722

Structure and mechanism of bacterial dehalogenases: different ways to cleave a carbonhalogen bond
Rene M de Jong and Bauke W Dijkstra
The dehalogenases make use of fundamentally different strategies to cleave carbonhalogen bonds. The structurally characterized haloalkane dehalogenases, haloacid dehalogenases and 4-chlorobenzoate-coenzyme A 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 has provided 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.
Addresses Department of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, NL-9747 AG, Groningen, The Netherlands e-mail: bauke@chem.rug.nl

Halogenated compounds are, however, not only of anthropogenic origin. Over 1500 organohalogens are known to be produced naturally [3,4]. They range from volatile compounds such as methylchloride to antibiotics such as vancomycin and chloramphenicol. Insight into the enzymes that incorporate halogens into organic compounds is rather limited [5]. This strongly contrasts with the large amount of detailed information on the 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 carbonhalogen bonds. X-ray structures of haloalkane dehalogenases, haloacid dehalogenases and 4-chlorobenzoyl-coenzyme A (CoA) dehalogenase have demonstrated the power of substitution mechanisms that proceed via a covalent aspartyl intermediate [69]. The recent structural characterization of a haloalcohol dehalogenase and a trans-3chloroacrylic acid dehalogenase has revealed the details of two other elegant catalytic strategies, which exploit catalytic mechanisms from homologous enzymes in a different chemical context ([10]; RM de Jong, W Brugman, GJ Poelarends, CP Whitman, BW Dijkstra, unpublished data). The available information on the various dehalogenases supports different views on the possible origins of their individual activities towards presumed anthropogenic halogenated substrates.

Current Opinion in Structural Biology 2003, 13:722730 This review comes from a themed issue on Catalysis and regulation Edited by Bauke W Dijkstra and Rowena G Matthews 0959-440X/$ see front matter 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2003.10.009

Abbreviations CaaD trans-3-chloroacrylic acid dehalogenase CoA coenzyme A 4-OT 4-oxalocrotonate tautomerase PCB polychlorinated biphenyl PDB Protein Data Bank SDR short-chain dehydrogenase/reductase

Haloalkane dehalogenases
Haloalkane dehalogenases cleave the carbonhalogen bond in halogenated aliphatic hydrocarbons (Figure 1a). The haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 was the rst dehalogenase for which the crystal structure was determined [11], but other structures have since become available [12,13]. 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 b sheet (only the second b strand is antiparallel) connected by a helices on both sides of the sheet. The cap domain is a helical. This fold is the hallmark of the superfamily of a/b-hydrolases, to which, besides the haloalkane dehalogenases, lipases, esterases, carboxypeptidases and acetylcholinesterases also belong [1416]. 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 (e.g. see [17,18]). In the rst step, the serine or cysteine of a Ser/Cys-His-Asp/Glu catalytic triad functions as a nucleophile that attacks the sp2-hybridized carbonyl
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Introduction
Since the beginning of the last 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 halogenated solvents, such as, for instance, 1,2-dichloroethane, have also been classied as probably carcinogenic to humans [1,2]. Because of these noxious properties, many of these organohalogens have now been banned from use and have been replaced by environmentally less harmful compounds.
Current Opinion in Structural Biology 2003, 13:722730

