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University of Groningen
Molecular basis of two novel dehalogenating activities in bacteria
de Jong, René Marcel
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Chapter 2: Structure and mechanism of
bacterial dehalogenases: Different ways to
cleave a carbon-halogen bond
René M. de Jong & Bauke W. Dijkstra
Abstract
Dehalogenases make use of fundamentally different strategies to cleave carbon-halogen bonds. The
structurally
characterized
haloalkane
dehalogenases,
haloacid
dehalogenases,
and
4-
chlorobenzoate-CoA dehalogenases use substitution mechanisms that proceed via a covalent
aspartyl intermediate. Recent X-ray crystallographic analysis of a haloalcohol dehalogenase and a
trans-3-chloroacrylic acid dehalogenase provide detailed insight into a different intramolecular
substitution mechanism and a hydratase-like mechanism, respectively. The available information on
the various dehalogenases supports different views on the possible evolutionary origins of their
activities.
Published in: Current Opinion in Structural Biology 2003, 13, 722-730.
23
Introduction
From the beginning of the past century, halogenated hydrocarbons have been
extensively applied in industry and agriculture. Decades after the start of their widespread
use, evidence started to accumulate that some of these xenobiotic halogenated
compounds are highly toxic. Notorious examples are dioxins and polychlorinated biphenyls
(PCBs), but also halogenated solvents like for instance 1,2-dichloroethane have been
classified as probably carcinogenic to humans (Safe, 1994; Salovsky et al., 2002).
Because of these noxious properties, many of these organohalogens have now been
banned from use and have been replaced by environmentally less harmful compounds.
Halogenated compounds are, however, not only of anthropogenic origin. Over
1500 organohalogens are known that are produced naturally (Odberg, 2002; Balschmitter,
2003). They range from volatile compounds such as methylchloride to antibiotics like
vancomycin and chloramphenicol. Insight into the enzymes that incorporate halogens into
organic compounds is rather limited (van Pée and Unversucht, 2003). This strongly
contrasts with the large amount of detailed information on enzymes that degrade
halogenated hydrocarbons, releasing the halogens as halide ions.
In this review we focus on bacterial dehalogenases. These enzymes make use of
a variety of distinctly different catalytic mechanisms to cleave carbon-halogen bonds. X-ray
structures of haloalkane dehalogenases, haloacid dehalogenases, and 4-chlorobenzoylCoA dehalogenase demonstrated the power of substitution mechanisms that proceed via a
covalent aspartyl intermediate (Verschueren et al., 1993; Benning et al., 1996; Hisano et
al., 1996; Ridder et al., 1999). Recent structural characterizations of a haloalcohol
dehalogenase and a trans-3-chloroacrylic acid dehalogenase reveal the details of two
other elegant catalytic strategies, which exploit catalytic mechanisms from homologous
enzymes in a different chemical context (de Jong et al., 2003; de Jong et al., submitted).
The available information on the various dehalogenases supports different views on the
possible origins of their individual activities towards presumed anthropogenic halogenated
substrates.
24
Figure 1. The bacterial degradation routes of four different halogenated compounds, in which the five
families of dehalogenases discussed in this chapter facilitate the enzymatic cleavage of the carbonhalogen bonds.
Dehalogenase families
Haloalkane dehalogenases
Haloalkane dehalogenases cleave the carbon-halogen bond in halogenated
aliphatic hydrocarbons (Figure 1A). The haloalkane dehalogenase from Xanthobacter
autotrophicus GJ10 was the first dehalogenase of which the crystal structure was
determined (Franken et al., 1991), but later also other structures have become available
(Newman et al., 1999; Oakley et al., 2002). The structure consists of two domains, a main
domain and a cap domain (Figure 2A). The main domain consists of a mostly parallel
eight-stranded β-sheet (only the second β-strand is antiparallel), connected by α-helices
on both sides of the β-sheet. The cap domain is α-helical. This fold is the hallmark of the
superfamily of α/β-hydrolases, to which, besides the haloalkane dehalogenases, also
lipases, esterases, carboxypeptidases, and acetylcholinesterases belong (Ollis et al.,
1992; Heikinheimo et al., 1999; Nardini & Dijkstra, 1999). These latter enzymes catalyze
the hydrolysis of ester and amide bonds via a two-step nucleophilic acyl substitution
mechanism similar to that of serine proteases (see e.g. Dodson and Wlodawer, 1998;
25
Jaeger et al., 1999). In the first step, the serine or cysteine of a Ser/Cys-His-Asp/Glu
catalytic triad functions as a nucleophile that attacks the sp2-hybridized carbonyl carbon
atom of the scissile ester/amide bond, resulting in a covalently bound ester intermediate.
