- Email: [email protected]
Biological dehalogenation and halogenation reactions
Chemosphere 52 (2003) 299–312 www.elsevier.com/locate/chemosphere
Review
Biological dehalogenation and halogenation reactions Karl-Heinz van Pee *, Susanne Unversucht Institut f€ur Biochemie, TU Dresden, D-01062 Dresden, Germany Received 19 February 2002; received in revised form 19 November 2002; accepted 19 November 2002
Abstract A large number of halogenated compounds is produced by chemical synthesis. Some of these compounds are very toxic and cause enormous problems to human health and to the environment. Investigations on the degradation of halocompounds by microorganisms have led to the detection of various dehalogenating enzymes catalyzing the removal of halogen atoms under aerobic and anaerobic conditions involving different mechanisms. On the other hand, more than 3500 halocompounds are known to be produced biologically, some of them in great amounts. Until 1997, only haloperoxidases were thought to be responsible for incorporation of halogen atoms into organic compounds. However, recent investigations into the biosynthesis of halogenated metabolites by bacteria have shown that a novel type of halogenating enzymes, FADH2 -dependent halogenases, are involved in biosyntheses of halogenated metabolites. In every gene cluster coding for the biosynthesis of a halogenated metabolite, isolated so far, one or several genes for FADH2 -dependent halogenases have been identified. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Dehalogenation; Dehalogenase; Halogenation; Halogenase; Haloperoxidase
Contents 1. 2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dehalogenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Hydrolytic dehalogenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Thiolytic dehalogenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Intramolecular substitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Dehydrohalogenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Dehalogenation by hydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Dehalogenation by methyltransfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Oxidative dehalogenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. Reductive dehalogenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8.1. Reductive dehalogenation under aerobic conditions. . . . . . . . . . . . . . . . . . . . 2.8.2. Anaerobic reductive dehalogenation (halorespiration) . . . . . . . . . . . . . . . . . . Halogenation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Halogenated metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Halogenating enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
*
Corresponding author. Tel.: +49-351-463-4494; fax: +49-351-463-35506/5508. E-mail address: [email protected] (K.-H. van Pee).
0045-6535/03/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0045-6535(03)00204-2
300 300 300 300 301 301 302 302 302 302 302 303 304 304 304
300
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
3.2.1. Haloperoxidases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Perhydrolases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. FADH2-dependent halogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Halogenated organic compounds are widely used as pharmaceuticals, herbicides, fungicides, insecticides, flame retardants, intermediates in organic synthesis, solvents etc. Additionally, many halogenated compounds are produced as by-products during chemical synthesis, such as dioxins, polychlorinated biphenyls and pentachlorophenol. Since many of these halocompounds show toxic or even carcinogenic effects and many of them are often difficult to degrade, many investigations were conducted to analyse the fate of these compounds in the environment. Studies on the degradation of different halogenated aliphatic and aromatic compounds led to the detection and elucidation of a variety of dehalogenases and dehalogenation mechanisms (Janssen et al., 1994; Fetzner, 1998). However, halogenated compounds are not only man-made, but also synthesized in nature by a large number of different organisms (Gribble, 1998). Until today, over 3500 halogen-containing metabolites have been isolated. Some of these halogenated metabolites have great importance as antibiotics such as chloramphenicol, 7-chlorotetracyclin, or vancomycin. However, until recently (van Pee, 1996), very little was known about the enzymes catalyzing the incorporation of halogen atoms during the biosynthesis of these compounds.
2. Dehalogenation 2.1. Hydrolytic dehalogenation The first bacterial dehalogenase was purified in 1960 by Jensen. This enzyme catalyzes the dechlorination of chloroacetate and 2-chloropropionic acid. Similar 2haloacid dehalogenases were subsequently isolated from many different bacteria such as Moraxella sp., Pseudomonas sp., and Xanthobacter autotrophicus (Van der Ploeg et al., 1991). 2-Haloacid dehalogenases catalyze a substitution of the halogen with water as the nucleophile resulting in the formation of an alcohol (Fig. 1). No other cofactor is required. The best studied hydrolytic halogenase is the haloalkane dehalogenase from the 1,2-dichloroethane-
Cl C
+
H2O
304 307 307 309 310
OH + HCl C
Fig. 1. Hydrolytic dehalogenation. In hydrolytic dehalogenation reactions, the halogen is displaced by water in a nucleophilic replacement reaction.
degrading bacterium Xanthobacter autotrophicus GJ10. The reaction mechanism of this enzyme was elucidated by X-ray crystallographic analysis (Verschueren et al., 1993) and site-directed mutagenesis (Pries et al., 1994). The enzyme belongs to the a=b hydrolase fold family and contains a catalytic triad consisting of one histidin and two aspartate residues which are involved in the nucleophilic substitution of the halide ion by water. The removal of the halide ion is additionally facilitated by two tryptophan residues (Fig. 2; Kennes et al., 1995). During degradation of 4-chlorobenzoic acid by Nocardia species, 4-hydroxybenzoic acid was isolated as an intermediate in the degradation pathway (Klages and Lingens, 1979). During investigation of the enzyme from Pseudomonas sp. CBS3, it could be shown that the hydroxyl group was derived from water and not from molecular oxygen (M€ uller et al., 1984). For hydrolytic substitution of chloride by a hydroxyl group at an aromatic system, activation by coenzyme A ester formation is required. The first step in 4-chlorobenzoate degradation, the formation of 4-chlorobenzoyl-coenzyme A, is catalyzed by a ligase. The second step, the actual dehalogenation reactions, is catalyzed by 4-chlorobenzoylcoenzyme A dehalogenase and 4-hydroxybenzoyl-coenzyme A is hydrolyzed by a thioesterase (Fig. 3; Luo et al., 2001).
2.2. Thiolytic dehalogenation Methylotrophic bacteria such as Methylophilus sp., Methylobacterium sp. and Hyphomicrobium sp. isolated with dichloromethane as growth substrate produce an inducible glutathione S-transferase which catalyzes the formation of an unstable S-chloromethyl glutathione intermediate. This intermediate is hydrolyzed to glutathione, chloride, and formaldehyde (Fig. 4; Leisinger
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
O
Trp175
C
OH
301
O C
S CoA
+ ATP, CoA-SH Cl
O - O
N
HN
N H
4-CBA:CoA ligase
H
H N
O
+ H2O - HCl dehalogenase O C
Asp124
His289
Cl
Trp 125
O - O H
Asp260
Cl
C C Cl H2 H HN 2
O
OH
C + H2O, - CoA-SH thioesterase
Trp175
OH
OH
Fig. 3. Hydrolytic dehalogenation of 4-chlorobenzoic acid (4-CBA) involving activation by formation of a coenzyme A ester.
N H Cl
Cl C CH 2 H2 O
- O
N
HN
H
HN
H N
O H
His289
Asp260
Trp 125
O
O
Asp124 Trp175
Cl
O
-
O
Asp260
HN
N H Cl C CH 2 H2 HN OH + NH O - O H N
His289
S CoA
Trp 125
2.3. Intramolecular substitution Haloalkohol dehalogenases, also known as halohydrin dehalogenases or halohydrin hydrogen–halide lyases which have been isolated from Arthrobacter sp. and other bacteria, catalyze the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides (Fig. 5; Van Hylckama Vileg et al., 2001). Although these enzymes show sequence homology with the family of short-chain dehydrogenases/reductases, they do not require NAD(P)þ as a coenzyme. They have the same two active site amino acid residues serine and tyrosine with the lysine residue of the short-chain dehydrogenases/reductases replaced by an arginine residue. These enzymes are also able to catalyze the reverse reaction, the halogenation of epoxides to haloalcohols (Nakamura et al., 1991).
Asp124
2.4. Dehydrohalogenation
Fig. 2. Mechanism of haloalkane dehalogenase. Two tryptophan residues are involved in removing the halogen atom.
et al., 1994; Marsh and Ferguson, 1997; Gisi et al., 1998).
Cl CH 2 + GSH - HCl Cl
The first two steps in the degradation of lindane (chexachlorocyclohexane) by Pseudomonas paucimobilis are catalyzed by a dehydrohalogenase. The elimination of hydrochloric acid catalyzed by this enzyme leads to
+ H2 O GS GS C Cl - HCl H2
CH2OH
O H C H
Fig. 4. Thiolytic dehalogenation of dichloromethane catalyzed by a glutathione transferase.
302
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
H H C Cl haloalcohol dehalogenase H C OH - HCl H C Cl H
+ H2O
H H C OH H C O H C H
Cl + H2O C C COOH H H
H H C O H C H C Cl H
haloalcohol dehalogenase - HCl
Cl
Cl
- HCl
Cl
Cl
Cl
Cl
- HCl
Cl Cl
H H C OH H C OH H C Cl H
Cl
Cl
Cl
Cl
+ H2O - HCl
HO
Cl
Cl
OH
+ H2O - HCl
O C C COOH H H2
epoxide hydrolase
Cl Cl
- HCl
Fig. 7. Dehalogenation by addition of water onto a double bond.
Fig. 5. Intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group catalyzed by haloalcohol dehalogenases. These enzymes can also catalyze the reverse reaction, the halogenation of the epoxides to haloalcohols.
Cl
Cl HC C COOH O H2 H
HO
Cl
Cl
Cl
Fig. 6. Dehalogenation of lindane as an example of dehydrohalogenation with formation of a double bond.
the formation of a double bond (Fig. 6; Imai et al., 1991). 2.5. Dehalogenation by hydration In a coryneform bacterium, isolated with 3-chloroacrylate as the carbon source, two dehalogenases have been detected. The dehalogenation reactions catalyzed by these enzymes, are proposed to proceed by addition of water to the double bond yielding an unstable intermediate from which malonate semialdehyde is formed (Fig. 7; Van Hylckama Vileg and Janssen, 1992). 2.6. Dehalogenation by methyltransfer Dehalogenation by methyltransfer was found in aerobic and strictly anaerobic methylotrophic bacteria using chloromethane or dichloromethane as the sole
carbon source (Meßmer et al., 1996; Vanelli et al., 1998). Coulter et al. (1999) described a methyltransferase that contains a corrinoid-bound cobalt atom. In the active state the cob(II)alamin is oxidized to the cob(III)alamin state by methylation by halomethane and is reduced by demethylation by the methyl group acceptor ion which can be a halide or bisulfide ion (Fig. 8).
2.7. Oxidative dehalogenation Oxidative dehalogenation reactions can be found in pathways for the degradation of haloaliphatic as well as haloaromatic compounds. These reactions are catalyzed by monooxygenases with a broad substrate specificity like methane monooxygenase from Methylococcus capsulatus Bath or Methylococcus trichosporium (Yokota et al., 1986; Fox et al., 1990) or by dioxygenases like the two-component 4-chlorophenylacetate 3,4-dioxygenase from Pseudomonas sp. CBS3 (Fig. 9; Schweizer et al., 1987). Lignin and manganese peroxidase from the lignindegrading basidiomycete Phanerochaete chrysosporium were also found to catalyze an oxidative dechlorination reaction of pentachlorophenol to produce tetrachloro1,4-benzoquinone (Reddy and Gold, 2000).
2.8. Reductive dehalogenation 2.8.1. Reductive dehalogenation under aerobic conditions During degradation of pentachlorophenol by Sphingomonas chlorophenolica under aerobic conditions, tetra-
E---cob(II)alamin CH3X
CH3Y
X
Y E---CH3cob(III)alamin
Fig. 8. Dehalogenation of chloromethane catalyzed by a cobalamin-containing methyltransferase; CH3 X can be CH3 Cl, CH3 Br or CH3 I, and Y can be Cl , Br , I or HS .
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
O OH C CH2 + O2 + NADH +
H+
4-chlorophenylacetate 3,4-dioxygenase
Cl
303
O OH C CH2
Cl
O H
OH H
- HCl O OH C CH2
OH OH Fig. 9. Oxidative dehalogenation of 4-chlorophenylacetate catalyzed by the two-component system of 4-chlorophenylacetate 3,4dioxygenase.