Structure and mechanism of bacterial dehalogenases de Jong and Dijkstra 723

Figure 1

(a) Cl Cl

Haloalkane dehalogenase + H2O HCl

Cl OH

Oxidation

Cl O

Haloacid dehalogenase

HO O
Glycolate

+ H2O HCl

1,2-dichloroethane

2-chloroethanol

2-chloroacetate

(b) Cl

OH Cl

Haloalcohol dehalogenase

Epoxide hydrolase

O
HCl

OH HO Cl

Haloalcohol dehalogenase

O HO
Glycidol

Epoxide hydrolase + H2O

OH HO
Glycerol

Cl
Epichlorohydrin

+ H2O

HCl

OH

1,3-dichloro-2-propanol

1-chloro-2,3-dipropanol

(c) Cl

Cl

Haloalkane dehalogenase + H 2O HCl

O OH Cl
Oxidation

O Cl
trans-3-chloroacrylate

trans-3-chloroacrylic acid dehalogenase + H 2O HCl

O H O O

Malonate semialdehyde decarboxylase CO2

H O

1,3-dichloropropene

1-chloro-3-hydroxypropene

Malonate semialdehyde

Acetaldehyde

(d) Cl

4-chlorobenzoate CoA ligase

O S

4-chlorobenzoyl-CoA dehalogenase

O S

4-hydroxybenzoyl-CoA thioesterase

O HO O
4-hydroxybenzoate
Current Opinion in Structural Biology

Cl O
+ CoA H2O + H2O HCl

HO
+ H2O CoA

CoA
4-chlorobenzoyl-CoA

CoA
4-hydroxybenzoyl-CoA

4-chlorobenzoate

Bacterial degradation pathways for the halogenated compounds discussed in this review: (a) 1,2-dichloroethane and 2-chloroacetate; (b) 1,3-dichloro-2-propanol; (c) 1,3-dichloropropene; (d) 4-chlorobenzoate.

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 halogenbearing sp3-hybridized carbon atom. This also leads to a covalently bound ester intermediate that can be easily hydrolyzed by attack of a histidine-activated water molecule on the Cg atom of the aspartate (Figure 2c) [6]. Thus, substitution of the serine/cysteine of an a/b-hydrolase by an aspartate confers on the enzyme an essential prerequisite to hydrolyze carbonhalogen bonds in haloalkanes. In addition, haloalkane dehalogenases contain a halidebinding site to facilitate the dehalogenation reaction. During the past few years, computational studies have unraveled details of the catalytic mechanism. In the
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enzyme, the nucleophilic aspartate is nearly entirely positioned in a near attack conformation, with the attacking oxygen of the aspartate in line with the carbonchlorine bond of the substrate, at a distance favorable for attack on the halogen-bearing carbon atom. However, the correct positioning contributes only 25% to the lowering of the transition state free energy [19,20]. The remaining 75% probably originates from contributions of the halide-binding residues to transition state stabilization [21] and from the half-hydrophilic/halfhydrophobic nature of the active site, which facilitates both the activation of the nucleophile and the departure of the leaving group. Computational quantication of these contributions is presently beyond reach [20].

Haloacid dehalogenases
Haloacid dehalogenases catalyze the hydrolysis of ahalogenated 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 [22,23], to
Current Opinion in Structural Biology 2003, 13:722730

724 Catalysis and regulation

Figure 2

(a)

(b)

(c) Halide-binding site

Cl R R O
B: Asp

Cl

Cl
+ H2O

R OH O
BH

O
B:

O H O H

Asp

Asp

R = CH2Cl (haloalkane dehalogenase)

R = COO (haloacid dehalogenase)

Current Opinion in Structural Biology

The structures and general mechanism of haloalkane and haloacid dehalogenases. (a) Structure of the haloalkane dehalogenase from X. autotrophicus [11], showing the nucleophilic aspartate and a bound chloride ion. (b) Dimeric structure of haloacid dehalogenase with a covalently bound intermediate [9]. (c) Schematic of the catalytic mechanism of haloalkane and haloacid dehalogenases.

which magnesium-dependent phosphatases and P-type ATPases also belong. Haloacid dehalogenases are dimers with two or three domains per subunit: a core domain with a Rossmann-fold-like six-stranded parallel b sheet anked by ve a helices, a subdomain consisting of a fourhelix bundle and, in some enzymes, a dimerization domain of two antiparallel a helices (Figure 2b) [8,24]. This fold is completely different from the a/b-hydrolase fold of the haloalkane dehalogenases. Like the latter enzymes, haloacid dehalogenases utilize an aspartatebased catalytic mechanism that proceeds via a covalent intermediate (Figure 2c), but there is no histidine to activate a nucleophilic water molecule. Also, the halidebinding site is very different [9]. How the water molecule is activated is not known, but it has been suggested that another aspartate in the active site fulls this function [9]. Intriguingly, at least one other unrelated haloacid dehalogenase family exists [25,26], which was shown to bypass the classic covalent ester intermediate (type II haloacid dehalogenases) [27]. This suggests that enzymes with different haloacid dehalogenating strategies have evolved from different enzyme precursors, but unfortunately crysCurrent Opinion in Structural Biology 2003, 13:722730

tallographic data that could provide structural insight into the mode of action of type II haloacid dehalogenases are still unavailable.