Subsequently, a water molecule activated by the His/Asp or His/Glu pair hydrolyzes this
ester intermediate in the second step.
Haloalkane dehalogenases use a very similar two-step catalytic mechanism,
except that the nucleophile is an aspartate instead of a serine/cysteine residue, which, in
an SN2 type substitution mechanism, attacks the halogen-bearing sp3-hybridized carbon
atom. This leads also to a covalently bound ester intermediate that can be easily
hydrolyzed by attack of a His-activated water molecule on the Cγ atom of the aspartate
(Figure 2C) (Verschueren et al., 1993). Thus, the substitution of the serine/cysteine of an
α/β-hydrolase by an aspartate confers on the enzyme an essential prerequisite to
hydrolyze carbon-halogen bonds in haloalkanes. In addition, haloalkane dehalogenases
contain a halide-binding site to facilitate the dehalogenation reaction.
Figure 2. A) Structure of haloalkane dehalogenase from X. autotrophicus (Franken et al., 1991),
showing the nucleophilic aspartate and a bound chloride ion. B) Dimeric structure of haloacid
dehalogenase with a covalently bound intermediate (Ridder et al., 1999). C) Schematic of the catalytic
mechanisms of haloalkane and haloacid dehalogenases.
26
During recent years, computational studies have unravelled the details of the
catalytic mechanism. In the enzyme, the nucleophilic aspartate is nearly entirely positioned
in a “near attack conformation”, with the attacking oxygen of the aspartate in line with the
C-Cl bond of the substrate, at a distance favorable for attack of the halogen-bearing
carbon atom. The correct positioning accounts, however, only for 25% to the lowering of
the transition state free energy (Shurki et al., 2002; Hur et al., 2003). The remaining 75%
probably originates from contributions of the halide-binding residues to transition state
stabilization (Boháč et al., 2002) and from the half-hydrophilic/half-hydrophobic nature of
the active site, which facilitates both the activation of the nucleophile and the departure of
the leaving group. Computational quantification of these contributions is presently beyond
reach (Hur et al., 2003).
Haloacid dehalogenases
Haloacid dehalogenases catalyze the hydrolysis of α-halogenated carboxylic
acids, such as 2-chloroacetate, which is an intermediate in the degradation of 1,2dichloroethane (Figure 1A). They are members of the haloacid dehalogenase (HAD)
superfamily (Koonin and Tatusov, 1994; Ridder et al., 1999), to which also magnesiumdependent phosphatases and P-type ATPases belong. The haloacid dehalogenases are
dimers with two- or three-domains per subunit: a core domain with a Rossmann-fold-like
six-stranded parallel β-sheet flanked by five α-helices, a sub-domain consisting of a fourhelix bundle, and, in some enzymes, a dimerization domain of two anti-parallel α-helices
(Figure 2B) (Hisano et al., 1996; Ridder et al., 1997). This fold is completely different from
the α/β-hydrolase fold of the haloalkane dehalogenases. Like the latter enzymes, the
haloacid dehalogenase utilizes an aspartate-based catalytic mechanism that proceeds via
a covalent intermediate (Figure 2C), but there is no histidine to activate a nucleophilic
water molecule. Also, the halide-binding site is very different (Ridder et al., 1999). How the
water molecule is activated is not known, but it has been suggested that another aspartate
in the active site fulfils this function (Ridder et al., 1999). Intriguingly, at least one other
unrelated haloacid dehalogenase family exists (Hill et al., 1999; Marchesi & Weightman,
2003), which was shown to bypass the classic covalent ester intermediate (Nardi-Dei et
al., 1999). This suggests that enzymes with different haloacid dehalogenating strategies
have evolved from different enzyme precursors, but unfortunately, crystallographic data
that could provide structural evidence on the mode of action of these type II haloacid
dehalogenases is still unavailable.