OH
OH
Cl
Cl
Cl
Cl
+ GSH - HCl
Cl
Cl
Cl
H SG
OH
O + H
-
S
C H2 Enzyme
OH Enzyme
Cl
Cl
Cl
+ GS-S C H2 H OH + GSH S
C H2 Enzyme + GS-SG
Fig. 10. Reductive dehalogenation of tetrachlorohydroquinone under aerobic conditions catalyzed by a glutathione transferase.
chlorohydroquinone is produced by a monooxygenase reaction catalyzed by pentachlorophenol hydroxylase. The thus formed tetrachlorohydroquinone is dehalogenated in a reductive dehalogenation reaction catalyzed by tetrachlorohydroquinone dehalogenase to trichlorohydroquinone (Fig. 10; Kiefer et al., 2002). A similar tetrachlorohydroquinone reductive dehalogenase system was also found in the white rot fungus Phanerochaete chrysosporium (Reddy and Gold, 2001). Tetrachlorohydroquinone dehalogenase is a member of the glutathione
S-transferase family like the enzyme involved in dichloromethane dehalogenation. Lignin peroxidases from white rot fungi such as Phanerochaete chrysosporium can also catalyze the reductive dehalogenation of carbon tetrachloride via a mechanism involving one electron oxidation of veratryl alcohol to the veratryl alcohol cation radical. This radical is reduced by oxalacetate resulting in a one-electron oxidation of oxalacetate. The remaining electron can then be used to catalyze reduction reactions such as the reductive dechlorination of carbon tetrachloride (Shad et al., 1993). A different type of reductive dehalogenation reaction is catalyzed by the NADH-dependent enzyme maleylacetate reductase which has been isolated from several Pseudomonas strains and Ralstonia eutropha (Fig. 11; M€ uller et al., 1996). 2.8.2. Anaerobic reductive dehalogenation (halorespiration) In halorespiration, reductive dehalogenation is coupled to energy metabolism. Here a halogenated compound, like tetrachloroethene and trichloroethene, serves as a terminal electron acceptor during the oxidation of an electron rich compound, like hydrogen or an organic substrate (Schuhmacher et al., 1997; Wohlfahrt and Diekert, 1997). The reductive dehalogenases isolated from several bacteria such as Dehalococcoides ethenogenes or Desulfitobacterium dehalogenans catalyzing these reactions contain a cobalamin cofactor and iron– sulfur clusters (Fig. 12; Van de Pas et al., 1999; Magnuson et al., 2000).
304
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
OH O OH C C CH2 CH2 C O + maleylacetate reductase C O + NAD + + HCl CH + NADH + H CH C Cl CH C C HO O HO O O
Fig. 11. NADH-dependent reductive dehalogenation of 2-chloromaleylacetate to 3-oxoadipate under aerobic conditions.
Cl
Cl
C C Cl Cl
corrinoid-dependent PCE reductive dehalogenase
Cl
Cl
-
C C Cl Cl - Cl
H
Cl
C C Cl Cl
.
H+
Cl
C C Cl Cl
Fig. 12. Corrinoid-dependent reductive dehalogenation of tetrachloroethene (PCE) under anaerobic conditions coupled to energy metabolism (halorespiration).
shown that the deschloro derivative did not show any antimicrobial activity against the different microorganisms tested in contrast to the chlorinated compound (Sancelme et al., 1994). 3.2. Halogenating enzymes Although a large number of halogenated organic compounds has been isolated, not much is known about the mechanisms by which fluorine, chlorine, bromine, or iodine atoms are incorporated into organic compounds. The elucidation of some biosynthetic pathways, like 7chlorotetracyclin (McCormick, 1997) or pyrrolnitrin biosynthesis (Kirner et al., 1998) showed that the halogenation reactions in these pathways must be catalyzed by enzymes with substrate specificity and regioselectivity (Fig. 14).
3. Halogenation 3.1. Halogenated metabolites The synthetic diversity of nature is also reflected in a large number of naturally produced halogenated compounds discovered in many different organisms. Until today, more than 3500 halogenated metabolites have been isolated from bacteria, fungi, marine algae, lichens, higher plants, mammals and insects (Gribble, 2001). Whereas brominated metabolites are predominant in the marine environment, chlorine-containing metabolites are preferentially produced by terrestrial organisms. Although fluorine is the most abundant halogen in EarthÕs crust, biologically produced fluorinated metabolites are quite rare (Murphy et al., 2003), as is the case with iodinated metabolites (Gribble, 1996). The chemical structures of naturally produced halogenated compounds show a high degree of variability, with compounds containing a single or several halogen atoms (Fig. 13). Halogen atoms in organic compounds are important for biological activity. Nogami et al. (1987) for example showed that the biological activity of several pyrrolomycins is dependent on number, type and position of halide ion in the organic compound, and for the antitumor compound rebeccamycin it could be
3.2.1. Haloperoxidases The first halogenating enzyme was detected in the fungus Caldariomyces fumago (Shaw and Hager, 1959), the producer of caldariomycin (Fig. 15). For the catalysis of halogenation reactions, this enzyme requires hydrogen peroxide and halide ions (only chloride, bromide and iodide) and was thus named chloroperoxidase. During characterization of chloroperoxidase, a photospectroscopic enzyme assay was developed that is based on the chlorination or bromination of monochlorodimedone, a synthetic compound with structural similarity to 2-chloro-1,3-cyclopentanedione, an intermediate in caldariomycin biosynthesis (Fig. 15; Hager et al., 1966). Using this assay many other chloro-, bromo-, and iodoperoxidases were subsequently detected and isolated from bacteria, fungi, marine algae, marine invertebrates, and mammals (Neidleman and Geigert, 1986). Biochemical characterization showed that chloroperoxidase from Caldariomyces fumago contains a heme group, is able to exhibit catalase activity and additionally catalyzes P450-type reactions (Dunford, 1999). Elucidation of the three-dimensional structure (Sundaramoorthy et al., 1998) and biomimetic studies
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312 OH OH RO HO NH 2 O
HO
O
O
O
chloroeremomycin: R =
OH OH O
Cl
OH
HOCH2
H NH NH N O H H H O NH2
OH OCH3 rebeccamycin
H
balhimycin: R = H
OH O
Cl O
H OH
O OH H
CH3 H
N H
N
O
H O Cl H N O N N H H O H NH H H
Cl H O
O
CH OH O 2 O Cl
O H
HO
H N
O
O
305
Cl
HO NH 2 O
N H
O
S
NH2
thienodolin Cl
N(CH3)2 OH NH2
Cl
HO
Cl
N H
O
Cl OH
Cl O
Cl
N H
OH Cl
7-chlorotetracycline
pyoluteorin
pentachloropseudilin
Fig. 13. Examples of naturally produced halocompounds.
CH3
O
Cl OH
CH3
O OH
Cl
NH2 OH
OH
OH O O
O
OH
CH3
H
OH
O
4-ketoanhydro-7-chlorotetracycline
4-ketoanhydrotetracycline Cl
NH2 OH O O
Cl H O
NH2 OH
CH3
H
N(CH3)2 OH
NH2 OH
OH
OH O O
O
4-aminoanhydro-7-chlorotetracycline
NH2 OH
O
OH OH O
O
7-chlorotetracycline
Fig. 14. Partial 7-chlorotetracycline biosynthetic pathway showing 4-ketoanhydrotetracycline, the substrate for the halogenating enzyme (McCormick, 1997).
(Wagenknecht and Woggon, 1997) revealed the reaction mechanisms showing that heme-type haloperoxidases produce free hypohalous acid as the halogenating agent in the presence of H2 O2 and halide ions (Fig. 16). A different type of haloperoxidases, originally isolated from a marine alga, was found to require vanadium ions for its halogenating activity (Vilter, 1984). Vanadium-containing haloperoxidases were subsequently
isolated from marine algae, lichen, and fungi (Almeida et al., 2001). Investigation of the reaction mechanism and elucidation of the X-ray structure of chloroperoxidase from the fungus Curvularia inaequalis showed that this type of haloperoxidases also produces hypohalous acids as the halogenating agent (Fig. 17; Franssen, 1994; Messerschmidt et al., 1997).
306
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
H
Cl
OH
HO
Cl H
Cl H O
O
caldariomycin
O
O
2-chloro-1,3-cyclopentanedione monochlorodimedone Cl
Cl H O
O
Cl
O
O
BPO H2O2 / Br
Br
O
O
Fig. 15. Chemical structures of caldariomycin, 2-chloro-1,3-cyclopentanedione, an intermediate in caldariomycin biosynthesis, and the synthetic compound monochlorodimedone. Halogenation of monocglorodimedone was used for many years as the standard assay in the search for halogenating enzymes.
H2C
H2C
CH2
CH2
CH2 His
Glu
Glu
Glu H2C O C
- O
His H
O C
- OH
O C
- O
His
H
H
O
+
-O
-O O H 3+
Fe
3+
S C H2 Cys
Fe S C H2 Cys -H2O
H2O2
Glu
Glu
H2C
H2C
CH2 His
H O -O
3+
Fe S C H2 Cys
+
CH2
O C
- O
His
O C
- O O
H2O
4+
Fe S
3+
Fe
C H2 Cys
S C H2 Cys
H2O -HOCl
Cl Glu
H2C
H2C
CH2
CH2 His
Glu
H2C
Glu
O C
His
- O H
Cl O 3+
Fe S C H2 Cys
-H2O
CH2
O C
- O H3O +
-O
His 3+
- O Cl
Cl
Fe S C H2 Cys
O C
H3O +
-O 3+
Fe S C H2 Cys
Fig. 16. Reaction mechanism of heme-containing haloperoxidases showing the formation of hypohalous acid. Only the heme ion without the porphyrin ring system is shown.
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
5+
ENZ- V native enzyme
H2O2
H
Br
5+
ENZ-V -OOH +
H
+
307
5+
ENZ-V -O Br H2O
H
+
5+
Br2
ENZ- V + HO Br native enzyme
Br3-
+A-H -H2O A-Br Fig. 17. Reaction mechanism of vanadium-containing haloperoxidases.
This fact makes it very unlikely that haloperoxidases and perhydrolases are involved in the biosyntheses of halogenated metabolites. Interestingly, whereas the involvement of haloperoxidases, like myeloperoxidase, in defense mechanisms, where unspecific halogenation occurs, has been well established (Albert et al., 2001), the involvement of haloperoxidases in biosyntheses of halogenated metabolites has not been shown so far. From these observations it can be concluded that other halogenating enzymes than haloperoxidases must exist.
3.2.2. Perhydrolases A further type of halogenating enzymes requiring hydrogen peroxide was isolated from halogenated metabolite-producing Pseudomonas and Streptomyces strains using the monochlorodimedone assay (van Pee, 1996). These halogenating enzymes contain neither a heme group nor any metal ions. Although they require hydrogen peroxide for their halogenating activity, they are not peroxidases. The three-dimensional structure of these halogenating enzymes shows a catalytic triad consisting of a serine, an aspartate and a histidine residue (Hecht et al., 1994) revealing that they belong to the a=b hydrolase fold enzyme family. In the presence of H2 O2 they can act as perhydrolases. During catalysis, an acyl-enzyme intermediate is formed by reaction of acetic or another short-chained carboxylic acid with the serine residue at the active site (Picard et al., 1997). In the presence of H2 O2 , perhydrolysis of the acyl-enzyme intermediate results in the formation of peracids, which can oxidize halide ions to hypohalous acids which then act as the halogenating agent (Fig. 18; Hofmann et al., 1998). Thus, heme- and vanadium-containing haloperoxidases and also perhydrolases are halogenating enzymes without any substrate specificity and regioselectivity.
3.3. FADH2 -dependent halogenases Dairi et al. (1995) cloned the biosynthetic gene cluster for 7-chlorotetracycline biosynthesis and in a complementation experiment they identified a gene region that contained the gene for the halogenating enzyme involved in 7-chlorotetracycline biosynthesis. The amino acid sequence deduced from the nucleotide sequence showed no similarity to haloperoxidases or perhydrolases. Cloning and sequencing of the pyrrolnitrin biosynthetic gene cluster revealed that it contains four open reading frames. When introduced into E. coli this bacterium produces pyrrolnitrin (Hammer et al., 1997).
Ser CH2 Asp
His
Asp
HO
CH2
His
H2O2
O H2O
+
H O C
Ser
Ser
C O
CH3
CH2 Asp
His HO
HO
O
CH3
C
CH3
O
O
H
+
Cl
+ H -O
+
H O C
CH3 + A
Cl + H2O
A
H
C
HOCl +
O
Fig. 18. Reaction mechanism perhydrolases.
O
CH3
308
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312 COOH
COOH
PrnA
NH2
N H
N H
Cl
tryptophan
NH2
7-chlorotryptophan PrnB
Cl PrnC
Cl
NH2 N H
NH2 N H
Cl
aminopyrrolnitrin
monodechloroaminopyrrolnitrin
PrnD
Cl
Cl
NO2 N H
pyrrolnitrin
Fig. 19. Pyrrolnitrin biosynthetic pathway.
