermediate degradability include dichloromethane, 1,2dichloroethane and the nematocides 1,2-dibromoethaneand 1,3-dichloropropene (Fig. 1).
With halogenated compounds, an obvious critical stepin a potential biodegradation pathway is dehalogenation.
Biochemical research with organisms that grow on halogenated compounds has shown that a broad range of
dehalogenases exists, both for aliphatic and aromaticcompounds. The enzymes represent different reaction© 2005 The Authors
Journal compilation © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd

Bacterial degradation of xenobiotic compounds 1869

degradable (aerobic)

difficult

CH3Cl

CH2Cl2

methylchloride

methyleneechloride

impossible

H

Cl

H
C

CHCl3

C

chloroform

ClCl

dichloroethylene

H2C COOH
chloroacetic
acid

Cl

Cl

Cl

CH2 CH2CH3 C Cl

Br

Br

CH2

CH2

1,2-dichloroethaneFig. 1. Degradability of some (halogenated)aliphatic and aromatic compounds. The substrates on the left have often found to be readily
degradable, and organisms that utilize them for
growth are easily isolated from soil or water.
The compounds on the right have repeatedly
been found to be recalcitrant and enrichment
cultures were negative. The other compounds(middle) may be biodegradable, but organisms
are not ubiquitous and success of enrichment
very much depends on conditions and choiceof inoculum.

Cl
1,1,1-trichloroethane

1,2-dibromoethane

CH3
H

Cl
C

C
H

H

Cl

Cl

CH2

R
toluenesevinyl chloride

Cl

CH

Cl

CH2

epichlorohydrin
OH
Cl

CH

Cl

Cl

Cl

Cl

CH2

CH

CH2

1,2,3-trichloropropane
Cl
Cl

Cl

hexachlorocyclohexane

H
C

Cl
Cl

CH3

1,3-dichloropropyleneClchlorobenzene

Cl

CH2

1,2-dichloropropane

CH

Cl

O
H2C

CH

Cl

Cl

C
Cl

trichloroethylene

naphthalene

Cl

Cl
Cl

pentachlorophenol

types, and they may or may not use cofactors for activity.
Several X-ray structures have been solved, and thesesuggest that most of these enzymes have evolved specifically to catalyse a dehalogenation reaction, and are notjust enzymes that carry out a certain reaction with anatural compound and fortuitously also cleave carbonhalogen bonds in a xenobiotic organohalogen compound
due to catalytic promiscuity. The substrate range of adehalogenase often determines whether or not a compound is degradable, although the accumulation of toxic
intermediates after an initial dehalogenation step may
also be the cause of poor metabolism, especially with
substrates carrying more than one halogen (van Hylckama Vlieg et al., 2000). In view of the key role of dehalogenases, several questions arise. How do newdehalogenases arise in nature? What are their catalytic
mechanisms and how do they influence the range of compounds that can be converted? How are these enzymes
distributed in environmental organisms?
This review addresses some of these issues, bydescribing examples of catalytic mechanisms of dehalogenases that were obtained from X-ray crystallography.
From these mechanisms, conserved functional sequencemotifs can be derived, and by using these in searches of
databases, an estimate is obtained about the abundanceof some key dehalogenases in bacteria. The results arecompared with the situation for alkane hydroxylases,
which are the key enzymes in the initial steps of alkane
metabolism. Sequence information and comparison of
catabolic gene clusters also shed light on the mechanisms
by which new catabolic activities may evolve and become
distributed.
Dehalogenase mechanisms
Five dehalogenases that are members of different protein
superfamilies have been studied by X-ray crystallography.
This has provided detailed insight in their catalytic mechanisms and evolutionary relationships. For several otherdehalogenases, structural and mechanistic insight has
been obtained by comparing their sequences with othermembers of the same phylogenetic family, often in combination with mutagenesis experiments. From this work, it
has become clear that most dehalogenases belong toprotein superfamilies that harbour both dehalogenases
and proteins that carry out completely different reactions(de Jong and Dijkstra, 2003; Fig. 2).
The first example is provided by the haloalkane dehalogenases, with three solved X-ray structures (DhlA, DhaA
and LinB in Table 1). These proteins possess a α/βhydrolase fold main domain, with a cap domain on top of
it and the active site located in between the two domains© 2005 The Authors
Journal compilation © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1868–1882

1870 D. B. Janssen, I. J. T. Dinkla, G. J. Poelarends and P. Terpstra
Trp175

Trp175

A
N
Cl
CH2

Cl

CH2

O

N

H N

O

N

H

O-

H

Cl

H
CH2

H

-

O

N

O

H N

H

H

B

His289

N

Ala9
CH3
C
C
O
O
N
C
H

Asp124

Tyr10

NH

H
C

Cl

C

O

C

H

N

H

C
H

-

N+

C
O

Phe175

C

O

C

H
O

H
Phe175

Asp8

H
O

H

O
Ser132

Tyr145

C H
H2 N
N H
+
O
H
H
O
C
H
H
C
O
Cl
H
R

H

O
Cl C
H
H
C
O
R
H
H

C

H

Ser132N

Leu178αGlu52
O

NH2
+

HN

-

O

αArg8
NH

H
H

N+

C

Arg149O

C
N
Leu178DO

C

Lys147

H

H

Cl- H

N

O

NH2

O

Tyr145C H
H2N + N

H

O

H
H

H

N

H
Asp8

Arg149O

CH3C

Lys147

C

O
H

C

H

Arg39
NH

H

Ser114

Trp125

O

Tyr10

O

H

N

Asp124

H

O

H

O

O-

NH2

O

H

C

CH3

H

Ser114H N
HO
H

His289

H
Ala9
C
CH3
C N
C
O
O H
N
C
H

Arg39

H

O

H

Asp260

N

H

H

O

C

-

O

Trp125

H
N

O

O

O
Asp260

O

H

O

H

Cl-

CH2

N

O

HN+

OH

O-

H2N+

αArg8
NH

HN+

H

OH

O-

O

αArg11

NH
O-

Cl

H2N+

N+

NH

H
H
O

O

αArg11

NH
-

Cl

NH2

O

H2N+

NH2

H

H

NH2

αGlu52O

H

αArg11

O

H
Cl

NH2

αGlu52O

βPr o1

H

H
N+

βPro1

NH2

H
N

NH

βPro1

Fig. 2. Common dehalogenase mechanisms for aliphatic organochlorine compounds.
A. Hydrolytic dehalogenation catalysed by an α/β-hydrolase fold type haloalkane dehalogenase (DhlA).
B. Hydrolytic dehalogenation catalysed by HAD-type haloacid dehalogenase (DhlB).
C. Dehalogenation by an SDR-type halohydrin dehalogenase (HheC).
D. Chloroacrylic acid dehalogenation by a 4-OT type chloroacrylic acid dehalogenase (CaaD).
© 2005 The Authors
Journal compilation © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1868–1882