27
4-Chlorobenzoyl-CoA dehalogenases
The 4-chlorobenzoyl-Coenzyme A (CoA) dehalogenase from Pseudomonas sp.
strain CBS-3 is a homotrimer, of which each subunit folds into two domains. The Nterminal domain contains a 10-stranded β-sheet, forming two nearly perpendicular layers,
which are flanked by α-helices (Figure 3A). The C-terminal domain is composed of three
amphiphilic α-helices, and is primarily involved in trimerization. Thorough characterization
of the enzyme has revealed the details of the mechanism by which a halogen is displaced
from the aromatic ring of 4-chlorobenzoate, a degradation product of PCBs (Figure 1D)
(Benning et al., 1996; Luo et al., 2001; Dong et al., 2002). The mechanism also proceeds
via a covalent aspartyl intermediate. First, the substrate is ligated to CoA by 4chlorobenzoate CoA ligase. The CoA-ligated product is then bound by the 4-chlorobenzylCoA dehalogenase, with the enolate anion of the thioester link of the CoA-ligated substrate
stabilized by two backbone NH groups. This induces a partially positive charge on the
halogen-bearing carbon atom, which makes it susceptible to nucleophilic attack by the
aspartate (Figure 3B) (Luo et al., 2001). The SNAr-type substitution results in a covalent
Meisenheimer intermediate, which was recently confirmed by Raman spectroscopic
measurements (Dong et al., 2002). Chloride is expelled upon restoration of the aromatic
ring system, producing a second arylated enzyme intermediate that is subsequently
hydrolyzed by attack on the Cγ atom of the nucleophilic aspartate by a histidine-activated
water molecule, yielding 4-hydroxybenzoyl-CoA (Zhang et al., 2001; Lau et al., 2002).
The stabilization of the acyl-CoA enolate anion intermediate is shared with other
hydratases, isomerases and thioesterases of the crotonase (or enoyl-CoA hydratase)
superfamily (Holden et al., 2001) but the nucleophilic aspartate is only present in the
dehalogenase. Like in haloalkane and haloacid dehalogenases, the aspartate confers to
the enzyme its unique capability to hydrolyze the bond between a halogen and an
aromatic carbon atom.
28
Figure 3. A) Trimeric structure of 4-chlorobenzoyl-coenzyme A dehalogenase (Benning et al., 1996),
showing the nucleophilic aspartate, a bound calcium ion, and the product 4-hydroxybenzoylcoenzyme A. B) Schematic of the catalytic mechanism of
4-chlorobenzoyl-coenzyme A
dehalogenase.
The enzymes discussed above illustrate the power of aspartate-mediated
substitution
mechanisms
to
hydrolyze
organohalogens.
However,
several
other
dehalogenases exist that use completely different catalytic strategies. For instance,
halorespiring anaerobic bacteria contain reductive dehalogenases, but unfortunately their
structures are not yet known (Wohlfarth and Diekert, 1997; Furukawa, 2003). In contrast,
X-ray structures of a haloalcohol dehalogenase and the trans-3-chloroacrylic acid
dehalogenase CaaD have recently provided detailed insight into two other fundamentally
different dehalogenation strategies.
Haloalcohol dehalogenases
Several bacteria contain enzymes that are able to displace a halogen from a
vicinal haloalcohol substrate. One such enzyme is the haloalcohol dehalogenase HheC
from Agrobacterium radiobacter AD1, which plays a role in the degradation of 1,3-dichloro2-propanol (Figure 1B). HheC is a homotetrameric enzyme (Figure 4A): the monomer is
homologous to that of members of the widespread short-chain dehydrogenase/reductase
(SDR) family. SDR-enzymes are redox enzymes that catalyze alcohol-ketone conversions
29
of a wide variety of alcohols, steroids and sugars (Filling et al., 2002; Oppermann et al.,
2003). They have the well-known dinucleotide-binding Rossmann fold (Rossmann et al.,
1974) and a Ser-Tyr-Lys/Arg catalytic triad. HheC shares the fold and catalytic triad of the
SDR-family, but lacks the characteristic dinucleotide-binding Gly-X-X-X-Gly-X-Gly motif
(Figure 4D) (van Hylckama Vlieg et al., 2001; de Jong et al., 2003). Instead, several larger
residues replace the smaller ones of the motif, thus filling up the space of the cofactorbinding site. In this way, a spacious halide-binding site is created (Figure 4B). The enzyme
uses an intramolecular SN2-type substitution mechanism, in which the secondary hydroxyl
group of the haloalcohol substrate is deprotonated by the tyrosine of the conserved SerTyr-Arg catalytic triad acting as a base (Figure 4C). The hydroxyl oxygen concomitantly
substitutes the vicinal halogen to yield the corresponding epoxide product, a proton and a
chloride ion. Thus, while the aspartate-dependent dehalogenases discussed above
catalyze a two-step hydrolysis of the carbon-halogen bond, the presence of a hydroxyl
function vicinal to the halogen allows the one-step, intramolecular substitution of the
halogen by the hydroxyl.