Biochemical and molecular genetic studies demonstrated that two of the four genes coded for two different halogenating enzymes (PrnA and PrnC) required for pyrrolnitrin biosynthesis (Fig. 19; Kirner et al., 1998). Neither PrnA nor PrnC shows any similarity to haloperoxidases or perhydrolases. However, whereas PrnA has also no similarity to the halogenase from the 7chlorotetracycline biosynthetic pathway, PrnC shows
NADH + FAD + H
+
significant similarity to this enzyme (Hammer et al., 1997; van Pee, 2001). Elucidation of the biosynthetic pathway for pyrrolnitrin biosynthesis in Pseudomonas fluorescens revealed that regioselective chlorination of tryptophan to 7-chlorotryptophan, catalyzed by tryptophan 7-halogenase (PrnA), is the first step in pyrrolnitrin biosynthesis. The second halogenase, monodechloroaminopyrrolnitrin 3-halogenase (PrnC), catalyzes the regioselective chlorination of monodechloroaminopyrrolnitrin in the 3-position of the pyrrole ring resulting in aminopyrrolnitrin formation (Fig. 19). Due to their different substrate specificities and regioselectivities, the two halogenases cannot substitute each other during pyrrolnitrin biosynthesis (Kirner et al., 1998). During purification of the tryptophan 7- and monodechloroaminopyrrlonitrin halogenase, it became evident that a second enzyme, whose gene was not part of the pyrrolnitrin biosynthetic gene cluster, was required for halogenating activity. This enzyme was identified as a flavin reductase. This flavin reductase produces FADH2 from FAD and NADH which is required by the halogenases. Thus the new type of halogenating enzymes was named FADH2 -dependent halogenases (Keller et al., 2000). In addition to FADH2 , halide ions (chloride or bromide) and oxygen are necessary for the halogenation reaction. There is obviously no specificity required between the flavin reductase and the halogenase, since the original flavin reductase from Pseudomonas fluorescens can be substituted by reductases from other bacteria, like E. coli or Thermus thermophilus (Keller et al., 2000).
Reductase
FADH2 + NAD
+
Halogenase + FADH2
Halogenase-FADH2
Halogenase-FADH2 + O2
Halogenase-FADHOOH
COOH
COOH Halogenase-FADHOOH N H
NH2
- FAD, H2O
N H
O
+
tryptophan
Cl , + H
COOH
COOH
Cl
N H
NH2
NH2
- H2 O
HO Cl
N H
NH2
7-chlorotryptophan Fig. 20. Hypothetical reaction mechanism of FADH2 -dependent halogenases.
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
309
Fig. 21. Alignment of the conserved region of FADH2 -dependent halogenases containing two tryptophan residues. PrnA-P and PrnAM: PrnA from Pseudomonas fluorescens and from Myxoccocus fulvus, respectively (Hammer et al., 1999); RebH: Halogenase from Saccharotrix aerocolonigenes (Sanchez et al., 2002); PrnC-P and PrnC-M: PrnC from Pseudomonas fluorescens and from Myxococcus fulvus; respectively (Hammer et al., 1999); Cts4: halogenase from Streptomyces aureofaciens (Dairi et al., 1995); PltA: halogenase A from Pseudomonas fluorescens Pf-5 (Nowak-Thompson et al., 1999); PltM: halogenase M from Pseudomonas fluorescens Pf-5 (NowakThompson et al., 1999); ere-ORF10: halogenase from Amycolatopsis orientalis (van Wageningen et al., 1998); BhaA: halogenase from Amycolatopsis mediterranei (Pelzer et al., 1999); ComH: halogenase from Streptomyces lavendulae (Chiu et al., 2001); AvilH: halogenase from Streptomyces viridochromogenes (Weitnauer et al., 2001); AdpC: halogenase from Anabena sp. 90 (Rouhiainen et al., 2000); Clm: halogenase from Streptomyces venezuelae (Piraee and Vining, 2001).
H2 CH3 H C H N C C C C OCH3 Cl O HC H C 2 Cl N S C Cl CH3 Fig. 22. Chemical structure of barbamide. In barbamide biosynthesis the involvement of a radical halogenation mechanism is discussed.
A postulated mechanism which takes the cofactor requirements of these halogenases into account is shown in Fig. 20. In this mechanism, we propose that activation of the organic substrate in a monooxygenase-like reaction involving a halogenase-flavin hydroperoxide intermediate is necessary to allow the incorporation of the halide as a nucleophile. Oxygen is removed by specific elimination of water resulting in reformation of a double bond. To allow regioselective incorporation of halide ions, they have to be specifically positioned at the active site. Interestingly, all FADH2 -dependent halogenases that have been detected so far contain two conserved tryptophan residues (Fig. 21). These two tryptophan residues might be involved in halide binding, as could be shown for haloalkane dehalogenases, where two tryptophan residues facilitate the removal of a halide ion from the halogenated substrate during the dehalogenation reaction (Fig. 2; Verschueren et al., 1993). Genes for FADH2 -dependent halogenases have been found in every biosynthetic gene cluster for halogenated metabolites that has been cloned and sequenced so far,
like 7-chlorotetracycline (Dairi et al., 1995), pyrrolnitrin (Hammer et al., 1997, 1999), chloroeremomycin (van Wageningen et al., 1998); balhimycin (Pelzer et al., 1999), pyoluteorin (Nowak-Thompson et al., 1999), rebeccamycin (Zehner et al., 2000; Sanchez et al., 2002), anabaenopeptilides (Rouhiainen et al., 2000), chloramphenicol (Piraee and Vining, 2001), avilamycin A (Weitnauer et al., 2001), and complestatin (Chiu et al., 2001). This clearly demonstrates that FADH2 -dependent halogenases play a major role in biosyntheses of halogenated metabolites. However, investigations on the biosynthesis of fluoroacetate showed that the incorporation of the fluoride ion is catalyzed by a different mechanism that involves nucleophilic substitution by fluoride of S-adenosylmethionine which is subsequently degraded to fluoroacetate (OÕHagan et al., 2002). A different halogenation mechanism must be involved in the biosynthesis of barbamide (Fig. 22) by the cyanobacetreium Lyngbya majuscula, since it could be shown by labelling experiments that a double bond, necessary for the reaction catalyzed by FADH2 -dependent halogenases, cannot exist during barbamide biosynthesis (Sitachitta et al., 2000). In this case a radical mechanism is discussed (Hartung, 1999). 4. Conclusions There is large variety in dehalogenating enzymes and mechanisms for the degradation of halogenated compounds. These enzymes and the organisms capable of growth on halogenated compounds using these
310
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
compounds either as carbon or energy source or in co-metabolic reactions, were detected by screening for growth or the ability to degrade specific halogenated compounds. Thus, it is not surprising that many different dehalogenating enzymes with varying substrate specificities were detected and isolated. However, in the case of halogenating enzymes the situation was quite different. Here usually a halogenated metabolite with biological activity was the starting point. Since these halogenated metabolites are produced by more or less complicated biosynthetic pathways, the substrates for the halogenating enzymes were only known in very few cases. Thus, looking for a halogenating enzyme meant to look for an enzymatic activity without a specific assay. Using the monochlorodimedone assay led to selection for unspecific enzymes that accepted a wide variety of substrates and therefore lacked substrate specificity–– haloperoxidases and perhydrolases. The detection of FADH2 -dependent halogenases that show substrate specificity and regioselectivity solved this problem only partially. The genes for FADH2 -dependent halogenases can now be detected rather easily using genetic probes, however, halogenating activity can still only be shown, if the natural substrate or one with a similar structure, is known and available. Although, this might slow down the detection of new halogenating enzymes, it can be predicted that specific enzymatic halogenation will develop an important synthetic tool for the production of fine chemicals and compounds with biological activity. The question, whether there is any connection between biological halogenation and dehalogenation, can still not be answered. There are no data available showing that biological halogenation led to the development of dehalogenating enzymes. Additionally, similarities between halogenating and dehalogenating enzymes have not yet been found. The only cases where a similarity in the reaction mechanism could be postulated, are the reversible intramolecular substitution reaction catalyzed by halohydrin dehalogenases (Van Hylckama Vileg et al., 2001), the halogenation reaction catalyzed by FADH2 -dependent halogenases, and the postulated involvement of two tryptophan residues in the halogenation reaction catalyzed by FADH2 -dependent halogenases as shown for haloalkane dehalogenase from Xanthobacter autrophicus and the transhalogenation reaction catalyzed by halomethane:bisulfide/halide ion methyltransferase (Coulter et al., 1999).
References Albert, C.J., Crowley, J.R., Hsu, F.-F., Thukkani, A.K., Ford, D.A., 2001. Reactive chlorinating species produced by myeloperoxidase target the vinyl ether bond in plasmalogens. J. Biol. Chem. 276, 23733–23741.
Almeida, M., Filipe, S., Humanes, M., Maia, M.F., Melo, R., Severino, N., Da Silva, J.A.L., da Silva, J.J.R.F., Wever, R., 2001. Vanadium haloperoxidases from brown algae of the Laminariaceae family. Phytochemistry 57, 633– 642. Chiu, H.-T., Hubbard, B.K., Shah, A.N., Eide, J., Fredenburg, R.A., Walsh, C.T., Khosla, C., 2001. Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster. Proc. Natl. Acad. Sci. USA 98, 8548–8553. Coulter, C., Hamilton, J.T.G., McRoberts, W.C., Kulakov, L., Larkin, M.J., Harper, D.B., 1999. Halomethane:bisulfide/ halide ion methyltransferase, an unusual corrinoid enzyme of environmental significance isolated from an aerobic methylotroph using chloromethane as the sole carbon source. Appl. Environ. Microbiol. 65, 4301–4312. Dairi, T., Nakano, T., Aisaka, K., Katsumata, R., Hasegawa, M., 1995. Cloning and nucleotide sequence of the gene responsible for chlorination of tetracycline. Biosci. Biotech. Biochem. 59, 1099–1106. Dunford, H.B., 1999. Heme peroxidases. Wiley–VCH, New York, pp. 323–348. Fetzner, S., 1998. Bacterial dehalogenation. Appl. Microbiol. Biotechnol. 50, 633–657. Fox, B.R., Borneman, J.G., Wackett, L.P., Lipscomb, J.D., 1990. Haloalkene oxidation by the soluble methane Monooxygenase from Methylosinus trichosporium OB3b. Appl. Environ. Microbiol. 29, 6419–6427. Franssen, M.C.R., 1994. Halogenation and oxidation reactions with haloperoxidases. Biocatalysis 10, 87–111. Gisi, D., Willi, L., Traber, H., Leisinger, T., Vuilleumier, S., 1998. Effects of bacterial host and dichloromethane dehalogenase on the competitiveness of methylotrophic bacteria growing with dichloromethane. Appl. Environ. Microbiol. 64, 1194–1202. Gribble, G.W., 1996. Naturally occurring organohalogen compounds––a comprehensive survey. In: Herz, G., Kirby, G.W., Moore, R.E., Steglich, W., Tamm, C. (Eds.), Progress in the Chemistry of Organic Natural Products. Springer, Berlin, pp. 1–498. Gribble, G.W., 1998. Naturally occurring organohalogen compounds. Acc. Chem. Res. 31, 141–152. Gribble, G.W., 2001. Diversity of naturally produced organohalogens––an overview. In: Second International Conference on Naturally Produced Organohalogens, Heidelber, Germany. Hager, L.P., Morris, D.R., Brown, F.S., Eberwein, H., 1966. Chloroperoxidase. II. Utilization of halogen anions. J. Biol. Chem. 241, 1769–1777. Hammer, P.E., Hill, D.S., Lam, S.T., van Pee, K.-H., Ligon, M.J., 1997. Four genes from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin. Appl. Environ. Microbiol. 63, 2147–2154. Hammer, P.E., Burd, W., Hill, D.S., Ligon, J.M., van Pee, K.-H., 1999. Conservation of the pyrrolnitrin biosynthetic gene cluster among six pyrrolnitrin-producing strains. FEMS Microbiol. Lett. 180, 39–44. Hartung, J., 1999. The biosynthesis of barbamide––A radical pathway for ‘‘biohalogenation’’? Angew. Chem. Int. Ed. 38, 1209–1211. Hecht, H.J., Sobek, H., Haag, T., Pfeifer, O., van Pee, K.-H., 1994. The metal-ion-free oxidoreductase from Streptomyces