Bacterial degradation of xenobiotic compounds 1871
(Verschueren et al., 1993). Their properties have recently
been reviewed (Janssen, 2004). The α/β-hydrolase
structural fold is also found in lipases, acetylcholinesterases, esterases, lactonases, epoxide hydrolases andothers, showing that the haloalkane dehalogenases
belong to a protein superfamily of which the members
carry out diverse reactions, mostly with non-halogenatedcompounds. The key residues for identification of new
haloalkane dehalogenase sequences are the catalytic
pentad amino acids: a nucleophilic aspartate, a conserved
histidine base close to the C-terminus, an acidic residue
located in the sequence between the Asp and His, andtwo residues involved in binding of the halide. Of these
five residues, the nucleophilic Asp, the His and one halidebinding Trp positioned next to the nucleophilic Asp are
fully conserved (Fig. 2A).
A rather common group of aliphatic dehalogenases are
the haloacid dehalogenases. These have been divided ingroup I and group II enzymes, of which the latter are wellcharacterized (Hisano et al., 1996; Ridder et al., 1999).
They define the so-called HAD superfamily of hydrolases.
The enzymes (DhlB, HAD-Ps in Table 1) have a nucleophilic aspartate close to the N-terminus and share struc-tural similarity with the phosphatase domain found inmany proteins, including eukaryotic transporter proteins.
The conserved nucleophilic aspartate is involved in forming a covalent intermediate by a nucleophilic substitution,
similar to what was found in haloalkane dehalogenases.
For database analysis, other important catalytic residues
are an arginine at a position (Nu + c. 30), which is involved
in halide binding, and a lysine further downstream thatacts as the catalytic base needed for cleavage of thecovalent intermediate (Fig. 2B).
A third family of aliphatic dehalogenases is formed by
the halohydrin dehalogenases, of which the structure has
been investigated recently (de Jong et al., 2003).The catalytic mechanism of these enzymes is somewhat similar
to that of members of the short-chain dehydrogenasereductase (SDR) superfamily of proteins. Both the dehalogenases and the SDR enzymes possess a conserved
catalytic triad for proton abstraction from the hydroxyl
group of the substrate (van Hylckama Vlieg et al., 2001).
In the case of SDR proteins, the negative charge developing on the hydroxyl oxygen is transferred via a hydrideto the NAD(P)+ cofactor; instead, in the dehalogenases it
is passed on to the halogen at a neighbouring carbon

Table 1. Number of positive hits obtained with protein sequences of dehalogenases and alkane hydroxylases as queries in Blast-P searches
against protein sequence databases.

Query
protein

GenBank
GI numberSuperfamilyFiltered byconserved
pattern or residues

No of identicalgenes foundin isolates

No. of homologues
in microbial
genomesNo. of homologues
in ArchaegenomesNo. ofhomologues
in Sargasso Sea

DcmA
DhlA
DhaA
LinB
DhlB
HAD-PsHheA

4825027296813114657
9789853
3122178
3122176
15213645

GST
α/β-HF
α/β-HF
α/β-HF
HAD
HAD
SDR

126H-W*
124D-W-G, 289H*
106D-W-G, 272H*
108D-W-G, 273F*
8D, 39R, 147K#
10D, 41R, 151K#
135S-X(7,17)-Y-X(3)-R*

2 (33–22%)
15 (50–26%)
28 (53–23%)
10 (69–25%)
39 (57–25%)
35 (55–23%)
6 (30–21%)

0
1 (30%)
1 (26%)
0
3 (26–24%)
4 (27–23%)
0

4 (30–25%)
35 (41–21%)
44 (49–22%)
43 (65–21%)
119 (52–23%)
115 (50–24%)
4 (31–21%)

HheB
HheC

1520911915213643

SDR
SDR

126S-X(7,17)-Y-X(3)-R*
132S-X(7,17)-Y-X(3)-R*

16 (45–19%)
6 (30–20%)

1 (27%)0

10 (53–21%)
5 (31–21%)

CaaD
LinA
CbzA
AtzA
TriA
PcpC
AlkB

10637969
51859616
239248432455822
42558845
22417110113639

4-OT
DH
ECH
DA
DA
GST
AH

9R-X-X-R*
25D, 73H#
90H, 145D#
60H-X-H, 243H, 327D*
76H-X-H, 251H, 287H*
12S-X-C*
269 N-Y-X-E-H-Y-G*

11 (> 96%)
12 (100%)
7 (> 98%)
3 (> 99%)
7 (49–39%)
12 (77–39%)
2 (99%),
3 (49–48%)
1 (98%)
2 (100–92%),
2 (49%)
0
3 (> 99%)
6 (86–51%)
4 (> 98%)
2 (> 99%)
1 (96%)
9 (100–77%)

0
4 (30–27%)
21 (41–26%)
134 (36–20%)
156 (36–21%)
2 (37–23%)
21 (50–32%)

00
1 (28%)
12 (31–27%)
21 (26–20%)
0
0

AlkM

2623971AH

281 N-Y-X-E-H-Y-G*

12 (90–62%)

1 (100%),20 (50–32%)

0

6 (35–29%)
3 (31–25%)
48 (37–27%)
20 (32–19%)
32 (34–21%)
49 (41–19%)
2 (82%),
49 (50–25%)
51 (56–28%)

Protein sequences (column 2) were compared by BlastP (Altschul et al., 1990; 1997) to the NCBI whole microbial database (295 proteomes,
April 2005) and to the NCBI environmental Sargasso sea proteins (Venter et al., 2004). Hits with an E-score < 0.01 were considered homologues.
Output lists were filtered by looking for conserved residues in a multiple alignment (#, column 4) or by using PHI-BlastP with a conserved pattern(* column 4). If uncertain, sequences in the low-similarity region were used as Blast queries against the Uniprot database to confirm their identity.
Results in columns 5–8 give the number of homologues followed by the percentage range of amino acid identities. In some cases very highscores are mentioned separately. AlkB, AlkM: only hits containing motif C (see text) were counted. AlkM scores 100% in one case in column 6
because the whole genome of the original host has been sequenced.
GST, glutathione transferase; α/β-HF, α/β-hydrolase fold family; HAD, haloacid dehalogenase; SDR, short chain dehydrogenase reductase; 4OT, 4-oxalocrotonate tautomerase; DH, dehydratase; ECH, enoyl CoA hydratase; DA, deaminase; AH, alkane hydroxylase. The abbreviations forthe proteins and their functions are given in the text.
© 2005 The Authors
Journal compilation © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1868–1882