4-chlorobenzoyl-CoA dehalogenases
4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp strain CBS-3 is a homotrimer in which each subunit folds into two domains. The N-terminal domain contains a tenstranded b sheet, forming two nearly perpendicular layers, which are anked by a helices (Figure 3a). The C-terminal domain is composed of three amphiphilic a 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) [7,28,29]. The mechanism also proceeds via a covalent aspartyl intermediate. First, the substrate is ligated to CoA by 4-chlorobenzoate CoA ligase. The CoA-ligated product is then bound by 4-chlorobenzoyl-CoA dehalogenase, with the enolate anion of the thioester link of the substrate stabilized by two backbone amide groups. This induces a partially
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Structure and mechanism of bacterial dehalogenases de Jong and Dijkstra 725

Figure 3

(a)

(b)
NH NH
O CoA S

Oxyanion hole
NH NH
O S CoA

Cl Cl O O

:B
O

:B

Asp

Asp

NH NH NH
O S CoA O

N H
CoA S

Cl

HO
O O

O H H

:B
O

HB

Asp

Asp
Current Opinion in Structural Biology

The structure and mechanism of 4-chlorobenzoyl-CoA dehalogenase. (a) Trimeric structure of 4-chlorobenzoyl-CoA dehalogenase [7], showing the nucleophilic aspartate, a bound calcium ion and the product 4-hydroxybenzoyl-CoA. (b) Schematic of the catalytic mechanism of 4-chlorobenzoyl-CoA dehalogenase.

positive charge on the halogen-bearing carbon atom, which makes it susceptible to nucleophilic attack by the aspartate (Figure 3b) [28]. This SNAr-type substitution results in a covalent Meisenheimer intermediate, as recently conrmed by Raman spectroscopic measurements [29]. Chloride is expelled upon restoration of the aromatic ring system, producing a second arylated enzyme intermediate that is subsequently hydrolyzed by attack on the Cg atom of the nucleophilic aspartate by a histidine-activated water molecule, yielding 4-hydroxybenzoyl-CoA [30,31]. The stabilization of the acyl-CoA enolate anion intermediate is shared with hydratases, isomerases and thioesterases of the crotonase (or enoyl-CoA hydratase) superfamily [32], but the nucleophilic aspartate is only present in the dehalogenase. As in haloalkane and haloacid dehalogenases, the aspartate confers on the enzyme its unique capability to hydrolyze the bond between a halogen and an aromatic carbon atom. The enzymes discussed above illustrate the power of aspartate-mediated substitution mechanisms to hydrowww.current-opinion.com

lyze 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 [33,34]. By contrast, the Xray structures of a haloalcohol dehalogenase and the trans3-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-dichloro-2-propanol (Figure 1b). HheC is a homotetrameric enzyme (Figure 4a); the monomer is homologous to members of the widespread short-chain dehydrogenase/reductase (SDR) family. SDR enzymes are redox enzymes that catalyze alcohol-ketone conversions of a wide variety of alcohols, steroids and sugars [35,36]. They have the well-known dinucleotidebinding Rossmann fold [37] and a Ser-Tyr-Lys/Arg
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726 Catalysis and regulation

Figure 4

(a)

(b)

(c)
Ser132 Tyr145 Arg149 Ser132 Tyr145 Arg149

O H H2 C Cl Cl
Halide-binding site

O O H O H H N H N N H H Cl Cl H2 C H O H H O H N H N N H H

(d)
HheC ---------HheA-AD2 ---------HheB-GP1 MANGRKREMA 1FMC -----MFNSD 1YBV ~DAIPGPLGPK 1AE1 ~VSMMNCNNEG 2AE2 -------MAG

-----MSTAI -----MVIAL NGRLAGKRVL NLRLDGKCAI SASLEGKVAL RWSLKGTTAL RWNLEGCTAL * ..