3-Chloroacrylic acid dehalogenases
Like 4-chlorobenzoyl-CoA dehalogenases, the chloroacrylic acid dehalogenases
displace a halogen from an sp2-hybridized carbon atom. The cis- and trans-3-chloroacrylic
acid dehalogenases from Pseudomonas pavonaceae 170 (Poelarends et al., 2001) play a
role in the degradation of 1,3-dichloropropene, a compound applied in agriculture to kill
plant-infecting nematodes (Figure 1C). After conversion of trans-1,3-dichloropropene by a
haloalkane dehalogenase, oxidation of the trans-1-chloro-3-hydroxypropene product yields
trans-3-chloroacrylate. The trans-3-chloroacrylic acid dehalogenase (CaaD) converts this
product into malonate semialdehyde, a chloride ion and a proton. Subsequent
decarboxylation of malonate semialdehyde yields acetaldehyde, which can be used as an
alternative growth substrate.
The X-ray structures of the native and an inactivated form of trans-3-chloroacrylic
acid dehalogenase (CaaD) have recently been solved (de Jong et al., submitted). The
enzyme is a trimer of αβ heterodimers (Figures 5A, 5B). Its fold is similar to that of 4oxalocrotonate tautomerase (4-OT), a member of the tautomerase superfamily, which
contains isomerases and tautomerases (Whitman, 2003). However, whereas in the
isomerases and tautomerases the catalytic Pro-1 is in a hydrophobic environment, in
CaaD the corresponding Pro-1β is in a hydrophilic active site near a buried glutamate. This
30
has a major effect on the pKa of the proline residue: in 4-OT the proline has a strongly
reduced pKa (Czerwinski et al., 2001), whereas in CaaD the pKa is normal. This has
consequences for the catalytic role of the proline: in the isomerases and tautomerases it
functions as a general base, but in CaaD it functions as a general acid.
Figure 4. A) Tetrameric structure of haloalcohol dehalogenase (de Jong et al., 2003) with a bound
chloride ion and a bound epoxide product. B) Detailed view of the halide binding site. Trp249* comes
from another subunit. Dashed lines indicate hydrogen bonds. C) Schematic of the catalytic
mechanism of haloalcohol dehalogenase. D) Sequence comparison of the haloalcohol dehalogenases
HheC, HheA, and HheB with 4 SDR-family members indicated by their PDB entry number. 1FMC is
an NAD-dependent enzyme, while 1YBV, 1AE1, and 2AE2 are NADP-dependent. An aspartate or
glutamate in one of the boxed positions is indicative of an NAD-dependent enzyme, whereas NADPdependent enzymes have 1 or 2 arginines or lysines in different positions. NAD-dependent SDR
subfamilies are defined among others by the position of the aspartate (position 1, 2 or 3 in the box).
31
Figure 5. A) Heterohexameric structure of trans-3-chloroacrylic acid dehalogenase with a covalently
bound malonyl adduct to Pro-1β (de Jong et al., in press). B) Structure of the trans-3-chloroacrylic
acid dehalogenase monomer with a covalently bound malonyl adduct to Pro-1β. C) Stereo view of the
active site showing the bound adduct. The red and pink main chains indicate chains from the α and β
subunits, respectively. D) Schematic of the catalytic mechanism of trans-3-chloroacrylic acid
dehalogenase.
32
The role of the Pro-1β in CaaD became clear from the crystal structure of CaaD
inactivated by the suicide substrate 3-bromopropynoate (Figure 5C) (de Jong et al.,
submitted). This compound inactivates the enzyme by forming a covalent malonyl adduct
with the proline residue. This modification is the result of the hydration of the carboncarbon triple bond of the inhibitor via a Michael addition, catalyzed by the active site
glutamate, which activates a water molecule, and by the proline, which donates a proton.