1872 D. B. Janssen, I. J. T. Dinkla, G. J. Poelarends and P. Terpstra
atom, which is displaced by an intramolecular substitution
mechanism, resulting in the formation of an epoxide(Fig. 2C, HheA, HheB, HheC). Thus, no covalent intermediate is formed during dechlorination catalysis by thisenzyme.
Another group of recently characterized dehalogenating
proteins is formed by the chloroacrylic acid dehalogenases (CaaD in Table 1), which are present in bacteria that
degrade the nematocide 1,3-dichloropropene. These proteins can dehalogenate a substrate in which the halogen
is bound to an sp2-hybridized carbon atom, and there arecis- and trans-specific enzymes (Poelarends and Whitman, 2004). Structures have recently been solved, andthey confirmed what was expected on basis of thesequence, i.e. that the enzymes belong to the tautomerase superfamily of proteins. The members of thissuperfamily are characterized by a ring-like hexameric
structure, which is formed by a trimer of dimers. In somemembers of the tautomerase superfamily, such as 4oxalocrotonate tautomerase (4-OT), the hexamers arecomposed of identical peptides, while in other members,
such as trans-3-CaaD, they are composed of different but
similar peptide chains. Still other members of the superfamily, like cis-3-CaaD, have a trimeric quaternary structure. In these dehalogenases, the catalytic mechanism
does not involve the formation of a covalent enzymesubstrate intermediate (de Jong et al., 2004). Instead, themechanism of trans-3-CaaD is similar to that of ahydratase, with a key role for the N-terminal proline ofthree of the subunits, which acts as the general acidcatalyst that protonates the C-2 carbon atom in trans-3chloroacrylic acid (Fig. 2D) (Azurmendi et al., 2004). In
CaaD, no clear halide-binding site has yet been detected
and it is possible that these enzymes mechanistically
carry out only a hydration reaction to form an unstablehalohydrin intermediate that rapidly dechlorinates.
For several other aliphatic dehalogenases, no structures have been solved, but mechanistic information
can be deduced from similarity to enzymes that are wellcharacterized (Leisinger et al., 1994). For example,
dichloromethane dehalogenase (DcmA) catalyses theconversion of dichloromethane to formaldehyde in a glutathione-dependent reaction. Catalysis was proposed toproceed via the formation of a reactive S-(chloromethyl)glutathione intermediate that stays bound to the
enzyme and decomposes by solvolysis, which may be
the rate-limiting step (Stourman et al., 2003). Sitedirected mutagenesis of DcmA demonstrated the essentiality of a serine residue in the N-terminal part of theprotein, a residue also shown to be critical for catalysis inθ-class glutathione transferases (Vuilleumier and Leisinger, 1996). This conserved catalytic serine may play arole in enhancing the nucleophilicity of the glutathione
thiol.

An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in thebacterial degradation of lindane (α-hexachlorocyclohexane). In a peculiar reaction, HCl is eliminated, converting
the substrate to pentachlorocyclohexene. A structure has
not been solved, but a mechanism was predicted on basisof the stereochemistry of the reaction and low but significant sequence similarity to scytalone dehydratase. The
reaction is proposed to involve abstraction of an axial
proton, with concomitant anti-elimination of a trans-axial
chloride from the adjacent carbon atom (Nagata et al.,
2001; Trantirek et al., 2001). A conserved His-Asp pair
was proposed to be involved in proton abstraction, but
details about halogen binding and stabilization are stilllacking.
If we consider aromatic compounds, it is again possible
to distinguish several phylogenetically and mechanisticallydifferent dehalogenases. The carbon-halogen bond
between a halogen and an arenic or vinylic carbon atom
is more difficult to cleave than the one between a halogen
and an sp3-hybridized carbon atom. Therefore, mosthaloaromatics are dehalogenated after ring cleavage.
However, a nucleophilic displacement of aromatic halogens occurs in 4-chlorobenzoate degradation. This is thefifth type of dehalogenase for which a structure has beensolved (Benning et al., 1998). The compound is first activated by conjugation to coenzyme A, after which a hydrolytic dehalogenase (CbzA) that belongs to the enoyl
hydratase superfamily, displaces the halogen by a nucleophilic addition-elimination mechanism. No distinct halidebinding site is present in the X-ray structure, but a nucleophilic aspartate and a histidine involved in catalysis wereidentified (Zhang et al., 2001). Another aromatic dehalogenase is tetrachlorohydroquinone dehalogenase. This
enzyme catalyses the replacement of chlorine by hydrogen in tetra- and trichlorohydroquinone during thedegradation of pentachlorophenol by Sphingbium chlorophenolicum (Kiefer and Copley, 2002). The protein(PcpC in Table 1) bears sequence and mechanistic similarity to glutathione transferases.
A specific hydrolytic dehalogenase (AtzA) is also
involved in the bacterial degradation of atrazine. This
enzyme is related to melamine deaminase (TriA) and theAtzA and TriA proteins differ only by nine amino acidsubstitutions (Seffernick et al., 2001).
Origin and distribution of dehalogenase genesequences
Analysis of the genetic organization of biodegradation pathways provides insight into the genetic processes that led
to their evolution. It appears that catabolic genes for xenobiotic compounds are often associated with transposable
elements and insertion sequences. They are also fre-© 2005 The Authors
Journal compilation © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1868–1882