VTNVKHFGGM VTHARHFAGP LTNADAYMGE ITGAGAGIGK VTGAGRGIGR VTGGSKGIGY VTGGSRGIGY .* *

GSALRLSEAG AAVEALTQDG ATVQVFEEEG EIAITFATAG EMAMELGRRG AIVEELAGLG GIVEELASLG . . *

HTVACHDESF YTVVCHDASF AEVFADHTDL ASVVVSDINCKVIVNYANS ARVYTCSRNASVYTCSRN*

35 35 50 42 61 52 40

Current Opinion in Structural Biology

The structure and mechanism of haloalcohol dehalogenase. (a) Tetrameric structure of haloalcohol dehalogenase [10] with a bound chloride ion and bound epoxide product. (b) Detailed view of the halide-binding site. Trp249 is contributed by a neighboring 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 four SDR family members indicated by their PDB entry number. 1FMC is an NAD-dependent enzyme, whereas 1YBV, 1AE1 and 2AE2 are NADP dependent. An aspartate or glutamate in one of the yellow boxed positions is indicative of an NAD-dependent enzyme, whereas NADP-dependent enzymes have one or two arginines or lysines in different positions. The seven NAD-dependent SDR subfamilies are, among others, defined by the position of the aspartate (position 1, 2 or 3 in the box). The asterisks and dots indicate strictly and partly conserved residues, respectively.

catalytic triad. HheC has the fold and catalytic triad of the SDR family, but lacks the characteristic dinucleotidebinding Gly-X-X-X-Gly-X-Gly motif (Figure 4d) [10,38]. Instead, several larger residues replace the smaller ones of the motif, thus lling up the cofactor-binding site. In this way, a spacious halide-binding site is created (Figure 4b). The enzyme uses an intramolecular SN2Current Opinion in Structural Biology 2003, 13:722730

type substitution mechanism, in which the secondary hydroxyl group of the haloalcohol substrate is deprotonated by the tyrosine of the conserved Ser-Tyr-Arg catalytic triad, which acts 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, whereas the aspartate-dependent
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Structure and mechanism of bacterial dehalogenases de Jong and Dijkstra 727

dehalogenases discussed above catalyze a two-step hydrolysis of the carbonhalogen bond, the presence of a hydroxyl function vicinal to the halogen allows the one-step intramolecular substitution of the halogen by the hydroxyl.

decomposes to form malonate semialdehyde, a chloride ion and a proton (Figure 5d). Thus, the conjugated system of the carboncarbon 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 unidentied hydratase activity in the tautomerase superfamily.

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 [39] play a role in the degradation of 1,3-dichloropropene, a compound used in agriculture to kill plantinfecting 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. 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 CaaD have recently been solved (RM de Jong, W Brugman, GJ Poelarends, CP Whitman, BW Dijkstra, unpublished data). The enzyme is a trimer of ab-heterodimers (Figure 5a,b). Its fold is similar to that of 4oxalocrotonate tautomerase (4-OT), a member of the tautomerase superfamily, which includes isomerases and tautomerases [40]. However, in isomerases and tautomerases the catalytic Pro1 is in a hydrophobic environment, whereas in CaaD the corresponding bPro1 is in a hydrophilic active site near a buried glutamate. This has a major effect on the pKa of the proline residue: in 4-OT the proline has a strongly reduced pKa [41], whereas in CaaD the pKa is normal. This has consequences for the catalytic role of the proline: in isomerases and tautomerases it functions as a general base, but in CaaD it functions as a general acid. The role of bPro1 in CaaD became clear from the crystal structure of CaaD inactivated by the suicide substrate 3bromopropynoate (Figure 5c) (RM de Jong, W Brugman, GJ Poelarends, CP Whitman, BW Dijkstra, unpublished data). This compound inactivates the enzyme by forming a covalent malonyl adduct with the proline residue. This modication is the result of the hydration of the carbon carbon 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. By contrast, the substrate trans-3-chloroacrylate is converted to an unstable halohydrin that
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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 [42]. 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 [43]. 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 it seems difcult to reconcile with the worldwide spread of some of the enzymes. Conserved gene clusters containing haloalkane-utilizing catabolic pathways have been isolated from bacterial strains from three different continents [44]. Although horizontal gene transfer and/or integrase-dependent gene acquisition can help the local spread in bacteria [45], 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 for parasitic microorganisms in a worldwide distribution mechanism [46]. 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 [47]. Dioxins were long believed to be exclusively produced by the recent industrial activity of mankind [48], but they have also entered the environment by natural processes [49] and by the pre-industrial domestic burning of peat [50].
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728 Catalysis and regulation