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312 aureofaciens has an a=b hydrolase fold. Nature Struct. Biol. 1, 532–537. Hofmann, B., T€ olzer, S., Pelletier, I., Altenbuchner, J., van Pee, K.-H., Hecht, H.J., 1998. Structural investigation of the cofactor-free chloroperoxidases. J. Mol. Biol. 279, 889–900. Imai, R., Nagata, Y., Fukuda, M., Takagi, M., Yano, K., 1991. Molecular cloning of a Pseudomonas paucimobilis gene encoding a 17-kilodalton polypeptide that eliminates HCl molecules from c-hexachlorocyclohexane. J. Bacteriol. 173, 6811–6819. Janssen, D.B., Pries, F., van der Ploeg, J.R., 1994. Genetics and biochemistry of dehalogenating enzymes. Ann. Rev. Microbiol. 48, 163–191. Jensen, H.L., 1960. Decomposition of chloroacetates and chloropropionates by bacteria. Acta Agric. Scand. 10, 83– 103. Keller, S., Wage, T., Hohaus, K., H€ olzer, M., Eichhorn, E., van Pee, K.-H., 2000. Purification and partial characterization of tryptophan 7-halogenase (PrnA) from Pseudomonas fluorescens. Angew. Chem. Int. Ed. 39, 2300–2302. Kennes, C., Pries, F., Krooshof, G.H., Bokma, E., Kingma, J., Janssen, D.B., 1995. Replacement of tryptophan residues in haloalkane dehalogenase reduces halide binding and catalytic activity. Eur. J. Biochem. 228, 403–407. Kiefer Jr., P.M., McCarthy, D.L., Copley, S.D., 2002. The reaction catalyzed by tetrachlorohydroquinone dehalogenase does not involve nucleophilic aromatic substitution. Biochemistry 41, 1308–1314. Kirner, S., Hammer, P.E., Hill, D.S., Altmann, A., Fischer, I., Weislo, L.J., Lanahan, M., van Pee, K.-H., Ligon, J.M., 1998. Functions encoded by pyrrolnitrin biosynthetic genes from Pseudomonas fluorescens. J. Bacteriol. 180, 1939–1943. Klages, U., Lingens, F., 1979. Degradation of 4-chlorobenzoic acid by Nocardia species. FEMS Microbiol. Lett. 6, 201– 203. Leisinger, T., Bader, R., Hermann, R., Schmid-Appert, M., Vuilleumier, S., 1994. Microbes, enzymes and genes involved in dichloromethane utilization. Biodegradation 5, 237–248. Luo, L., Taylor, K.L., Xiang, H., Wie, Y., Zhang, W., Dunaway-Mariano, D., 2001. Role of active site binding interactions in 4-chlorobenzoyl-coenzyme A dehalogenase catalysis. Biochemistry 40, 15684–15692. Magnuson, J.K., Romine, M.F., Burris, D.R., Kingsley, M.T., 2000. Trichloroethene reductive dehalogenase from Dehalococcoides ethenogenes: sequence of tceA and substrate range characterization. Appl. Environ. Microbiol. 66, 5141– 5147. Marsh, A., Ferguson, D.M., 1997. Knowledge-based modelling of a bacterial dichloromethane dehalogenase. Prot. Struct. Funct. Genet. 28, 217–226. McCormick, J.R.D., 1997. Tetracyclines. In: Gottlieb, D., Shaw, P.D. (Eds.), Antibiotics, vol. 2. Springer, Berlin, pp. 113–122. Messerschmidt, P., Prade, L., Wever, R., 1997. Implications for the catalytic mechanism of the vanadium-containing enzyme chloroperoxidase from the fungus Curvularia inaequalis by X-ray structures of the native and peroxide form. Biol. Chem. 378, 309–315. Meßmer, M., Reinhardt, S., Wohlfarth, G., Diekert, G., 1996. Studies on methylchloride dehalogenase and O-demethylase
311
in cell extracts of the homoacetogen strain MC based on newly developed coupled enzyme assay. Arch. Microbiol. 165, 18–25. M€ uller, R., Thiele, J., Klages, U., Lingens, F., 1984. Incorporation of [18 O] water into 4-hydroxybenzoic acid in the reaction of 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3. Biochem. Biophys. Res. Commun. 124, 178–182. M€ uller, D., Schl€ omann, M., Reineke, W., 1996. Maleylacetate reductases in chloroaromatic-degrading bacteria using the modified ortho pathway: comparison of catalytic properties. J. Bacteriol. 178, 298–300. Murphy, C.D., Schaffrath, C., OÕHagan, D., 2003. Fluorinated natural products: the biosynthesis of fluoroacetate and 4fluorothreonine in Streptomyces cattleya. Chemosphere, this issue. PII: S0045-6535(03)00191-7. Nakamura, T., Yu, F., Mizunashi, W., Watanabe, I., 1991. Microbial transformation of prochiral 1,3-dichloro-2propanol into optically active 3-chloro-1,2-propanediole. Agric. Biol. Chem. 55, 1931–1933. Neidleman, S.L., Geigert, J., 1986. Biohalogenation: Principles, Basic Roles and Applications. Ellis Horwood, Chichester. Nogami, T., Shigihara, Y., Matsuda, N., Takahashi, Y., Naganawa, H., Nakamura, H., Hamada, M., Muraoka, Y., Takita, T., Iitaka, Y., Takeuchi, T., 1987. Neopyrrolomycin, a new chlorinated phenylpyrrole antibiotic. J. Antibiotics 53, 1192–1194. Nowak-Thompson, B., Chaney, N., Wing, J.S., Gould, S.J., Loper, J.E., 1999. Characterization of the pyoluteorin biosynthetic gene cluster of Pseudomonas fluorescens Pf-5. J. Bacteriol. 181, 2166–2174. OÕHagan, D., Schaffrath, C., Cobb, S.L., Hamilton, J.T.G., Murphy, C.D., 2002. Biosynthesis of an organofluorine molecule. Nature 416, 279. Pelzer, S., S€ ußmuth, R., Heckmann, D., Recktenwald, J., Huber, P., Jung, G., Wohlleben, W., 1999. Identification and analysis of the balhimycin biosynthetic gene cluster and its use for manipulating glycopeptide biosynthesis in Amycolatopsis mediterranei DSM5908. Antimicrob. Agents Chemother. 43, 1565–1573. Picard, M., Gross, J., L€ ubbert, E., T€ olzer, S., Krauss, S., van Pee, K.-H., Berkessel, A., 1997. Metal-free bacterial haloperoxidases as unusual hydrolases: activation of H2 O2 by the formation of peracetic acid. Angew. Chem. Int. Ed. 36, 1196–1199. Piraee, M., Vining, L.C., 2001. Streptomyces venezuelae halogenase ACCESSION AY026946. Pries, F., Kingma, J., Pentenga, M., van Pouderoyen, G., Jeronimus-Stratingh, C.M., Bruins, A.P., Janssen, D.B., 1994. Site-directed mutagenesis and oxygen isotope incorporation studies of the nucleophilic aspartate of haloalkane dehalogenase. Biochemistry 33, 1242–1247. Reddy, G.V.B., Gold, M.H., 2000. Degradation of pentachlorophenol by Phanerochaete chrysosporium: intermediates and reactions involved. Microbiology 146, 405–413. Reddy, G.V., Gold, M.H., 2001. Purification and characterization of glutathione conjugate reductase: a component of the tetrachlorohydroquinone reductive dehalogenase system from Phanerochaete chrysosporium. Arch. Biochem. Biophys. 391, 271–277.
312
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
Rouhiainen, L., Paulin, L., Suomalainen, S., Hyytiainen, H., Buikema, W., Haselkorn, R., Sivonen, K., 2000. Genes encoding synthetases of cyclic depsipeptides, anabaenopeptilides, in Anabaena strain 90. Mol. Microbiol. 37, 156–167. Sancelme, M., Fabre, S., Prudhomme, M., 1994. Antimicrobial activities of indolocarbazole and bis-indole protein kinase C inhibitors. J. Antibiotics 47, 792–798. Sanchez, C., Butovich, I.A., Brana, A.F., Rohr, J., Mendez, C., Salas, J., 2002. The biosynthetic gene cluster for the antitumor rebeccamycin from Saccharothrix aerocolonigenes ATCC39243: characterization and generation of indolocarbazole derivatives. Chem. Biol. 9, 519–531. Schuhmacher, W., Holliger, C., Zehnder, A.J.B., Hagen, W.R., 1997. Redox chemistry of cobalamin and iron–sulfur cofactors in the tetrachloroethene reductase of Dehalobacter restrictus. FEBS Lett. 409, 421–425. Schweizer, D., Markus, A., Seez, M., Ruf, H.H., Lingens, F., 1987. Purification and some properties of component B of the 4-chlorophenylacetate 3,4-dioxygenase from Pseudomonas sp. strain CBS3. J. Biol. Chem. 262, 9340–9346. Shad, M.M., Grover, T.A., Sust, S.D., 1993. Reduction of CCl4 to the trichloromethyl radical by lignin peroxidase H2 from Phanerochaete chrysosporium. Biochem. Biophys. Res. Commun. 191, 887–892. Shaw, P.D., Hager, L.P., 1959. Biological chlorination. IV. Peroxidative nature of enzymatic chlorination. J. Am. Chem. Soc. 81, 6527–6528. Sitachitta, N., Marquez, B.L., Williamson, R.T., Rossi, J., Roberts, M.A., Gerwick, W.H., Nguyen, V.A., Willis, C.L., 2000. Biosynthetic pathway and origin of the chlorinated methyl group in barbamide and dechlorobarbamide, metabolites from the marine cyanobacterium Lyngbya majuscula. Tetrahedron 56, 9103–9113. Sundaramoorthy, M., Terner, J., Poulos, T.L., 1998. Stereochemistry of the chloroperoxidase active site: crystallographic and molecular-modelling studies. Chem. Biol. 5, 461–473. Van de Pas, B.A., Smidt, H., Hagen, W.R., van der Oost, J., Schraa, G., Stams, A.J., de Vos, W.M., 1999. Purification and molecular characterization of ortho-chlorophenol reductive dehalogenase, a key enzyme of halorespiration in Desulfitobacterium dehalogenans. J. Biol. Chem. 274, 20287–20292. Van der Ploeg, J., van Hall, G., Janssen, D.B., 1991. Characterization of the haloacid dehalogenase from Xanthobacter autothrophicus GJ10 and sequencing of the dhlB gene. J. Bacteriol. 173, 7925–7933. Van Hylckama Vileg, J.E.T., Janssen, D.B., 1992. Bacterial degradation of 3-chloroacrylic acid and the characterization
of cis- and trans-specific dehalogenases. Biodegradation 2, 139–150. Van Hylckama Vileg, J.E.T., Tang, L., Spelberg, J.H., Smilda, T., Poelarends, G.J., Bosma, T., van Merode, A.E.J., Fraaije, M.W., Janssen, D.B., 2001. Halohydrin dehalogenases are structurally and mechanistically related to short-chain dehalogenases/reductases. J. Bacteriol. 183, 5058–5066. Vanelli, T., Studer, A., Kertesz, M., Leisinger, T., 1998. Chloromethane metabolism by Methylobacterium sp. strain CM4. Appl. Environ. Microbiol. 64, 1933–1936. van Pee, K.-H., 1996. Biosynthesis of halogenated metabolites by bacteria. Ann. Rev. Microbiol. 50, 375–399. van Pee, K.-H., 2001. Microbial biosynthesis of halometabolites. Arch. Microbiol. 175, 250–258. van Wageningen, A.M.A., Kirkpatrick, P.N., Williams, D.H., Harris, B.R., Kershaw, J.K., Lennard, N.J., Jones, M., Jones, S.J.M., Solenberg, P.J., 1998. Sequencing and analysis of genes involved in the biosynthesis of a vancomycin group antibiotic. Chem. Biol. 5, 155–162. Verschueren, K.H.G., Seljee, F., Rozeboom, H.J., Kalk, K.H., Dijkstra, B.W., 1993. Crystallographic analysis of the catalytic mechanism of haloalkane dehalogenase. Nature 363, 693–698. Vilter, H., 1984. Peroxidases from Phaeophyceae: a vanadium (V)-dependent peroxidase from Ascophyllum nodosum. Phytochemistry 23, 1387–1390. Wagenknecht, H.-A., Woggon, W.-D., 1997. Identification of intermediates in the catalytic cycle of chlororperoxidase. Chem. Biol. 4, 367–372. Weitnauer, G., M€ uhlenweg, A., Trefzer, A., Hoffmeister, D., S€ ußmuth, R.D., Jung, G., Welzel, K., Vente, A., Girreser, U., Bechthold, A., 2001. Biosynthesis of the orthosomycin antibiotic avilamycin A: deductions from the molecular analysis of the avi biosynthetic gene cluster of Streptomyces viridochromogenes T€ u57 and production of new antibiotics. Chem. Biol. 8, 569–581. Wohlfahrt, G., Diekert, G., 1997. Anaerobic dehalogenases. Curr. Opin. Biotechnol. 8, 290–295. Yokota, T., Fuse, H., Omori, T., Minora, Y., 1986. Microbial dehalogenation of haloalkanes mediated by oxygenases or halidohydrolase. Agric. Biol. Chem. 50, 453–460. Zehner, S., Wynands, I., Schmid, C., T€ olzer, S., Kuschel, M., Barth, G., van Pee, K.-H., 2000. Design of PCR-primers for the detection of halogenase-encoding regions in bacteria. In: 100th General Meeting of the American Society for Microbiology, Los Angeles, USA.