Bacterial degradation of xenobiotic compounds 1873
quently located on transmissible plasmids. One striking
example of a mobile element that has assisted catabolic
genes in their dissemination is IS1071. This insertion element flanks the haloacetate dehalogenase gene dehH2
on plasmid pUO1 in Moraxella sp. strain B (Kawasaki et al.,1992), the haloalkane dehalogenase gene dhaA on thechromosome in P. pavonaceae 170 (Poelarends et al.,2000a), the atrazine degradative genes atzA, atzB and
atzC on plasmid pADP-1 in Pseudomonas sp. ADP (Wackett, 2004), the aniline degradative genes on plasmid pTDN1
in Pseudomonas putida UCC22 (Fukumori and Saint,1997), and presumably also the p-sulfobenzoate degradative genes on plasmids pTSA and pPSB in Comamonas
testosteroni strains T-2 and PSB-4 respectively (Junker
and Cook, 1997). These observations clearly indicate thatgene mobilization between and within replicons is an important process during genetic adaptation. It also suggeststhat genes that are involved in biodegradation of xenobiotics were recruited from a ‘pre-industrial’ gene pool by
integration, transposition, homologous recombination andmobilization. Association of dehalogenase sequences withmobile genetic elements has also been observed in othercases, for example with haloacetate dehalogenases (Slater
et al., 1985; Thomas et al., 1992; van der Ploeg et al.,1995), with dichloromethane dehalogenase (SchmidAppert et al., 1997), and with γ-hexachlorocyclohexane
dehalogenase (Dogra et al., 2004).
Although insight in the sequence of events that led tothe current genetic make-up of biodegradation pathways
is lacking, the general nature of some of the processes
involved is understood. Less is known about the origin of
the structural genes that encode critical enzymes, suchas dehalogenases, and the degree of divergence that
occurred during evolution of the current sequences. The
possibility to rapidly evolve new enzyme selectivities bylaboratory evolution is known since the 1970s, most notably through the work of P.H. Clarke and coworkers who
showed that the substrate specificity of Pseudomonas
aeruginosa amidase can be changed by mutagenesis andselection on plates (Betz et al., 1974; Paterson and
Clarke, 1979). This approach was called experimental
enzyme evolution and is conceptually similar to directed
evolution.
Theoretically, it is possible that a current gene for a
specific critical (dehalogenase) reaction was alreadypresent in the pre-industrial gene pool. Alternatively, there
could be a short evolutionary pathway that led from an
unknown pre-existing gene to the gene as we currently
find it in a biodegradation pathway. It has even beensuggested that a new sequence for an enzyme acting on
a synthetic compound could evolve through the activationof an unused alternative open reading frame of a preexisting internal repetitious coding sequence (Ohno,
1984). Here it should be noted that the similarity of adehalogenase to members of an enzyme superfamily thatcatalyse other reactions generally does not provide information about the process of adaptation to xenobiotic
compounds. The level of sequence similarity that exists
between a dehalogenase and other proteins in a phylogenetic family is usually less than 50% (Table 1). Therefore,
the time of divergence should be much earlier than acentury ago, and the process of divergence thus cannot
be related to the introduction of industrial chemicals into
the environment. If the dehalogenases and other critical
enzymes that occur in catabolic pathways have undergone recent mutations, there should be closely relatedsequences in nature that differ from the current enzymes
by only a few mutations. No such primitive dehalogenase
has yet been detected, with the notable exception of TriA,
the enzyme that dehalogenates the herbicide atrazine(vide infra).
Another issue is the function of the pre-industrial dehalogenase or dehalogenase-like sequences from which
the current catabolic systems with their activated andmobilized genes originate. The original genes may havebeen involved in the dehalogenation of naturally occurring
halogenated compounds, of which there are many
(Gribble, 1998). Such proteins may fortuitously also havebeen active with a xenobiotic halogenated substrate, justbecause of their lack of substrate specificity. Alternatively,
the evolutionary precursor of a dehalogenase may havecatalysed a different reaction that has some mechanistic
similarity to dehalogenation, in which case the original
enzyme may or may not have shown some dehalogenation activity due to catalytic promiscuity. Finally, the precursor genes for dehalogenases may have been silent or
cryptic genes, with no clear function for the pre-industrial
host (Hall et al., 1983). In all cases, the gene could havebecome functional in a dehalogenation pathway as aresult of the fortuitous ability to catalyse dehalogenationof a xenobiotic compound, possibly after acquisition ofsome mutations.
One way to obtain information about the evolutionary
origin of dehalogenase genes is to compare the dehalogenase sequences that have been detected in different
bacterial cultures. If closely related sequences arepresent, this would make it possible to identify sequence
differences and to determine the effect of the mutations
on substrate selectivity. Another approach is to search forsequences that are closely related to dehalogenases indatabases of sequenced genomes. These organisms
have, with some recent exceptions, no known history of
degradation of halogenated compounds. Currently thewhole sequence of more than 300 bacterial genomes isavailable. If we find closely related sequences in these
databases, they could define evolutionary ancestors of thecurrent dehalogenases. A further potential source of newdehalogenase sequences could come from metagenome© 2005 The Authors
Journal compilation © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1868–1882

1874 D. B. Janssen, I. J. T. Dinkla, G. J. Poelarends and P. Terpstra
libraries. Several of these libraries have been prepared,
and some have been used for massive sequencing (Venter et al., 2004), whereas others have been explored forthe presence of new biotransformation enzymes.
Analysis of sequence databases such as Pfam and of
the literature revealed that in several cases the samedehalogenase sequence has repeatedly been detected inorganisms that have been isolated from different geographical locations. Thus, identical or almost identicalhaloalkane dehalogenases, dichloromethane dehalogenases and atrazine chlorohydrolases have been detected
in isolates from different areas (Table 1). This suggeststhat either these sequences have been recruited from thepre-industrial gene pool only once, after which they
became distributed by horizontal transmission, or they
have been repeatedly recruited from an identical preindustrial sequence. By itself, the worldwide distribution ofidentical sequences is common in the bacterial world, as
100% sequence identity is normal for specific genes
within different strains of the same species. However, it iscurrently unknown by what mechanisms a possible process of global distribution of recently evolved catabolic
genes may occur.
The structural and mechanistic analysis of dehalogenases makes it possible to define sequence fingerprints thatallow the identification of genes that are phylogeneticallyrelated in genomic databases. The current version of the
NCBI databases lists more than 250 proteome sequencesof bacteria, and 31 archaeal proteomes. When these weresearched for 15 different dehalogenase sequences thathave been detected in organisms isolated on halogenatedcompounds as a carbon source, it appeared that not asingle closely related counterpart of dehalogenase genes
was present in these sequenced bacterial genomes(Table 1). Even close homologues are absent, including
for those sequences that have repeatedly been detected
in identical form in independently isolated bacteria. For
example, both dichloromethane dehalogenase and 1,2dichloroethane dehalogenase have been detected inessentially identical form in more than 10 independently
isolated bacterial cultures that originate from different
geographical locations, but no close homologue is present
in the sequenced microbial genomes. Similarly, Blast
searches with dehalogenase sequences against thesequences of environmental DNA obtained in the Sargasso Sea sequencing project (Venter et al., 2004) again
indicated that no close homologues are present in the 1.2
million new genes that were discovered in this massive
DNA sequencing project (Table 1). Thus, sequences thathave repeatedly been acquired by batch enrichment were
not detected in the environmental DNA. Marchesi andWeightman (2003), working with haloacid dehalogenases(HAD superfamily), also described that a bias is introduced by enrichment and isolation. Both for group I deh-alogenases and for group II (HAD) enzymes, the diversity
and phylogenetic grouping of dehalogenase sequences
obtained via pure cultures significantly deviated from what
was observed by analysis of DNA directly isolated fromthe samples that were used for enrichment, such as activated sludge.
At the same time, it appears that more distantly
related dehalogenase sequences are quite common.
For example, large numbers of putative haloalkane dehalogenase and haloacid dehalogenase sequences arepresent in the whole-genome database and in the
Sargasso Sea database (Table 1). The identification ofthese sequences is based on detailed insight in structure–function relationships and the fact that several
homologous sequences in these protein families havebeen detected that indeed encode functional dehalogenases. The exact activity of the proteins encoded byputative dehalogenase sequences is difficult to predict.
Jesenska and colleagues (2002) have shown that atleast one active dehalogenase is encoded by theM. tuberculosis genome, but its physiological function is
unclear. In the HAD superfamily of enzymes, at least
eight different sequences have been found to encode
functional haloacid dehalogenases, and the environmental diversity may be large (Marchesi and Weightman,2003). In other cases, e.g. with CaaD and hexachlorocyclohexane dechlorinase (LinA), it is more difficult to predict which of the homologues do encode dehalogenating
enzymes because the sequence similarity is low and theresidues involved in dehalogenation have been less wellidentified.
Haloalkane dehalogenase from Xanthobacter
autotrophicus (DhlA)
Haloalkane dehalogenase (DhlA) was originally discovered in X. autotrophicus GJ10, a nitrogen-fixing hydrogen
bacterium that was enriched with 1,2-dichloroethane as
the sole carbon source. Subsequently, identical DhlAencoding genes have been discovered in several otherstrains of X. autotrophicus, isolated in the Netherlands
and in Germany, and in isolates of Ancylobacter aquaticus
obtained with 1,2-dichloroethane or chloroethylvinyl ether
as the growth substrate (van den Wijngaard et al., 1992).
Recently, a strain of Xanthobacter flavus was isolated inSouth Korea, and this organism also possessed an identical dehalogenase (Song et al., 2004). In fact, DhlA is still
the only known hydrolytic haloalkane dehalogenase that
operates in 1,2-dichloroethane degrading bacteria; novariants have been described, and at least 12 identicalcopies of this gene have been obtained from different
environmental isolates. The question arises: where
does this enzyme that has such a remarkable activity
with a very stable xenobiotic compound come from? Did© 2005 The Authors
Journal compilation © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1868–1882