Figure 5

(a)

(b)

(c)

(d)
OH O

Arg8

Arg8

Arg8

+
O-

+
Arg11

+
Arg11

+
O H H
+

OO

+
Cl H H O

OO

Arg11

Cl

+
Cl H H H N

Cl

O H

H N H

H H

Glu52

O H O-

Pro1

Glu52
OH O

Pro1

Glu52
OH O

Pro1

Current Opinion in Structural Biology

The structure and mechanism of trans-3-chloroacrylic acid dehalogenase. (a) Heterohexameric structure of CaaD with a covalently bound malonyl adduct to bPro1 (RM de Jong, W Brugman, GJ Poelarends, CP Whitman, BW Dijkstra, unpublished data). (b) Structure of the CaaD monomer with a covalently bound malonyl adduct to bPro1. (c) Stereo view of the active site, showing the bound adduct. The red and pink mainchains indicate chains from the b and a subunits, respectively. (d) Schematic of the catalytic mechanism of CaaD.

Although all dehalogenases that have been structurally characterized to date are members of existing enzyme superfamilies, their sequences are highly divergent from superfamily members without dehalogenase activity. However, a structure-based sequence alignment of
Current Opinion in Structural Biology 2003, 13:722730

haloalcohol dehalogenases and SDR enzymes demonstrates a signicant sequence identity of up to 3035% [10]. Two families of haloalcohol dehalogenases can be discerned that are related to different coenzyme-based SDR subfamilies, indicating that the two dehalogenase
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Structure and mechanism of bacterial dehalogenases de Jong and Dijkstra 729

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 [51]. Strikingly, the closest relatives of 3-chloroacrylic acid dehalogenase, the tautomerase 4OT and its homologue YwhB, have a promiscuous dehalogenating activity towards trans-3-chloroacrylic acid [40]. It would be interesting to see whether some existing members of the SDR family display low haloalcohol dehalogenation activity.

enzyme catalyst generated via adaptive mutation. Biochemistry 1996, 35:8103-8109. 8. Hisano T, Hata Y, Fujii T, Liu JQ, Kurihara T, Esaki N, Soda K: Crystal structure of L-2-haloacid dehalogenase from Pseudomonas sp YL - An a/b hydrolase structure that is different from the a/b hydrolase fold. J Biol Chem 1996, 271:20322-20330. Ridder IS, Rozeboom HJ, Kalk KH, Dijkstra BW: Crystal structures of intermediates in the dehalogenation of haloalkanoates by L-2-haloacid dehalogenase. J Biol Chem 1999, 274:30672-30678.

9.