Review
Biological dehalogenation and halogenation reactions Karl-Heinz van Pee *, Susanne Unversucht Institut f€ur Biochemie, TU Dresden, D-01062 Dresden, Germany Received 19 February 2002; received in revised form 19 November 2002; accepted 19 November 2002
Abstract A large number of halogenated compounds is produced by chemical synthesis. Some of these compounds are very toxic and cause enormous problems to human health and to the environment. Investigations on the degradation of halocompounds by microorganisms have led to the detection of various dehalogenating enzymes catalyzing the removal of halogen atoms under aerobic and anaerobic conditions involving different mechanisms. On the other hand, more than 3500 halocompounds are known to be produced biologically, some of them in great amounts. Until 1997, only haloperoxidases were thought to be responsible for incorporation of halogen atoms into organic compounds. However, recent investigations into the biosynthesis of halogenated metabolites by bacteria have shown that a novel type of halogenating enzymes, FADH2 -dependent halogenases, are involved in biosyntheses of halogenated metabolites. In every gene cluster coding for the biosynthesis of a halogenated metabolite, isolated so far, one or several genes for FADH2 -dependent halogenases have been identified. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Dehalogenation; Dehalogenase; Halogenation; Halogenase; Haloperoxidase
Contents 1. 2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dehalogenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Hydrolytic dehalogenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Thiolytic dehalogenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Intramolecular substitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Dehydrohalogenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Dehalogenation by hydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Dehalogenation by methyltransfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Oxidative dehalogenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. Reductive dehalogenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8.1. Reductive dehalogenation under aerobic conditions. . . . . . . . . . . . . . . . . . . . 2.8.2. Anaerobic reductive dehalogenation (halorespiration) . . . . . . . . . . . . . . . . . . Halogenation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Halogenated metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Halogenating enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
*
Corresponding author. Tel.: +49-351-463-4494; fax: +49-351-463-35506/5508. E-mail address: [email protected] (K.-H. van Pee).
0045-6535/03/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0045-6535(03)00204-2
300 300 300 300 301 301 302 302 302 302 302 303 304 304 304
300
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
3.2.1. Haloperoxidases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Perhydrolases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. FADH2-dependent halogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Halogenated organic compounds are widely used as pharmaceuticals, herbicides, fungicides, insecticides, flame retardants, intermediates in organic synthesis, solvents etc. Additionally, many halogenated compounds are produced as by-products during chemical synthesis, such as dioxins, polychlorinated biphenyls and pentachlorophenol. Since many of these halocompounds show toxic or even carcinogenic effects and many of them are often difficult to degrade, many investigations were conducted to analyse the fate of these compounds in the environment. Studies on the degradation of different halogenated aliphatic and aromatic compounds led to the detection and elucidation of a variety of dehalogenases and dehalogenation mechanisms (Janssen et al., 1994; Fetzner, 1998). However, halogenated compounds are not only man-made, but also synthesized in nature by a large number of different organisms (Gribble, 1998). Until today, over 3500 halogen-containing metabolites have been isolated. Some of these halogenated metabolites have great importance as antibiotics such as chloramphenicol, 7-chlorotetracyclin, or vancomycin. However, until recently (van Pee, 1996), very little was known about the enzymes catalyzing the incorporation of halogen atoms during the biosynthesis of these compounds.
2. Dehalogenation 2.1. Hydrolytic dehalogenation The first bacterial dehalogenase was purified in 1960 by Jensen. This enzyme catalyzes the dechlorination of chloroacetate and 2-chloropropionic acid. Similar 2haloacid dehalogenases were subsequently isolated from many different bacteria such as Moraxella sp., Pseudomonas sp., and Xanthobacter autotrophicus (Van der Ploeg et al., 1991). 2-Haloacid dehalogenases catalyze a substitution of the halogen with water as the nucleophile resulting in the formation of an alcohol (Fig. 1). No other cofactor is required. The best studied hydrolytic halogenase is the haloalkane dehalogenase from the 1,2-dichloroethane-
Cl C
+
H2O
304 307 307 309 310
OH + HCl C
Fig. 1. Hydrolytic dehalogenation. In hydrolytic dehalogenation reactions, the halogen is displaced by water in a nucleophilic replacement reaction.
degrading bacterium Xanthobacter autotrophicus GJ10. The reaction mechanism of this enzyme was elucidated by X-ray crystallographic analysis (Verschueren et al., 1993) and site-directed mutagenesis (Pries et al., 1994). The enzyme belongs to the a=b hydrolase fold family and contains a catalytic triad consisting of one histidin and two aspartate residues which are involved in the nucleophilic substitution of the halide ion by water. The removal of the halide ion is additionally facilitated by two tryptophan residues (Fig. 2; Kennes et al., 1995). During degradation of 4-chlorobenzoic acid by Nocardia species, 4-hydroxybenzoic acid was isolated as an intermediate in the degradation pathway (Klages and Lingens, 1979). During investigation of the enzyme from Pseudomonas sp. CBS3, it could be shown that the hydroxyl group was derived from water and not from molecular oxygen (M€ uller et al., 1984). For hydrolytic substitution of chloride by a hydroxyl group at an aromatic system, activation by coenzyme A ester formation is required. The first step in 4-chlorobenzoate degradation, the formation of 4-chlorobenzoyl-coenzyme A, is catalyzed by a ligase. The second step, the actual dehalogenation reactions, is catalyzed by 4-chlorobenzoylcoenzyme A dehalogenase and 4-hydroxybenzoyl-coenzyme A is hydrolyzed by a thioesterase (Fig. 3; Luo et al., 2001).
2.2. Thiolytic dehalogenation Methylotrophic bacteria such as Methylophilus sp., Methylobacterium sp. and Hyphomicrobium sp. isolated with dichloromethane as growth substrate produce an inducible glutathione S-transferase which catalyzes the formation of an unstable S-chloromethyl glutathione intermediate. This intermediate is hydrolyzed to glutathione, chloride, and formaldehyde (Fig. 4; Leisinger
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
O
Trp175
C
OH
301
O C
S CoA
+ ATP, CoA-SH Cl
O - O
N
HN
N H
4-CBA:CoA ligase
H
H N
O
+ H2O - HCl dehalogenase O C
Asp124
His289
Cl
Trp 125
O - O H
Asp260
Cl
C C Cl H2 H HN 2
O
OH
C + H2O, - CoA-SH thioesterase
Trp175
OH
OH
Fig. 3. Hydrolytic dehalogenation of 4-chlorobenzoic acid (4-CBA) involving activation by formation of a coenzyme A ester.
N H Cl
Cl C CH 2 H2 O
- O
N
HN
H
HN
H N
O H
His289
Asp260
Trp 125
O
O
Asp124 Trp175
Cl
O
-
O
Asp260
HN
N H Cl C CH 2 H2 HN OH + NH O - O H N
His289
S CoA
Trp 125
2.3. Intramolecular substitution Haloalkohol dehalogenases, also known as halohydrin dehalogenases or halohydrin hydrogen–halide lyases which have been isolated from Arthrobacter sp. and other bacteria, catalyze the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides (Fig. 5; Van Hylckama Vileg et al., 2001). Although these enzymes show sequence homology with the family of short-chain dehydrogenases/reductases, they do not require NAD(P)þ as a coenzyme. They have the same two active site amino acid residues serine and tyrosine with the lysine residue of the short-chain dehydrogenases/reductases replaced by an arginine residue. These enzymes are also able to catalyze the reverse reaction, the halogenation of epoxides to haloalcohols (Nakamura et al., 1991).
Asp124
2.4. Dehydrohalogenation
Fig. 2. Mechanism of haloalkane dehalogenase. Two tryptophan residues are involved in removing the halogen atom.
et al., 1994; Marsh and Ferguson, 1997; Gisi et al., 1998).
Cl CH 2 + GSH - HCl Cl
The first two steps in the degradation of lindane (chexachlorocyclohexane) by Pseudomonas paucimobilis are catalyzed by a dehydrohalogenase. The elimination of hydrochloric acid catalyzed by this enzyme leads to
+ H2 O GS GS C Cl - HCl H2
CH2OH
O H C H
Fig. 4. Thiolytic dehalogenation of dichloromethane catalyzed by a glutathione transferase.
302
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
H H C Cl haloalcohol dehalogenase H C OH - HCl H C Cl H
+ H2O
H H C OH H C O H C H
Cl + H2O C C COOH H H
H H C O H C H C Cl H
haloalcohol dehalogenase - HCl
Cl
Cl
- HCl
Cl
Cl
Cl
Cl
- HCl
Cl Cl
H H C OH H C OH H C Cl H
Cl
Cl
Cl
Cl
+ H2O - HCl
HO
Cl
Cl
OH
+ H2O - HCl
O C C COOH H H2
epoxide hydrolase
Cl Cl
- HCl
Fig. 7. Dehalogenation by addition of water onto a double bond.
Fig. 5. Intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group catalyzed by haloalcohol dehalogenases. These enzymes can also catalyze the reverse reaction, the halogenation of the epoxides to haloalcohols.
Cl
Cl HC C COOH O H2 H
HO
Cl
Cl
Cl
Fig. 6. Dehalogenation of lindane as an example of dehydrohalogenation with formation of a double bond.
the formation of a double bond (Fig. 6; Imai et al., 1991). 2.5. Dehalogenation by hydration In a coryneform bacterium, isolated with 3-chloroacrylate as the carbon source, two dehalogenases have been detected. The dehalogenation reactions catalyzed by these enzymes, are proposed to proceed by addition of water to the double bond yielding an unstable intermediate from which malonate semialdehyde is formed (Fig. 7; Van Hylckama Vileg and Janssen, 1992). 2.6. Dehalogenation by methyltransfer Dehalogenation by methyltransfer was found in aerobic and strictly anaerobic methylotrophic bacteria using chloromethane or dichloromethane as the sole
carbon source (Meßmer et al., 1996; Vanelli et al., 1998). Coulter et al. (1999) described a methyltransferase that contains a corrinoid-bound cobalt atom. In the active state the cob(II)alamin is oxidized to the cob(III)alamin state by methylation by halomethane and is reduced by demethylation by the methyl group acceptor ion which can be a halide or bisulfide ion (Fig. 8).
2.7. Oxidative dehalogenation Oxidative dehalogenation reactions can be found in pathways for the degradation of haloaliphatic as well as haloaromatic compounds. These reactions are catalyzed by monooxygenases with a broad substrate specificity like methane monooxygenase from Methylococcus capsulatus Bath or Methylococcus trichosporium (Yokota et al., 1986; Fox et al., 1990) or by dioxygenases like the two-component 4-chlorophenylacetate 3,4-dioxygenase from Pseudomonas sp. CBS3 (Fig. 9; Schweizer et al., 1987). Lignin and manganese peroxidase from the lignindegrading basidiomycete Phanerochaete chrysosporium were also found to catalyze an oxidative dechlorination reaction of pentachlorophenol to produce tetrachloro1,4-benzoquinone (Reddy and Gold, 2000).
2.8. Reductive dehalogenation 2.8.1. Reductive dehalogenation under aerobic conditions During degradation of pentachlorophenol by Sphingomonas chlorophenolica under aerobic conditions, tetra-
E---cob(II)alamin CH3X
CH3Y
X
Y E---CH3cob(III)alamin
Fig. 8. Dehalogenation of chloromethane catalyzed by a cobalamin-containing methyltransferase; CH3 X can be CH3 Cl, CH3 Br or CH3 I, and Y can be Cl , Br , I or HS .
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
O OH C CH2 + O2 + NADH +
H+
4-chlorophenylacetate 3,4-dioxygenase
Cl
303
O OH C CH2
Cl
O H
OH H
- HCl O OH C CH2
OH OH Fig. 9. Oxidative dehalogenation of 4-chlorophenylacetate catalyzed by the two-component system of 4-chlorophenylacetate 3,4dioxygenase.