The reactivity of the triple bond is increased by electrostatic interactions of its conjugated
carboxylate group with two arginine residues. In the case of 3-bromopropynoate the
product rearranges to a reactive acyl bromide, which forms a covalent bond with the
deprotonated proline. In contrast, the substrate trans-3-chloroacrylate is converted to an
instable halohydrin that decomposes to form malonate semialdehyde, a chloride ion and a
proton (Figure 5D).
Thus, the conjugated system of the carbon-carbon double bond and the
carboxylate group enables the dehalogenase to hydrate the substrate via a conjugate
addition reaction, followed by spontaneous dehalogenation of the product. The active site
glutamate and proline thus form the basis of a previously unidentified hydratase activity in
the tautomerase superfamily.
On the evolution of dehalogenating activities
The structural characterization of several bacterial dehalogenases has provided
detailed insight into their evolutionary relationship to other enzyme families. Some
dehalogenases may have evolved from superfamily members with distinctly different
activities, analogous to the creation of low-level crotonase activity in 4-chlorobenzoyl-CoA
dehalogenase by a double glutamate mutation (Xiang et al., 1999). Alternatively,
dehalogenation activity may emerge alongside an original enzyme activity. This is
illustrated by the bacterial muconate lactonizing enzymes, some of which can also
dehalogenate chlorinated muconates. A few amino acid substitutions can account for this
dehalogenating activity (Kajander et al., 2003). Although these case studies nicely
illustrate that minimal changes can drastically alter enzyme activities, they do not provide
compelling evidence on the origins of the various isolated dehalogenases.
The recent adaptation of a precursor enzyme in response to large concentrations
of a xenobiotic halogenated substrate may seem a reasonable mechanism for the
emergence of these activities, but seems difficult to reconcile with the worldwide spread of
some of the enzymes. Conserved gene clusters containing haloalkane-utilizing catabolic
33
pathways have been isolated from bacterial strains from three different continents
(Poelarends et al., 2000). Although horizontal gene transfer and/or integrase-dependent
gene acquisition can help in the local spread in bacteria (Poelarends et al., 2000), a global
diffusion by this mechanism seems unlikely. However, the presence of haloalkane
dehalogenases in various parasitic Mycobacterium strains, which colonize both animal
tissues and the free environment, could suggest a possible role of parasitic
microorganisms in a worldwide distribution mechanism (Jesenská et al., 2000).
The global distribution of these enzymes could also support a pre-industrial origin.
Many
different
organohalogens
occur
naturally,
suggesting
that
corresponding
dehalogenating activities exist in nature. Even in the case of highly toxic dioxins an
anaerobic bacterium has been isolated that can grow on them (Bunge et al., 2003).
Dioxins were long believed to be exclusively produced by the recent industrial activity of
mankind (Alcock and Jones, 1996), but they have entered the environment also by natural
processes (Hoekstra et al., 1999) and by the pre-industrial domestic burning of peat
(Meharg and Killham, 2003).
Although all dehalogenases that have been structurally characterized up to now
are members of existing enzyme superfamilies, their sequences are highly divergent from
superfamily members without dehalogenase activity. However, a structure-based
sequence alignment of haloalcohol dehalogenases and SDR enzymes demonstrates a
significant sequence identity of up to 30 to 35 % (de Jong et al., 2003). Two families of
haloalcohol dehalogenases can be discerned that are related to different coenzyme-based
SDR subfamilies, indicating that the two dehalogenase families independently originated
from two different NAD-binding precursors, rather than NAD(P)H-binding precursors
(Figure 4D). It is conceivable, that the haloalcohol could have bound in the active site of
such an SDR enzyme, whereupon the catalytic tyrosine fortuitously facilitated the
intramolecular substitution of the halogen by deprotonating the intramolecular hydroxyl
group. The dehalogenase activity could have been improved as a result of evolutionary
optimization of this rudimentary promiscuous activity. Catalytic promiscuity has been
discovered in various other enzyme superfamilies that use similar structural features in a
different chemical context (O'Brien and Herschlag, 1999). Strikingly, the closest relatives of
3-chloroacrylic acid dehalogenase, the tautomerase 4-OT and its homologue YwhB, have
a promiscuous dehalogenating activity towards trans-3-chloroacrylic acid (Whitman, 2003).
It would be interesting to see whether some existing members of the SDR family display a
low haloalcohol dehalogenation activity.
34
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