10. de Jong RM, Tiesinga JJW, Rozeboom HJ, Kalk KH, Tang L,  Janssen DB, Dijkstra BW: Structure and mechanism of a bacterial haloalcohol dehalogenase: a new variation of the short-chain dehydrogenase/reductase fold without an NAD(P)H binding site. EMBO J 2003, 22:4933-4944. The rst structure of a haloalcohol dehalogenase, and its complexes with substrate and product analogues allowed a detailed comparison with the SDR enzyme family. 11. Franken SM, Rozeboom HJ, Kalk KH, Dijkstra BW: Crystal structure of haloalkane dehalogenase - an enzyme to detoxify halogenated alkanes. EMBO J 1991, 10:1297-1302. 12. Newman J, Peat TS, Richard R, Kan L, Swanson PE, Affholter JA, Holmes IH, Schindler JF, Unkefer CJ, Terwilliger TC: Haloalkane dehalogenases: structure of a Rhodococcus enzyme. Biochemistry 1999, 38:16105-16114. 13. Oakley AJ, Prokop Z, Bohac M, Kmuncek J, Jedlicka T, Monincova M, Kuta-Smatanova I, Nagata Y, Damborsky J, Wilce MCJ: Exploring the structure and activity of haloalkane dehalogenase from Sphingomonas paucimobilis UT26: evidence for product- and water-mediated inhibition. Biochemistry 2002, 41:4847-4855. 14. Ollis DL, Cheah E, Cygler M, Dijkstra B, Frolow F, Franken SM, Harel M, Remington SJ, Silman I, Schrag J et al.: The a/bhydrolase fold. Protein Eng 1992, 5:197-211. 15. Heikinheimo P, Goldman A, Jeffries C, Ollis DL: Of barn owls and bankers: a lush variety of a/b hydrolases. Structure Fold Des 1999, 7:R141-R146. 16. Nardini M, Dijkstra BW: a/b Hydrolase fold enzymes: the family keeps growing. Curr Opin Struct Biol 1999, 9:732-737. 17. Dodson G, Wlodawer A: Catalytic triads and their relatives. Trends Biochem Sci 1998, 23:347-352. 18. Jaeger KE, Dijkstra BW, Reetz MT: Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol 1999, 53:315-351. ` 19. Shurki A, Strajbl M, Villa J, Warshel A: How much do enzymes  really gain by restraining their reacting fragments? J Am Chem Soc 2002, 124:4097-4107. Detailed computations analyzing the inuence of the position of the catalytic aspartate with respect to the substrate on catalysis by haloalkane dehalogenase. 20. Hur S, Kahn K, Bruice TC: Comparison of formation of reactive conformers for the S(N)2 displacements by CH3CO2S in water and by Asp124-CO2S in a haloalkane dehalogenase. Proc Natl Acad Sci U S A 2003, 100:2215-2219. 21. Bohac M, Nagata Y, Prokop Z, Prokop M, Monincova M, Tsuda M, Koca J, Damborsky J: Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis. Biochemistry 2002, 41:14272-14280. 22. Koonin EV, Tatusov RL: Computer-analysis of bacterial haloacid dehalogenases denes a large superfamily of hydrolases with diverse specicity - application of an iterative approach to database search. J Mol Biol 1994, 244:125-132. 23. Ridder IS, Dijkstra BW: Identication of the Mg2R-binding site in the P-type ATPase and phosphatase members of the HAD (haloacid dehalogenase) superfamily by structural similarity to the response regulator protein CheY. Biochem J 1999, 339:223-226. Current Opinion in Structural Biology 2003, 13:722730

Conclusions
Crystal structures have revealed that the various dehalogenases belong to widespread enzyme superfamilies. Subtle differences in the active site or in the catalytic residues, rather than the common reaction specicities of the superfamilies, make possible dehalogenase activity. One can, however, only guess at the origin of these activities, although, in the case of the haloalcohol dehalogenases, structure-based sequence alignments suggest that these enzymes have independently evolved from two NAD-dependent SDR enzymes.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1. Safe SH: Polychlorinated-biphenyls (PCBs) - Environmental impact, biochemical and toxic responses, and implications for risk assessment. Crit Rev Toxicol 1994, 24:87-149. Salovsky P, Shopova V, Dancheva V, Yordanov Y, Marinov E: Early pneumotoxic effects after oral administration of 1,2dichloroethane. J Occup Environ Med 2002, 44:475-480.

2.

3. Oberg G: The natural chlorine cycle - tting the scattered  pieces. Appl Microbiol Biotechnol 2002, 58:565-581. This review reports that chlorinated organic compounds are not all xenobiotic, that many organisms are able to convert chloride to organic chlorine, that the vast majority of naturally produced organic chlorine is neither persistent nor toxic, and that organic chlorine is as abundant as or even more abundant than chloride in soil. 4. Ballschmiter K: Pattern and sources of naturally produced organohalogens in the marine environment: biogenic formation of organohalogens. Chemosphere 2003, 52:313-324. van Pee KH, Unversucht S: Biological dehalogenation and halogenation reactions. Chemosphere 2003, 52:299-312. Verschueren KHG, Seljee F, Rozeboom HJ, Kalk KH, Dijkstra BW: Crystallographic analysis of the catalytic mechanism of haloalkane dehalogenase. Nature 1993, 363:693-698. Benning MM, Taylor KL, Liu RQ, Yang G, Xiang H, Wesenberg G, Dunaway-Mariano D, Holden HM: Structure of 4-chlorobenzoyl coenzyme A dehalogenase determined to 1.8 A resolution: an

5. 6.

7.

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730 Catalysis and regulation

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