OH
OH
Cl
Cl
Cl
Cl
+ GSH - HCl
Cl
Cl
Cl
H SG
OH
O + H
-
S
C H2 Enzyme
OH Enzyme
Cl
Cl
Cl
+ GS-S C H2 H OH + GSH S
C H2 Enzyme + GS-SG
Fig. 10. Reductive dehalogenation of tetrachlorohydroquinone under aerobic conditions catalyzed by a glutathione transferase.
chlorohydroquinone is produced by a monooxygenase reaction catalyzed by pentachlorophenol hydroxylase. The thus formed tetrachlorohydroquinone is dehalogenated in a reductive dehalogenation reaction catalyzed by tetrachlorohydroquinone dehalogenase to trichlorohydroquinone (Fig. 10; Kiefer et al., 2002). A similar tetrachlorohydroquinone reductive dehalogenase system was also found in the white rot fungus Phanerochaete chrysosporium (Reddy and Gold, 2001). Tetrachlorohydroquinone dehalogenase is a member of the glutathione
S-transferase family like the enzyme involved in dichloromethane dehalogenation. Lignin peroxidases from white rot fungi such as Phanerochaete chrysosporium can also catalyze the reductive dehalogenation of carbon tetrachloride via a mechanism involving one electron oxidation of veratryl alcohol to the veratryl alcohol cation radical. This radical is reduced by oxalacetate resulting in a one-electron oxidation of oxalacetate. The remaining electron can then be used to catalyze reduction reactions such as the reductive dechlorination of carbon tetrachloride (Shad et al., 1993). A different type of reductive dehalogenation reaction is catalyzed by the NADH-dependent enzyme maleylacetate reductase which has been isolated from several Pseudomonas strains and Ralstonia eutropha (Fig. 11; M€ uller et al., 1996). 2.8.2. Anaerobic reductive dehalogenation (halorespiration) In halorespiration, reductive dehalogenation is coupled to energy metabolism. Here a halogenated compound, like tetrachloroethene and trichloroethene, serves as a terminal electron acceptor during the oxidation of an electron rich compound, like hydrogen or an organic substrate (Schuhmacher et al., 1997; Wohlfahrt and Diekert, 1997). The reductive dehalogenases isolated from several bacteria such as Dehalococcoides ethenogenes or Desulfitobacterium dehalogenans catalyzing these reactions contain a cobalamin cofactor and iron– sulfur clusters (Fig. 12; Van de Pas et al., 1999; Magnuson et al., 2000).
304
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
OH O OH C C CH2 CH2 C O + maleylacetate reductase C O + NAD + + HCl CH + NADH + H CH C Cl CH C C HO O HO O O
Fig. 11. NADH-dependent reductive dehalogenation of 2-chloromaleylacetate to 3-oxoadipate under aerobic conditions.
Cl
Cl
C C Cl Cl
corrinoid-dependent PCE reductive dehalogenase
Cl
Cl
-
C C Cl Cl - Cl
H
Cl
C C Cl Cl
.
H+
Cl
C C Cl Cl
Fig. 12. Corrinoid-dependent reductive dehalogenation of tetrachloroethene (PCE) under anaerobic conditions coupled to energy metabolism (halorespiration).
shown that the deschloro derivative did not show any antimicrobial activity against the different microorganisms tested in contrast to the chlorinated compound (Sancelme et al., 1994). 3.2. Halogenating enzymes Although a large number of halogenated organic compounds has been isolated, not much is known about the mechanisms by which fluorine, chlorine, bromine, or iodine atoms are incorporated into organic compounds. The elucidation of some biosynthetic pathways, like 7chlorotetracyclin (McCormick, 1997) or pyrrolnitrin biosynthesis (Kirner et al., 1998) showed that the halogenation reactions in these pathways must be catalyzed by enzymes with substrate specificity and regioselectivity (Fig. 14).
3. Halogenation 3.1. Halogenated metabolites The synthetic diversity of nature is also reflected in a large number of naturally produced halogenated compounds discovered in many different organisms. Until today, more than 3500 halogenated metabolites have been isolated from bacteria, fungi, marine algae, lichens, higher plants, mammals and insects (Gribble, 2001). Whereas brominated metabolites are predominant in the marine environment, chlorine-containing metabolites are preferentially produced by terrestrial organisms. Although fluorine is the most abundant halogen in EarthÕs crust, biologically produced fluorinated metabolites are quite rare (Murphy et al., 2003), as is the case with iodinated metabolites (Gribble, 1996). The chemical structures of naturally produced halogenated compounds show a high degree of variability, with compounds containing a single or several halogen atoms (Fig. 13). Halogen atoms in organic compounds are important for biological activity. Nogami et al. (1987) for example showed that the biological activity of several pyrrolomycins is dependent on number, type and position of halide ion in the organic compound, and for the antitumor compound rebeccamycin it could be
3.2.1. Haloperoxidases The first halogenating enzyme was detected in the fungus Caldariomyces fumago (Shaw and Hager, 1959), the producer of caldariomycin (Fig. 15). For the catalysis of halogenation reactions, this enzyme requires hydrogen peroxide and halide ions (only chloride, bromide and iodide) and was thus named chloroperoxidase. During characterization of chloroperoxidase, a photospectroscopic enzyme assay was developed that is based on the chlorination or bromination of monochlorodimedone, a synthetic compound with structural similarity to 2-chloro-1,3-cyclopentanedione, an intermediate in caldariomycin biosynthesis (Fig. 15; Hager et al., 1966). Using this assay many other chloro-, bromo-, and iodoperoxidases were subsequently detected and isolated from bacteria, fungi, marine algae, marine invertebrates, and mammals (Neidleman and Geigert, 1986). Biochemical characterization showed that chloroperoxidase from Caldariomyces fumago contains a heme group, is able to exhibit catalase activity and additionally catalyzes P450-type reactions (Dunford, 1999). Elucidation of the three-dimensional structure (Sundaramoorthy et al., 1998) and biomimetic studies
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312 OH OH RO HO NH 2 O
HO
O
O
O
chloroeremomycin: R =
OH OH O
Cl
OH
HOCH2
H NH NH N O H H H O NH2
OH OCH3 rebeccamycin
H
balhimycin: R = H
OH O
Cl O
H OH
O OH H
CH3 H
N H
N
O
H O Cl H N O N N H H O H NH H H
Cl H O
O
CH OH O 2 O Cl
O H
HO
H N
O
O
305
Cl
HO NH 2 O
N H
O
S
NH2
thienodolin Cl
N(CH3)2 OH NH2
Cl
HO
Cl
N H
O
Cl OH
Cl O
Cl
N H
OH Cl
7-chlorotetracycline
pyoluteorin
pentachloropseudilin
Fig. 13. Examples of naturally produced halocompounds.
CH3
O
Cl OH
CH3
O OH
Cl
NH2 OH
OH
OH O O
O
OH
CH3
H
OH
O
4-ketoanhydro-7-chlorotetracycline
4-ketoanhydrotetracycline Cl
NH2 OH O O
Cl H O
NH2 OH
CH3
H
N(CH3)2 OH
NH2 OH
OH
OH O O
O
4-aminoanhydro-7-chlorotetracycline
NH2 OH
O
OH OH O
O
7-chlorotetracycline
Fig. 14. Partial 7-chlorotetracycline biosynthetic pathway showing 4-ketoanhydrotetracycline, the substrate for the halogenating enzyme (McCormick, 1997).
(Wagenknecht and Woggon, 1997) revealed the reaction mechanisms showing that heme-type haloperoxidases produce free hypohalous acid as the halogenating agent in the presence of H2 O2 and halide ions (Fig. 16). A different type of haloperoxidases, originally isolated from a marine alga, was found to require vanadium ions for its halogenating activity (Vilter, 1984). Vanadium-containing haloperoxidases were subsequently
isolated from marine algae, lichen, and fungi (Almeida et al., 2001). Investigation of the reaction mechanism and elucidation of the X-ray structure of chloroperoxidase from the fungus Curvularia inaequalis showed that this type of haloperoxidases also produces hypohalous acids as the halogenating agent (Fig. 17; Franssen, 1994; Messerschmidt et al., 1997).
306
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
H
Cl
OH
HO
Cl H
Cl H O
O
caldariomycin
O
O
2-chloro-1,3-cyclopentanedione monochlorodimedone Cl
Cl H O
O
Cl
O
O
BPO H2O2 / Br
Br
O
O
Fig. 15. Chemical structures of caldariomycin, 2-chloro-1,3-cyclopentanedione, an intermediate in caldariomycin biosynthesis, and the synthetic compound monochlorodimedone. Halogenation of monocglorodimedone was used for many years as the standard assay in the search for halogenating enzymes.
H2C
H2C
CH2
CH2
CH2 His
Glu
Glu
Glu H2C O C
- O
His H
O C
- OH
O C
- O
His
H
H
O
+
-O
-O O H 3+
Fe
3+
S C H2 Cys
Fe S C H2 Cys -H2O
H2O2
Glu
Glu
H2C
H2C
CH2 His
H O -O
3+
Fe S C H2 Cys
+
CH2
O C
- O
His
O C
- O O
H2O
4+
Fe S
3+
Fe
C H2 Cys
S C H2 Cys
H2O -HOCl
Cl Glu
H2C
H2C
CH2
CH2 His
Glu
H2C
Glu
O C
His
- O H
Cl O 3+
Fe S C H2 Cys
-H2O
CH2
O C
- O H3O +
-O
His 3+
- O Cl
Cl
Fe S C H2 Cys
O C
H3O +
-O 3+
Fe S C H2 Cys
Fig. 16. Reaction mechanism of heme-containing haloperoxidases showing the formation of hypohalous acid. Only the heme ion without the porphyrin ring system is shown.
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
5+
ENZ- V native enzyme
H2O2
H
Br
5+
ENZ-V -OOH +
H
+
307
5+
ENZ-V -O Br H2O
H
+
5+
Br2
ENZ- V + HO Br native enzyme
Br3-
+A-H -H2O A-Br Fig. 17. Reaction mechanism of vanadium-containing haloperoxidases.
This fact makes it very unlikely that haloperoxidases and perhydrolases are involved in the biosyntheses of halogenated metabolites. Interestingly, whereas the involvement of haloperoxidases, like myeloperoxidase, in defense mechanisms, where unspecific halogenation occurs, has been well established (Albert et al., 2001), the involvement of haloperoxidases in biosyntheses of halogenated metabolites has not been shown so far. From these observations it can be concluded that other halogenating enzymes than haloperoxidases must exist.
3.2.2. Perhydrolases A further type of halogenating enzymes requiring hydrogen peroxide was isolated from halogenated metabolite-producing Pseudomonas and Streptomyces strains using the monochlorodimedone assay (van Pee, 1996). These halogenating enzymes contain neither a heme group nor any metal ions. Although they require hydrogen peroxide for their halogenating activity, they are not peroxidases. The three-dimensional structure of these halogenating enzymes shows a catalytic triad consisting of a serine, an aspartate and a histidine residue (Hecht et al., 1994) revealing that they belong to the a=b hydrolase fold enzyme family. In the presence of H2 O2 they can act as perhydrolases. During catalysis, an acyl-enzyme intermediate is formed by reaction of acetic or another short-chained carboxylic acid with the serine residue at the active site (Picard et al., 1997). In the presence of H2 O2 , perhydrolysis of the acyl-enzyme intermediate results in the formation of peracids, which can oxidize halide ions to hypohalous acids which then act as the halogenating agent (Fig. 18; Hofmann et al., 1998). Thus, heme- and vanadium-containing haloperoxidases and also perhydrolases are halogenating enzymes without any substrate specificity and regioselectivity.
3.3. FADH2 -dependent halogenases Dairi et al. (1995) cloned the biosynthetic gene cluster for 7-chlorotetracycline biosynthesis and in a complementation experiment they identified a gene region that contained the gene for the halogenating enzyme involved in 7-chlorotetracycline biosynthesis. The amino acid sequence deduced from the nucleotide sequence showed no similarity to haloperoxidases or perhydrolases. Cloning and sequencing of the pyrrolnitrin biosynthetic gene cluster revealed that it contains four open reading frames. When introduced into E. coli this bacterium produces pyrrolnitrin (Hammer et al., 1997).
Ser CH2 Asp
His
Asp
HO
CH2
His
H2O2
O H2O
+
H O C
Ser
Ser
C O
CH3
CH2 Asp
His HO
HO
O
CH3
C
CH3
O
O
H
+
Cl
+ H -O
+
H O C
CH3 + A
Cl + H2O
A
H
C
HOCl +
O
Fig. 18. Reaction mechanism perhydrolases.
O
CH3
308
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312 COOH
COOH
PrnA
NH2
N H
N H
Cl
tryptophan
NH2
7-chlorotryptophan PrnB
Cl PrnC
Cl
NH2 N H
NH2 N H
Cl
aminopyrrolnitrin
monodechloroaminopyrrolnitrin
PrnD
Cl
Cl
NO2 N H
pyrrolnitrin
Fig. 19. Pyrrolnitrin biosynthetic pathway.