Bacterial degradation of xenobiotic compounds 1875
it pre-exist at the time industrial production of 1,2dichloroethane started, or did it evolve from an ancestor
that converted some halogenated or non-halogenatedcompound?
Insight into this issue has been obtained by inspectionof the wild-type sequence and by experimental evolution
methods. The N-terminal part of the cap domain of thedehalogenase harbours two short tandem sequence
repeats (at the DNA level: one perfect 15 bp repeat andone 9 bp repeat carrying one substitution). These repeats
occurred in a segment of the protein that was observed
to be the target of substitutions, deletions and generationof new repeats when mutants were selected that possessed an enhanced activity toward 1-chlorohexane(Pries et al., 1994). Thus, experimental evolution leads
to duplications and other mutations in a part ofthe sequence where the wild-type already harbours
sequence repeats. This observation led to the hypothesisthat the sequence repeats in the wild-type dhlA gene
were the result of recent evolutionary events that caused
adaptation to 1,2-dichloroethane. An insertion is detectedin the same region of the cap domain when a sequencealignment is performed between DhlA and its most similar
homologue present in the bacterial genomes database,which independently confirms that the sequence encoding the N-terminal part of the cap domain is a target foradaptive mutations (Fig. 3).
Assuming that the short repeats present in the cap
domain sequence of the wild-type dehalogenase wereindeed generated recently in a pre-industrial dehalogenase, one can postulate a DhlA sequence for this hypothetical primitive dehalogenase (Pikkemaat and Janssen,2002) (Fig. 3B). The primitive dhlA sequence predicted bythis so-called retro genetics approach has been constructed in vitro, an it appeared to encode a protein withsignificant activity toward bromoalkanes, but was completely inactive toward 1,2-dichloroethane. Thus, theancestral dehalogenase may have been a debrominating
enzyme. Subsequently, we created with the incremental
truncation method (ITCHY) a library of derivatives of theprimitive DhlA that carried random direct repeats in thesame region where the wild-type sequence harboured
repeats. Some of the repeat-carrying variants indeed had
evolved activity with 1,2-dichloroethane. All these mutations influenced the region of the cap domain of haloalkane dehalogenase proximal to the tryptophan that
donates a hydrogen bond to the leaving halide (Trp175)(Pikkemaat and Janssen, 2002).
These results are all in agreement with the hypothesisthat the current 1,2-dichloroethane dehalogenase has
evolved by a short evolutionary pathway from a preexisting (pre-industrial) haloalkane dehalogenase that
was active with brominated but not with chlorinatedcompounds. Association with transmissible plasmids andother mobile genetic elements has facilitated the worldwide spread of the evolved dehalogenase.
Rhodococcus haloalkane dehalogenase (DhaA)
Another abundant type of haloalkane dehalogenase is theone from Rhodococcus erythropolis (DhaA). Mutually
identical copies of the dhaA genes have been detected indifferent organisms, of which the taxonomy has beenrather confusing due to errors in classification. The firstdhaA sequence was determined by Kulakova and colleagues (1997), using a strain of Rhodococcus (Rhodococcus rhodochrous NCIMB 13064) that was isolatedon 1-chlorobutane. Later sequencing of several otherhaloalkane dehalogenase genes from 1-chlorobutane-, 1chlorohexane- and 1,6-dichlorohexane-degrading grampositive organisms, some of which had been isolated
before strain NCIMB 13064, showed that these possessed identical dhaA sequences. Poelarends andcolleagues (2000b) have reclassified several organisms
and using 16S rRNA gene sequencing they showedthat R. erythropolis Y2 (England), R. rhodochrous
NCIMB13064 (N. Ireland), Corynebacterium sp. strain
m15 (Japan), Arthrobacter strain HA1 (Switzerland),strain GJ70 (the Netherlands, originally called Acinetobacter) and strain TB2 (USA) should all be classified as
R. erythropolis. All these strains possessed the same
haloalkane dehalogenase, and in all cases the dehalogenase gene was preceded by the same invertase genesequence and a regulatory gene, and on the downstreamside an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene. Thus, this catabolic cluster ishighly conserved and distributed worldwide in closely
related gram-positive bacteria. The structure of this DhaA
type haloalkane dehalogenase was solved by X-ray crystallography, using a protein with two substitutions (A172V
and A292G) compared with the original DhaA sequenceof strain NCIMB 13064 (Newman et al., 1999). In a recent
directed evolution study, the substrate specificity of DhaA
has been modified to enhance conversion of the highly
recalcitrant chemical 1,2,3-trichloropropopane (Bosma
et al., 2002).
This reservoir of haloalkane dehalogenase-producing
rhodococci may have been the source for catabolic pathways that are active with more exotic haloalkanes (Fig. 4).
In the first place, enrichment of 1,2-dibromoethane
degrading bacteria has yielded, after much patience, aculture that can slowly grow with 1,2-dibromoethane assole carbon source. This Mycobacterium strain produces
a haloalkane dehalogenase that is almost identical to
DhaA, but there are three substitutions (C176F, P248S,
Y272F), and, most remarkably, on the C-terminal side the
enzyme is 14 amino acids longer due to an in-frame fusionof the 3′ end of the dehalogenase gene with 42 bases that© 2005 The Authors
Journal compilation © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1868–1882