Biochemical and molecular genetic studies demonstrated that two of the four genes coded for two different halogenating enzymes (PrnA and PrnC) required for pyrrolnitrin biosynthesis (Fig. 19; Kirner et al., 1998). Neither PrnA nor PrnC shows any similarity to haloperoxidases or perhydrolases. However, whereas PrnA has also no similarity to the halogenase from the 7chlorotetracycline biosynthetic pathway, PrnC shows
NADH + FAD + H
+
significant similarity to this enzyme (Hammer et al., 1997; van Pee, 2001). Elucidation of the biosynthetic pathway for pyrrolnitrin biosynthesis in Pseudomonas fluorescens revealed that regioselective chlorination of tryptophan to 7-chlorotryptophan, catalyzed by tryptophan 7-halogenase (PrnA), is the first step in pyrrolnitrin biosynthesis. The second halogenase, monodechloroaminopyrrolnitrin 3-halogenase (PrnC), catalyzes the regioselective chlorination of monodechloroaminopyrrolnitrin in the 3-position of the pyrrole ring resulting in aminopyrrolnitrin formation (Fig. 19). Due to their different substrate specificities and regioselectivities, the two halogenases cannot substitute each other during pyrrolnitrin biosynthesis (Kirner et al., 1998). During purification of the tryptophan 7- and monodechloroaminopyrrlonitrin halogenase, it became evident that a second enzyme, whose gene was not part of the pyrrolnitrin biosynthetic gene cluster, was required for halogenating activity. This enzyme was identified as a flavin reductase. This flavin reductase produces FADH2 from FAD and NADH which is required by the halogenases. Thus the new type of halogenating enzymes was named FADH2 -dependent halogenases (Keller et al., 2000). In addition to FADH2 , halide ions (chloride or bromide) and oxygen are necessary for the halogenation reaction. There is obviously no specificity required between the flavin reductase and the halogenase, since the original flavin reductase from Pseudomonas fluorescens can be substituted by reductases from other bacteria, like E. coli or Thermus thermophilus (Keller et al., 2000).
Reductase
FADH2 + NAD
+
Halogenase + FADH2
Halogenase-FADH2
Halogenase-FADH2 + O2
Halogenase-FADHOOH
COOH
COOH Halogenase-FADHOOH N H
NH2
- FAD, H2O
N H
O
+
tryptophan
Cl , + H
COOH
COOH
Cl
N H
NH2
NH2
- H2 O
HO Cl
N H
NH2
7-chlorotryptophan Fig. 20. Hypothetical reaction mechanism of FADH2 -dependent halogenases.
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
309
Fig. 21. Alignment of the conserved region of FADH2 -dependent halogenases containing two tryptophan residues. PrnA-P and PrnAM: PrnA from Pseudomonas fluorescens and from Myxoccocus fulvus, respectively (Hammer et al., 1999); RebH: Halogenase from Saccharotrix aerocolonigenes (Sanchez et al., 2002); PrnC-P and PrnC-M: PrnC from Pseudomonas fluorescens and from Myxococcus fulvus; respectively (Hammer et al., 1999); Cts4: halogenase from Streptomyces aureofaciens (Dairi et al., 1995); PltA: halogenase A from Pseudomonas fluorescens Pf-5 (Nowak-Thompson et al., 1999); PltM: halogenase M from Pseudomonas fluorescens Pf-5 (NowakThompson et al., 1999); ere-ORF10: halogenase from Amycolatopsis orientalis (van Wageningen et al., 1998); BhaA: halogenase from Amycolatopsis mediterranei (Pelzer et al., 1999); ComH: halogenase from Streptomyces lavendulae (Chiu et al., 2001); AvilH: halogenase from Streptomyces viridochromogenes (Weitnauer et al., 2001); AdpC: halogenase from Anabena sp. 90 (Rouhiainen et al., 2000); Clm: halogenase from Streptomyces venezuelae (Piraee and Vining, 2001).
H2 CH3 H C H N C C C C OCH3 Cl O HC H C 2 Cl N S C Cl CH3 Fig. 22. Chemical structure of barbamide. In barbamide biosynthesis the involvement of a radical halogenation mechanism is discussed.
A postulated mechanism which takes the cofactor requirements of these halogenases into account is shown in Fig. 20. In this mechanism, we propose that activation of the organic substrate in a monooxygenase-like reaction involving a halogenase-flavin hydroperoxide intermediate is necessary to allow the incorporation of the halide as a nucleophile. Oxygen is removed by specific elimination of water resulting in reformation of a double bond. To allow regioselective incorporation of halide ions, they have to be specifically positioned at the active site. Interestingly, all FADH2 -dependent halogenases that have been detected so far contain two conserved tryptophan residues (Fig. 21). These two tryptophan residues might be involved in halide binding, as could be shown for haloalkane dehalogenases, where two tryptophan residues facilitate the removal of a halide ion from the halogenated substrate during the dehalogenation reaction (Fig. 2; Verschueren et al., 1993). Genes for FADH2 -dependent halogenases have been found in every biosynthetic gene cluster for halogenated metabolites that has been cloned and sequenced so far,
like 7-chlorotetracycline (Dairi et al., 1995), pyrrolnitrin (Hammer et al., 1997, 1999), chloroeremomycin (van Wageningen et al., 1998); balhimycin (Pelzer et al., 1999), pyoluteorin (Nowak-Thompson et al., 1999), rebeccamycin (Zehner et al., 2000; Sanchez et al., 2002), anabaenopeptilides (Rouhiainen et al., 2000), chloramphenicol (Piraee and Vining, 2001), avilamycin A (Weitnauer et al., 2001), and complestatin (Chiu et al., 2001). This clearly demonstrates that FADH2 -dependent halogenases play a major role in biosyntheses of halogenated metabolites. However, investigations on the biosynthesis of fluoroacetate showed that the incorporation of the fluoride ion is catalyzed by a different mechanism that involves nucleophilic substitution by fluoride of S-adenosylmethionine which is subsequently degraded to fluoroacetate (OÕHagan et al., 2002). A different halogenation mechanism must be involved in the biosynthesis of barbamide (Fig. 22) by the cyanobacetreium Lyngbya majuscula, since it could be shown by labelling experiments that a double bond, necessary for the reaction catalyzed by FADH2 -dependent halogenases, cannot exist during barbamide biosynthesis (Sitachitta et al., 2000). In this case a radical mechanism is discussed (Hartung, 1999). 4. Conclusions There is large variety in dehalogenating enzymes and mechanisms for the degradation of halogenated compounds. These enzymes and the organisms capable of growth on halogenated compounds using these
310
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
compounds either as carbon or energy source or in co-metabolic reactions, were detected by screening for growth or the ability to degrade specific halogenated compounds. Thus, it is not surprising that many different dehalogenating enzymes with varying substrate specificities were detected and isolated. However, in the case of halogenating enzymes the situation was quite different. Here usually a halogenated metabolite with biological activity was the starting point. Since these halogenated metabolites are produced by more or less complicated biosynthetic pathways, the substrates for the halogenating enzymes were only known in very few cases. Thus, looking for a halogenating enzyme meant to look for an enzymatic activity without a specific assay. Using the monochlorodimedone assay led to selection for unspecific enzymes that accepted a wide variety of substrates and therefore lacked substrate specificity–– haloperoxidases and perhydrolases. The detection of FADH2 -dependent halogenases that show substrate specificity and regioselectivity solved this problem only partially. The genes for FADH2 -dependent halogenases can now be detected rather easily using genetic probes, however, halogenating activity can still only be shown, if the natural substrate or one with a similar structure, is known and available. Although, this might slow down the detection of new halogenating enzymes, it can be predicted that specific enzymatic halogenation will develop an important synthetic tool for the production of fine chemicals and compounds with biological activity. The question, whether there is any connection between biological halogenation and dehalogenation, can still not be answered. There are no data available showing that biological halogenation led to the development of dehalogenating enzymes. Additionally, similarities between halogenating and dehalogenating enzymes have not yet been found. The only cases where a similarity in the reaction mechanism could be postulated, are the reversible intramolecular substitution reaction catalyzed by halohydrin dehalogenases (Van Hylckama Vileg et al., 2001), the halogenation reaction catalyzed by FADH2 -dependent halogenases, and the postulated involvement of two tryptophan residues in the halogenation reaction catalyzed by FADH2 -dependent halogenases as shown for haloalkane dehalogenase from Xanthobacter autrophicus and the transhalogenation reaction catalyzed by halomethane:bisulfide/halide ion methyltransferase (Coulter et al., 1999).
References Albert, C.J., Crowley, J.R., Hsu, F.-F., Thukkani, A.K., Ford, D.A., 2001. Reactive chlorinating species produced by myeloperoxidase target the vinyl ether bond in plasmalogens. J. Biol. Chem. 276, 23733–23741.
Almeida, M., Filipe, S., Humanes, M., Maia, M.F., Melo, R., Severino, N., Da Silva, J.A.L., da Silva, J.J.R.F., Wever, R., 2001. Vanadium haloperoxidases from brown algae of the Laminariaceae family. Phytochemistry 57, 633– 642. Chiu, H.-T., Hubbard, B.K., Shah, A.N., Eide, J., Fredenburg, R.A., Walsh, C.T., Khosla, C., 2001. Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster. Proc. Natl. Acad. Sci. USA 98, 8548–8553. Coulter, C., Hamilton, J.T.G., McRoberts, W.C., Kulakov, L., Larkin, M.J., Harper, D.B., 1999. Halomethane:bisulfide/ halide ion methyltransferase, an unusual corrinoid enzyme of environmental significance isolated from an aerobic methylotroph using chloromethane as the sole carbon source. Appl. Environ. Microbiol. 65, 4301–4312. Dairi, T., Nakano, T., Aisaka, K., Katsumata, R., Hasegawa, M., 1995. Cloning and nucleotide sequence of the gene responsible for chlorination of tetracycline. Biosci. Biotech. Biochem. 59, 1099–1106. Dunford, H.B., 1999. Heme peroxidases. Wiley–VCH, New York, pp. 323–348. Fetzner, S., 1998. Bacterial dehalogenation. Appl. Microbiol. Biotechnol. 50, 633–657. Fox, B.R., Borneman, J.G., Wackett, L.P., Lipscomb, J.D., 1990. Haloalkene oxidation by the soluble methane Monooxygenase from Methylosinus trichosporium OB3b. Appl. Environ. Microbiol. 29, 6419–6427. Franssen, M.C.R., 1994. Halogenation and oxidation reactions with haloperoxidases. Biocatalysis 10, 87–111. Gisi, D., Willi, L., Traber, H., Leisinger, T., Vuilleumier, S., 1998. Effects of bacterial host and dichloromethane dehalogenase on the competitiveness of methylotrophic bacteria growing with dichloromethane. Appl. Environ. Microbiol. 64, 1194–1202. Gribble, G.W., 1996. Naturally occurring organohalogen compounds––a comprehensive survey. In: Herz, G., Kirby, G.W., Moore, R.E., Steglich, W., Tamm, C. (Eds.), Progress in the Chemistry of Organic Natural Products. Springer, Berlin, pp. 1–498. Gribble, G.W., 1998. Naturally occurring organohalogen compounds. Acc. Chem. Res. 31, 141–152. Gribble, G.W., 2001. Diversity of naturally produced organohalogens––an overview. In: Second International Conference on Naturally Produced Organohalogens, Heidelber, Germany. Hager, L.P., Morris, D.R., Brown, F.S., Eberwein, H., 1966. Chloroperoxidase. II. Utilization of halogen anions. J. Biol. Chem. 241, 1769–1777. Hammer, P.E., Hill, D.S., Lam, S.T., van Pee, K.-H., Ligon, M.J., 1997. Four genes from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin. Appl. Environ. Microbiol. 63, 2147–2154. Hammer, P.E., Burd, W., Hill, D.S., Ligon, J.M., van Pee, K.-H., 1999. Conservation of the pyrrolnitrin biosynthetic gene cluster among six pyrrolnitrin-producing strains. FEMS Microbiol. Lett. 180, 39–44. Hartung, J., 1999. The biosynthesis of barbamide––A radical pathway for ‘‘biohalogenation’’? Angew. Chem. Int. Ed. 38, 1209–1211. Hecht, H.J., Sobek, H., Haag, T., Pfeifer, O., van Pee, K.-H., 1994. The metal-ion-free oxidoreductase from Streptomyces
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312 aureofaciens has an a=b hydrolase fold. Nature Struct. Biol. 1, 532–537. Hofmann, B., T€ olzer, S., Pelletier, I., Altenbuchner, J., van Pee, K.-H., Hecht, H.J., 1998. Structural investigation of the cofactor-free chloroperoxidases. J. Mol. Biol. 279, 889–900. Imai, R., Nagata, Y., Fukuda, M., Takagi, M., Yano, K., 1991. Molecular cloning of a Pseudomonas paucimobilis gene encoding a 17-kilodalton polypeptide that eliminates HCl molecules from c-hexachlorocyclohexane. J. Bacteriol. 173, 6811–6819. Janssen, D.B., Pries, F., van der Ploeg, J.R., 1994. Genetics and biochemistry of dehalogenating enzymes. Ann. Rev. Microbiol. 48, 163–191. Jensen, H.L., 1960. Decomposition of chloroacetates and chloropropionates by bacteria. Acta Agric. Scand. 10, 83– 103. Keller, S., Wage, T., Hohaus, K., H€ olzer, M., Eichhorn, E., van Pee, K.-H., 2000. Purification and partial characterization of tryptophan 7-halogenase (PrnA) from Pseudomonas fluorescens. Angew. Chem. Int. Ed. 39, 2300–2302. Kennes, C., Pries, F., Krooshof, G.H., Bokma, E., Kingma, J., Janssen, D.B., 1995. Replacement of tryptophan residues in haloalkane dehalogenase reduces halide binding and catalytic activity. Eur. J. Biochem. 228, 403–407. Kiefer Jr., P.M., McCarthy, D.L., Copley, S.D., 2002. The reaction catalyzed by tetrachlorohydroquinone dehalogenase does not involve nucleophilic aromatic substitution. Biochemistry 41, 1308–1314. Kirner, S., Hammer, P.E., Hill, D.S., Altmann, A., Fischer, I., Weislo, L.J., Lanahan, M., van Pee, K.-H., Ligon, J.M., 1998. Functions encoded by pyrrolnitrin biosynthetic genes from Pseudomonas fluorescens. J. Bacteriol. 180, 1939–1943. Klages, U., Lingens, F., 1979. Degradation of 4-chlorobenzoic acid by Nocardia species. FEMS Microbiol. Lett. 6, 201– 203. Leisinger, T., Bader, R., Hermann, R., Schmid-Appert, M., Vuilleumier, S., 1994. Microbes, enzymes and genes involved in dichloromethane utilization. Biodegradation 5, 237–248. Luo, L., Taylor, K.L., Xiang, H., Wie, Y., Zhang, W., Dunaway-Mariano, D., 2001. Role of active site binding interactions in 4-chlorobenzoyl-coenzyme A dehalogenase catalysis. Biochemistry 40, 15684–15692. Magnuson, J.K., Romine, M.F., Burris, D.R., Kingsley, M.T., 2000. Trichloroethene reductive dehalogenase from Dehalococcoides ethenogenes: sequence of tceA and substrate range characterization. Appl. Environ. Microbiol. 66, 5141– 5147. Marsh, A., Ferguson, D.M., 1997. Knowledge-based modelling of a bacterial dichloromethane dehalogenase. Prot. Struct. Funct. Genet. 28, 217–226. McCormick, J.R.D., 1997. Tetracyclines. In: Gottlieb, D., Shaw, P.D. (Eds.), Antibiotics, vol. 2. Springer, Berlin, pp. 113–122. Messerschmidt, P., Prade, L., Wever, R., 1997. Implications for the catalytic mechanism of the vanadium-containing enzyme chloroperoxidase from the fungus Curvularia inaequalis by X-ray structures of the native and peroxide form. Biol. Chem. 378, 309–315. Meßmer, M., Reinhardt, S., Wohlfarth, G., Diekert, G., 1996. Studies on methylchloride dehalogenase and O-demethylase
311
in cell extracts of the homoacetogen strain MC based on newly developed coupled enzyme assay. Arch. Microbiol. 165, 18–25. M€ uller, R., Thiele, J., Klages, U., Lingens, F., 1984. Incorporation of [18 O] water into 4-hydroxybenzoic acid in the reaction of 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3. Biochem. Biophys. Res. Commun. 124, 178–182. M€ uller, D., Schl€ omann, M., Reineke, W., 1996. Maleylacetate reductases in chloroaromatic-degrading bacteria using the modified ortho pathway: comparison of catalytic properties. J. Bacteriol. 178, 298–300. Murphy, C.D., Schaffrath, C., OÕHagan, D., 2003. Fluorinated natural products: the biosynthesis of fluoroacetate and 4fluorothreonine in Streptomyces cattleya. Chemosphere, this issue. PII: S0045-6535(03)00191-7. Nakamura, T., Yu, F., Mizunashi, W., Watanabe, I., 1991. Microbial transformation of prochiral 1,3-dichloro-2propanol into optically active 3-chloro-1,2-propanediole. Agric. Biol. Chem. 55, 1931–1933. Neidleman, S.L., Geigert, J., 1986. Biohalogenation: Principles, Basic Roles and Applications. Ellis Horwood, Chichester. Nogami, T., Shigihara, Y., Matsuda, N., Takahashi, Y., Naganawa, H., Nakamura, H., Hamada, M., Muraoka, Y., Takita, T., Iitaka, Y., Takeuchi, T., 1987. Neopyrrolomycin, a new chlorinated phenylpyrrole antibiotic. J. Antibiotics 53, 1192–1194. Nowak-Thompson, B., Chaney, N., Wing, J.S., Gould, S.J., Loper, J.E., 1999. Characterization of the pyoluteorin biosynthetic gene cluster of Pseudomonas fluorescens Pf-5. J. Bacteriol. 181, 2166–2174. OÕHagan, D., Schaffrath, C., Cobb, S.L., Hamilton, J.T.G., Murphy, C.D., 2002. Biosynthesis of an organofluorine molecule. Nature 416, 279. Pelzer, S., S€ ußmuth, R., Heckmann, D., Recktenwald, J., Huber, P., Jung, G., Wohlleben, W., 1999. Identification and analysis of the balhimycin biosynthetic gene cluster and its use for manipulating glycopeptide biosynthesis in Amycolatopsis mediterranei DSM5908. Antimicrob. Agents Chemother. 43, 1565–1573. Picard, M., Gross, J., L€ ubbert, E., T€ olzer, S., Krauss, S., van Pee, K.-H., Berkessel, A., 1997. Metal-free bacterial haloperoxidases as unusual hydrolases: activation of H2 O2 by the formation of peracetic acid. Angew. Chem. Int. Ed. 36, 1196–1199. Piraee, M., Vining, L.C., 2001. Streptomyces venezuelae halogenase ACCESSION AY026946. Pries, F., Kingma, J., Pentenga, M., van Pouderoyen, G., Jeronimus-Stratingh, C.M., Bruins, A.P., Janssen, D.B., 1994. Site-directed mutagenesis and oxygen isotope incorporation studies of the nucleophilic aspartate of haloalkane dehalogenase. Biochemistry 33, 1242–1247. Reddy, G.V.B., Gold, M.H., 2000. Degradation of pentachlorophenol by Phanerochaete chrysosporium: intermediates and reactions involved. Microbiology 146, 405–413. Reddy, G.V., Gold, M.H., 2001. Purification and characterization of glutathione conjugate reductase: a component of the tetrachlorohydroquinone reductive dehalogenase system from Phanerochaete chrysosporium. Arch. Biochem. Biophys. 391, 271–277.
312
K.-H. van Pee, S. Unversucht / Chemosphere 52 (2003) 299–312
Rouhiainen, L., Paulin, L., Suomalainen, S., Hyytiainen, H., Buikema, W., Haselkorn, R., Sivonen, K., 2000. Genes encoding synthetases of cyclic depsipeptides, anabaenopeptilides, in Anabaena strain 90. Mol. Microbiol. 37, 156–167. Sancelme, M., Fabre, S., Prudhomme, M., 1994. Antimicrobial activities of indolocarbazole and bis-indole protein kinase C inhibitors. J. Antibiotics 47, 792–798. Sanchez, C., Butovich, I.A., Brana, A.F., Rohr, J., Mendez, C., Salas, J., 2002. The biosynthetic gene cluster for the antitumor rebeccamycin from Saccharothrix aerocolonigenes ATCC39243: characterization and generation of indolocarbazole derivatives. Chem. Biol. 9, 519–531. Schuhmacher, W., Holliger, C., Zehnder, A.J.B., Hagen, W.R., 1997. Redox chemistry of cobalamin and iron–sulfur cofactors in the tetrachloroethene reductase of Dehalobacter restrictus. FEBS Lett. 409, 421–425. Schweizer, D., Markus, A., Seez, M., Ruf, H.H., Lingens, F., 1987. Purification and some properties of component B of the 4-chlorophenylacetate 3,4-dioxygenase from Pseudomonas sp. strain CBS3. J. Biol. Chem. 262, 9340–9346. Shad, M.M., Grover, T.A., Sust, S.D., 1993. Reduction of CCl4 to the trichloromethyl radical by lignin peroxidase H2 from Phanerochaete chrysosporium. Biochem. Biophys. Res. Commun. 191, 887–892. Shaw, P.D., Hager, L.P., 1959. Biological chlorination. IV. Peroxidative nature of enzymatic chlorination. J. Am. Chem. Soc. 81, 6527–6528. Sitachitta, N., Marquez, B.L., Williamson, R.T., Rossi, J., Roberts, M.A., Gerwick, W.H., Nguyen, V.A., Willis, C.L., 2000. Biosynthetic pathway and origin of the chlorinated methyl group in barbamide and dechlorobarbamide, metabolites from the marine cyanobacterium Lyngbya majuscula. Tetrahedron 56, 9103–9113. Sundaramoorthy, M., Terner, J., Poulos, T.L., 1998. Stereochemistry of the chloroperoxidase active site: crystallographic and molecular-modelling studies. Chem. Biol. 5, 461–473. Van de Pas, B.A., Smidt, H., Hagen, W.R., van der Oost, J., Schraa, G., Stams, A.J., de Vos, W.M., 1999. Purification and molecular characterization of ortho-chlorophenol reductive dehalogenase, a key enzyme of halorespiration in Desulfitobacterium dehalogenans. J. Biol. Chem. 274, 20287–20292. Van der Ploeg, J., van Hall, G., Janssen, D.B., 1991. Characterization of the haloacid dehalogenase from Xanthobacter autothrophicus GJ10 and sequencing of the dhlB gene. J. Bacteriol. 173, 7925–7933. Van Hylckama Vileg, J.E.T., Janssen, D.B., 1992. Bacterial degradation of 3-chloroacrylic acid and the characterization
of cis- and trans-specific dehalogenases. Biodegradation 2, 139–150. Van Hylckama Vileg, J.E.T., Tang, L., Spelberg, J.H., Smilda, T., Poelarends, G.J., Bosma, T., van Merode, A.E.J., Fraaije, M.W., Janssen, D.B., 2001. Halohydrin dehalogenases are structurally and mechanistically related to short-chain dehalogenases/reductases. J. Bacteriol. 183, 5058–5066. Vanelli, T., Studer, A., Kertesz, M., Leisinger, T., 1998. Chloromethane metabolism by Methylobacterium sp. strain CM4. Appl. Environ. Microbiol. 64, 1933–1936. van Pee, K.-H., 1996. Biosynthesis of halogenated metabolites by bacteria. Ann. Rev. Microbiol. 50, 375–399. van Pee, K.-H., 2001. Microbial biosynthesis of halometabolites. Arch. Microbiol. 175, 250–258. van Wageningen, A.M.A., Kirkpatrick, P.N., Williams, D.H., Harris, B.R., Kershaw, J.K., Lennard, N.J., Jones, M., Jones, S.J.M., Solenberg, P.J., 1998. Sequencing and analysis of genes involved in the biosynthesis of a vancomycin group antibiotic. Chem. Biol. 5, 155–162. Verschueren, K.H.G., Seljee, F., Rozeboom, H.J., Kalk, K.H., Dijkstra, B.W., 1993. Crystallographic analysis of the catalytic mechanism of haloalkane dehalogenase. Nature 363, 693–698. Vilter, H., 1984. Peroxidases from Phaeophyceae: a vanadium (V)-dependent peroxidase from Ascophyllum nodosum. Phytochemistry 23, 1387–1390. Wagenknecht, H.-A., Woggon, W.-D., 1997. Identification of intermediates in the catalytic cycle of chlororperoxidase. Chem. Biol. 4, 367–372. Weitnauer, G., M€ uhlenweg, A., Trefzer, A., Hoffmeister, D., S€ ußmuth, R.D., Jung, G., Welzel, K., Vente, A., Girreser, U., Bechthold, A., 2001. Biosynthesis of the orthosomycin antibiotic avilamycin A: deductions from the molecular analysis of the avi biosynthetic gene cluster of Streptomyces viridochromogenes T€ u57 and production of new antibiotics. Chem. Biol. 8, 569–581. Wohlfahrt, G., Diekert, G., 1997. Anaerobic dehalogenases. Curr. Opin. Biotechnol. 8, 290–295. Yokota, T., Fuse, H., Omori, T., Minora, Y., 1986. Microbial dehalogenation of haloalkanes mediated by oxygenases or halidohydrolase. Agric. Biol. Chem. 50, 453–460. Zehner, S., Wynands, I., Schmid, C., T€ olzer, S., Kuschel, M., Barth, G., van Pee, K.-H., 2000. Design of PCR-primers for the detection of halogenase-encoding regions in bacteria. In: 100th General Meeting of the American Society for Microbiology, Los Angeles, USA.