1876 D. B. Janssen, I. J. T. Dinkla, G. J. Poelarends and P. Terpstra

A::IIMNACLMTD∆FVTQPADGFTA::MT

WT DhlA::FTA

DWGGFLGLTLPMADPSRFKRLIIMNACLMTDPVTQPAFSAFVTQPADGFTAWKS HBDWGGFLGLTL-P-MADPSRFKRLIIMNACLMTDPVTQPAFSAFVTQPADGFTAWK::::..:: . : ::::.:::.:: .
:.
:
:.:::
DWGGILGLGIVPDMADR--FERLIVMNTAI---PIGE--------SPGPGFEAWKC

experimental
enzymeevolution
haloalkane
dehalogenase(DhlA)

chlorohexane-evolved
DhlA

hypothetical primitive
dehalogenase“retrogenetics”
ITCHY mutagenesis

DCE-evolved
primitive DhlAD
WT DhlA
primitive

IIMNACLMTDPVTQPAFSAF VTQPADGFTAWKYIIMNACLMTDP
VTQPADGF#AWKY

DBE DCE(U/mg)
2.4
2.9
1.3 <0.01

D2
1C12
3B2

IIMNACLMTDP
CLMTDPVTQPADGFTAWKYIIMNACLMTDPVMNACLMTDPVTQPADGFTAWKYIIMNACLMTDPVT RLMTDPVTQPADGFTAWKY

4.8
4.7
5.6

0.2
0.5
0.3

Fig. 3. Evolution of 1,2-dichloroethane dehalogenase activity. The N-terminal part of the cap domain of wild-type DhlA harbours two short tandem
sequence repeats (shown as arrows), which might be signs of recent genetic adaptation to 1,2-dichloroethane. This idea is supported by thefollowing observations. First, as shown in panel A, changes are observed in this part of the cap domain (shown in boxes, three tandem duplications,a large deletion and two substitutions) when DhlA is forced to evolve dehalogenase activity toward 1-chlorohexane, a substrate not used by thewild-type enzyme (experimental enzyme evolution; Pries et al., 1994). Second, in a pairwise sequence alignment the closest homologue of DhlAin the database (a putative protein from Erythrobacter litoralis HTCC2594) shows a gap precisely in this region of the cap domain (panel B).
Third, from the current 1,2-dichloroethane dehalogenase sequence (DhlA) a pre-industrial sequence (primitive) was proposed and constructed.
The encoded protein is inactive with 1,2-dichloroethane (DCE), but does convert 1,2-dibromoethane (DBE) (panel C). When the primitive
dehalogenase is subjected to ITCHY mutagenesis, a technique that allows the introduction of random repeats and deletions in the N-terminal
part of the cap domain, some mutants carrying repeats (D2, lC12 and 3B2) had evolved 1,2-dichloroethane dehalogenase activity (Pikkemaat
and Janssen, 2002) (panel D). On the basis of these findings, we propose that a primitive dehalogenase with a shorter stretch of sequence in
the N-terminal region of the cap (as observed in the E. litoralis putative dehalogenase) was recruited from the pre-industrial environmental genepool, and evolved into the current DhlA by a short evolutionary pathway that included generation of short duplications and substitutions.

encode the last 14 amino acids of a halohydrin dehalogenase (hheB gene) (Poelarends et al., 1999). Thus, thischimeric dehalogenase is 307 amino acids long insteadof the 293 amino acids of the standard Rhodocccus
enzyme. The fusion does not seem to be necessary forthe catalytic activity of the protein: 1,2-dibromoethane isan excellent substrate for both DhaA variants. Instead, it

happens to be there as a fortuitous result of a gene fusion
event that occurred during the evolution of the haloalkane
dehalogenase gene region.
Other striking differences between the organization of
the Mycobacterium GP1 dhaA region and that of the original Rhodococcus exist in the regulatory gene that
represses DhaA formation in R. erythropolis when no© 2005 The Authors
Journal compilation © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1868–1882

Bacterial degradation of xenobiotic compounds 1877
Mycobacterium sp. strain GP1

intM

invA

D
dhaR

fusiondhaA

R. rhodochrous NCIMB13064

IS 2112

invA

dhaR

dhaA

adhA

aldA

dhaA

tnpA

IS 1071source1 kb
intP

P. pavonaceae 170

Fig. 4. Evolution and distribution of DhaA-type haloalkane dehalogenase sequences. The proposed starting point is the widely distributed genecluster detected in rhodococci (middle). Adaptation to 1,2-dibromoethane (Mycobacterium GP1) involves inactivation of the regulatory gene(dhaR), association with an integrase sequence (intM), and loss of the alcohol and aldehyde dehydrogenase genes (adhA and aldA respectively).
Recombination sites are shown by vertical arrows. Somewhere during these processes, a fortuitous fusion with a segment of a hheB gene has
occurred. Adaptation to 1,3-dichloropropene (P. pavonaceae) is proposed to involve loss of the regulatory gene, loss of the dehydrogenase genes,
association with an integrase type sequence (intP) and with an insertion element carrying a transposase gene (IS1071), and mobilization to the
genome of a gram-negative Pseudomonas (Poelarends et al., 2000a).

inducer is present (Poelarends et al., 2000a). This regulatory gene is inactivated in Mycobacterium strain GP1 bya small deletion, which is required to allow gene expression because 1,2-dibromoethane does not act as an
inducer. Here, we see another sign of recent pathway
evolution: constitutive expression of a structural gene dueto the absence of a functional regulatory gene. Dehalogenation of a xenobiotic organohalogen compound requires
a catalytic protein that can convert the compound: thedehalogenase. Regulated expression requires a second
protein that recognizes and responds to the xenobiotic
chemical: the regulatory protein. Constitutive expressionof dehalogenase genes has been demonstrated for atrazine chlorohydrolase (AtzA), 1,2-dichloroethane dehalogenase (DhlA), L-2-haloacid dehalogenase (DhlB),trans-3-CaaD, γ-hexachlorocyclohexane dehydrochlorinase (LinA) and 1,3,4,6-tetrachloro-1,4,-cyclohexadiene
chlorohydrolase (LinB), suggesting that evolution isrecent and has not yet led to systems for regulating gene
expression.
Also in the 1,3-dichloropropene degrader Pseudomonas pavonaceae a dhaA-type haloalkane dehalogenase
gene is present, indicating that it has recently been transferred from the Gram-positive Rhodococcus to a Gramnegative bacterium. In the Pseudomonas the whole regulatory gene has been lost (Fig. 4; Poelarends et al.,2000a). Furthermore, the alcohol dehydrogenase andaldehyde dehydrogenase genes have disappeared. However, the haloalkane dehalogenase gene is associatedwith a putative integrase sequence, which is also the casein Mycobacterium sp. GP1. It is well possible that geneacquisition by integrases plays an important role duringacquisition of new catabolic gene clusters for xenobiotic
compounds. It is tempting to speculate that the fortuitous
fusion that we see between the dhaA gene and a segmentof the hheB gene in strain GP1 has something to do withthe activity of an integron-like gene acquisition system.
Atrazine chlorohydrolase (AtzA)The bacterial degradation of the herbicide atrazine by
Pseudomonas ADP starts with a hydrolytic dechlorination,
catalysed by an enzyme called atrazine chlorohydrolase(AtzA) (Fig. 5). Identical atrazine chlorohydrolase genes
have been obtained from different sources, including an
Arthrobacter from China (Cai et al., 2003), strains from
France (Rousseaux et al., 2001), an undescribed βproteobacterium strain CDB21 from Japan of which thesequence was deposited by Iwasaki and colleagues(EMBL Accession number AB194097), and four other isolates from different locations including an Alcaligenes, a
Ralstonia and an Agrobacterium sp. (de Souza et al.,1998). The protein belongs to the amidohydrolase superfamily, which also houses the next two enzymes of theatrazine catabolic pathway: hydroxyatrazine ethylaminohydrolase (AtzB) and N-isopropylammelide N-isopropylaminohydrolase (AtzC) (de Souza et al., 1996). Other
members of the amidohydrolase superfamily are triazine
deaminase, hydantoinase, melamine deaminase, cytosine
deaminase and phosphotriesterase. All these (αβ)8 barrel
proteins show rather low sequence identities in pairwise
comparisons, with the exception of AtzA and melamine© 2005 The Authors
Journal compilation © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1868–1882

1878 D. B. Janssen, I. J. T. Dinkla, G. J. Poelarends and P. Terpstra
could play an important role in the evolution of new
enzyme activities (Aharoni et al., 2005).

(2,4,6-triamino-1,3,5-triazine) deaminase (TriA), which are
98% identical (Seffernick et al., 2001). Thus, AtzA and TriA
are closely related, indicating very recent evolutionary
divergence. Both atrazine and melamine are xenobiotic
compounds, but melamine has been in use for a longer
period of time than atrazine. Accordingly, it was proposedthat TriA may have functioned as a precursor for AtzA. The
proteins differ only by nine out of 475 amino acids, andusing gene shuffling a number of hybrids has beenobtained from which determinants of the substrate specificity could be identified (Raillard et al., 2001). It appearedthat a wide range of activities could be obtained in sucha library of hybrids, but leaving group specificity was
mainly determined by residue 328. An Asn at this position
favoured dechlorination, whereas an Asp gave an enzymethat could hydrolytically displace a broader range ofsubstituents from the 2-position of 2-substituted 4,6dialkylamino-triazines, albeit with a relatively low catalytic
rate. Dechlorination activity may thus have evolved fromdeamination activity.
An overlap in these two activities was indeed observed
for another member of the amidohydrolase superfamily,
AtzB, which is the second enzyme in the normal atrazine
degradation pathway (Fig. 5). AtzB is catalytically promiscuous and can not only catalyse a deamination reaction,
but also is able to catalyse the dechlorination of the substrate analogue 2-chloro-4-amino-6-hydroxy-S-triazine,
which is an intermediate in an alternative atrazine degradation pathway (Fig. 5) (Boundy-Mills et al., 1997).
These observations suggest that atrazine hydrolase
and melamine deaminase very recently diverged from a
pre-existing hydrolase that mainly acted as a deaminaseor may have had both dehalogenase and deaminase
activity. It was proposed that the possibility to achieve
variations in substrate range in a key enzyme by an organism is a measure of the potential to recruit new catabolic
activities (Wackett, 2004. In general, catalytic promiscuity

Cl
N
N
H

H2O

OH

HCl

N
N

N
N
H

N

N
N
H

N
N

NH3

H2O

N
N
H

OH
N

TriA
N
H

N
H

N
N

N
H

melamine
Cl

OH

H2O
N
HO

HCl

N
N

N
NH2

OH

H2NEt

N

atrazine
NH2

Aliphatic hydrocarbons are introduced into the environment in large quantities both by human activities and by
natural processes. Plants, for instance, can produce(odd-length) n-alkanes as part of mixtures of waxes.
Thus, they cannot be regarded as xenobiotic compounds,
even though most cases of contamination of surface soils
with high levels of alkanes are caused by industrial
processing of petroleum. In many different environments
bacteria have been exposed to these compounds, andone would expect that evolution of enzymes that canhydroxylate alkanes has occurred over a longer periodof time than with dehalogenases that act on exotic
compounds.
The degradation of alkanes usually starts with the oxidation of the terminal carbon atom yielding an alcohol.
Conversion of medium- and long-chain alkanes is often
performed by integral membrane hydroxylases of which
the best described member is AlkB of P. putida GPo1 (alsoreferred to as Pseudomonas oleovorans GPo1) that converts C5 to C12 alkanes. Both the sequence and genetic
organization of the gene encoding AlkB, as well as thebiochemical characteristics of the protein have been studied extensively (van Beilen et al., 1994). AlkB contains six
membrane-spanning helices and forms an ω-hydroxylase
system that also consists of a rubredoxin (AlkG) and arubredoxin reductase (AlkT) involved in electron transport.
AlkB belongs to a large superfamily of proteins that
also includes non-haem integral membrane desaturases,
epoxidases, acetylenases, conjugases, ketolases, decarbonylases and methyl oxidases. These proteins all containeight conserved histidines that are located on the cytoplasmic site of the protein and are essential for positioning

N

AtzB

AtzA

N
H

H2O

Alkane hydroxylase (AlkB, AlkM)

AtzB

HO

N
N

HO

N

N
H

Fig. 5. Degradation of atrazine by AtzA and
AtzB. These hydrolases both are members of
the amidohydrolase superfamily. AtzA is highly
similar to TriA, the enzyme that deaminates theamino analogue of atrazine called melamine.
There are only nine substitutions and a shortevolutionary pathway may be involved in adaptation of TriA to atrazine (Seffernick et al.,2001). AtzB catalyses the second step in atrazine degradation, which is a deamination reaction, but it can also catalyse a dechlorination
reaction with the atrazine soil metabolite 2chloro-4,6-diamino-s-triazine to yield ammelide(Boundy-Mills et al., 1997). These observations
suggest that dechlorination and deamination
activities are evolutionarily closely related.

NH22-chloro-4-amino-6-hydroxys-triazine© 2005 The Authors
Journal compilation © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1868–1882

Bacterial degradation of xenobiotic compounds 1879of the Fe ions in the di-iron active site. The conserved
histidines are situated in three motifs (HX3−4H, HX2−3HH
and H/QX2−3HH) and are here referred to as motif A, B
and D (Shanklin et al., 1994). In alkane hydroxylases anadditional histidine-containing motif (NYXEHYG), referred
to as motif C, was identified (Smits et al., 1999). The
genes encoding alkane oxidation in P. putida GPo1 arelocated on the OCT-plasmid in two operons. The alkBFGHJKL operon encodes the alkane hydroxylase (AlkB),
two rubredoxins, an aldehyde dehydrogenase, an alcohol
dehydrogenase, an acyl-CoA synthetase and an outermembrane protein of unknown function. The alkST operon
encodes a regulator of expression and a rubredoxin
reductase.
A similar well-characterized alkane hydroxylase system
is present in Acinetobacter sp. strain ADP1 (Ratajczak
et al., 1998). It converts long-chain alkanes ranging from
C12 to C16 and the sequence has the same conserved
motifs for iron binding. As just mentioned, the alk genesof strain GPo1 are plasmid-localized, but in strain ADP1
the alkane hydroxylase genes are present at different
locations on the chromosome. This likely correlates withthe presence or absence of selection pressure in theenvironment from which the organisms were isolated:strain GPo1 was isolated from oil, while strain ADP1 has
no record of exposure to high levels of alkanes before or
during its isolation. On the other hand, both AlkB and AlkM
expression are regulated, which is evolutionary moreadvanced that what was observed with several dehalogenases, which are often constitutively expressed.
Using the alkB gene sequence it was possible todevelop probes that could be applied for detecting genes
related to alkB in bacteria isolated from oil-contaminatedenvironments or from uncontaminated soils. Many
positives have been found, in some cases with a highsequence identity (> 70%) to the alkB gene (Sotsky et al.,
1994; Smits et al., 1999; Vomberg and Klinner, 2000;
Whyte et al., 2002a). One of the alkane hydroxylase
sequences obtained in this way belongs to Pseudomonas
aerofaciens and shows 95% identity to AlkB. Other homologues of alkB were detected in known alkane-degradingstrains such as P. putida P1 and Alcanivorax borkumensis
AP1. Thus, the alkB gene or close homologues appear tooccur quite often, and nine different alkane-degrading
cultures have been described to possess highly similar
alkane hydroxylases (Table 1). For the P. aeruginosa
PAO1 alkB variant more than 10 identical sequences were
found in P. aeruginosa strains from clinical and soil
samples.
Alkane hydroxylase systems that are less related to
AlkB or AlkM also occur, for example in gram-positivebacteria. van Beilen and colleagues (2002) detected quite
diverse alkane hydroxylases in organisms from a trickle
bed reactor that degraded alkanes. Diversity was also

observed by Whyte and colleagues (2002b), who studied
the hydroxylase system of an alkane-degrading R. rhodochrous and a R. erythropolis strain. The organisms
contain three to five distinguished alkane hydroxylase
systems that all are only distantly related to AlkB, butalmost identical between the two different Rhodococcus
strains. There are also variations in genetic organization,and one of the Rhodococcus systems has all three alkanehydroxylase components clustered in a single putative
operon.
Whole genome sequencing of bacteria revealed a significant number of putative alkane hydroxylases. Both for
AlkB of P. putida GPo1 and for AlkM, hits were obtainedwith sequence identity percentages up to 50%. Using
growth complementation assays in different hosts, thefunctionality of more than 18 different putative alkanehydroxylases was proven, including those from Burkholderia cepacia RR10, P. aeruginosa PAO1 and Mycobacterium tuberculosis H37Rv (van Beilen et al., 1994; 2003;
Smits et al., 2002). When searching for putative alkanehydroxylase genes in environmental DNA (Venter et al.,2004), a large number of alkB and alkM homologues wereagain detected, including two sequences that were 82%
similar to alkB. These are the closest homologues of anyof the sequences tested in Table 1. Apparently, alkanehydroxylases are highly abundant in the microbial population in the Sargasso Sea. Of these alkane hydroxylases,
apart from the two hits just mentioned, only a few wereclosely related to AlkB or AlkM or other alk homologues
that have been detected in isolates. This suggests that alarge number of alkane hydroxylase sequences arepresent in the environment. The conservation of the catalytically important motifs described above suggests thatmost of these sequences encode functional enzymes.
The above observations indicate that the diversity offunctional alkane hydroxylases is much broader than thediversity of dehalogenases that act on xenobiotic substrates. Thus, whereas very few solutions have evolved forthe incorporation of dehalogenase genes in catabolic
pathways for nematocides such as 1,2-dibromoethaneand 1,2-dichloroethane, several different alkane hydroxylase genes have been recruited in operons for alkane
degradation. This correlates with the more widespreadoccurrence of alkane degraders and the easier degradability of n-alkanes as compared with many halogenatedcompounds.
Conclusions: accession and exploration ofsequence space
One of the striking observations concerning the distribution and evolution of key catabolic genes is that identicalsequences have repeatedly been detected in organismsthat are enriched on xenobiotic halogenated substrates as© 2005 The Authors
Journal compilation © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 1868–1882

1880 D. B. Janssen, I. J. T. Dinkla, G. J. Poelarends and P. Terpstraa carbon source. This holds for dichloromethane dehalogenase (DcmA), haloalkane dehalogenases (DhlA, DhaA,
LinB) and atrazine chlorohydrolase (AtzA). Probably, thenumber of solutions that nature has found to degrade
these compounds is very small, and horizontal distribution
occurs faster than generation of new pathways. Indeed,
the dehalogenase genes are often associated with integrase genes, invertase genes, or insertion elements, andthey are usually localized on mobile plasmids. Recent
mobilization of a dehalogenase gene across the gramborder has been suggested in at least one case.
It remains unknown to what degree the current active
dehalogenases differ from their proposed recent evolutionary ancestors. Only for 1,2-dichloroethane dehalogenase and atrazine chlorohydrolase short evolutionary
pathways have been suggested that could describe thegeneration of a functional dehalogenase from a closely
related precursor sequence that in pre-industrial times
may have acted on a different substrate. The diversity ofsuch specialized dehalogenases that act on xenobiotic
compounds seems to be more restricted than that of
enzymes converting alkanes, which are easier to degradeand for which organisms are more common.
Even though the same dehalogenase sequences aredetected in organisms that are isolated in different geographical areas, and in some cases even on different
substrates, they are not the most abundant dehalogenase
sequences identified in whole genome sequencing
projects and massive random sequencing. Thus, itappears that enrichment techniques explore a different
segment of sequence space than massive sequencing ofenvironmental DNA. The presence of large numbers of
unexplored functional sequences in genomic databases
suggests that the biotransformation scope of microbial
systems has an enormous potential for further growth.
Acknowledgements
We thank the present and former colleagues of our labs forcontributing with discussions and experimental work to our
research on dehalogenase mechanisms and enzyme evolution.

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