Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 183-193
doi:10.1111/j.1745-7270.2008.00390.x
Catalytic mechanisms, basic
roles, and biotechnological and environmental significance of halogenating
enzymes
Xianping Chen1,2
and Karl-Heinz van P?e2*
1 The Biomedical Engineering Centre, Guilin
University of Electronic Technology, Guilin 541004, China
2 The Institute
of Biochemistry, Dresden University of Technology, Dresden 01062, Germany
Received: July 13,
2007
Accepted: December
10, 2007
*Corresponding
author: Tel, 49-351-46334494; Fax, 49-351-46335506; E-mail,
The
understanding of enzymatic incorporation of halogen atoms into organic
molecules has increased during the last few years. Two novel types of
halogenating enzymes, flavin-dependent halogenases and a-ketoglutarate-dependent
halogenases, are now known to play a significant role in enzyme-catalyzed
halogenation. The recent advances on the halogenating enzymes RebH, SyrB2, and
CytC3 have suggested some new mechanisms for enzymatic halogenations. This
review concentrates on the occurrence, catalytic mechanisms, and
biotechnological applications of the halogenating enzymes that are currently
known.
Keywords haloperoxidases; flavin-dependent halogenases; aKG-dependent halogenases;
fluorinase; genetic algorithm
Over 4500 halogenated natural products are known to be produced by
living organisms [1]. These products display distinct physiological or
biochemical roles, for example, thyroxine functions as a hormone in mammals
[2], 4-chloroindolyl-3-acetic acid is a plant growth hormone [3], and
thienodolin also acts as a plant growth regulator [4,5]. Several
halometabolites, particularly those of marine origin, appear to have a
defensive role [6], and some are medically valuable and include antibiotics
(chlortetracycline and vancomycin), antitumor agents (rebeccamycin and
calicheamycin), human thyroid hormone (thyroxine) [2], and anti-HIV agents
(chloropeptin I, ambigol A) [7,8]. Commonly, the halogen that is incorporated
into a particular organic substrate is determined by the relative amount of
halide present in the surrounding environment. For natural organohalogen
compounds found in the marine environment, bromine (Br) mostly dominates over
chlorine (Cl), but for natural organohalogens found in the terrestrial
environment, Cl dominates over Br [9]. The mechanism of enzymatic halogenation
has become a hot topic to organic and medicinal chemists. This is because the
mechanisms represent potential novel pathways to both new halogenated synthetic
compounds and modified natural products. Herein we will focus on the catalytic mechanisms, basic roles, and
biocatalytic potential of halogenating enzymes.
Haloperoxidases
For the catalysis of halogenation reactions, haloperoxidases require
hydrogen peroxide (H2O2) and halide ions (Cl–, Br–, or I–, but not
F–) and are thus named chloroperoxidases (CPO). CPO might also use
chlorite (ClO2–) instead of Cl– and H2O2 to form the halogenated
products [9]. Haloperoxidases differ by the metal ion associated with the
prosthetic group and mostly contain either heme iron or a vanadate co-factor
for their halogenating activity [5,10]. Biochemical characterization showed
that CPO (EC 1.11.1.10) from Caldariomyces fumago contains a heme group,
is able to show catalytic activity, and additionally catalyzes P450-type
reactions [11]. Elucidation of the 3-D structure [12] revealed the reaction
mechanisms (Fig. 1) showing that heme-type haloperoxidases produce free
hypohalous acids (HOX; X=Cl–, Br–, or I–) as the
halogenating agent [13,14]. Recently, a second fungal haloperoxidase, Agrocybe
aegerita peroxidase (AaP) (EC 1.11.1.16), of the heme-thiolate type has
been discovered in the agaric mushroom A. aegerita. The AaP has
strong brominating as well as weak chlorinating and iodating activities, and
catalyzes both benzylic and aromatic hydroxylations (e.g., of toluene and
naphthalene) [14]. Several other heme peroxidases (in addition to CPO) possess
halogenating side activities, for example, lignin [15], manganese [16], soybean
[17], and horseradish [18] peroxidase show brominating and iodating activities
[14]. There are several human/animal heme peroxidases that can oxidize halides,
for example, the flavin-heme CPO from the marine polychaete Notomastus
lobatus [19], myeloperoxidase [20] and eosinophil peroxidase [21] from
human leukocytes, as well as bovine lactoperoxidase [22], and human thyroid
peroxidase [23].Vanadium-containing haloperoxidases have been isolated from marine
algae, lichen, and fungi [24], and also produce hypohalous acids as the
halogenating agent [5,13,25]. A quantum mechanics/molecular mechanics study of
the rest state of the vanadium-dependent CPO (EC 1.11.1.-) from Curvularia
inaequalis and of the early intermediates of the halide oxidation was
reported recently [26]. The investigation of different protonation states
indicates that the enzyme likely consists of an anionic H2VO4– vanadate moiety where one hydroxyl group is in the axial position.
The hydrogen peroxide directly attacks the axial hydroxyl group, resulting in
the formation of a hydrogen peroxide intermediate. This intermediate is
promptly protonated to yield a peroxo species (Fig. 2) [26]. The most
likely protonation states of the peroxo co-factor are neutral forms HVO2(O2) with a hydroxyl group either H-bonded to Ser402 or coordinated to Arg360. The calculations strongly
suggest that the hydrogen peroxide binding might not involve an initial
protonation of the vanadate co-factor, and the halide oxidation might take
place with the preliminary formation of a peroxovanadate/halogen adduct (Fig.
2). Subsequently, the halogen reacts with the peroxo moiety, yielding a
hypohalogen vanadate [26]. The use of haloperoxidases as halogenating
biocatalysts is limited because they have in common a lack of both substrate
specificity and regioselectivity.
Flavin-dependent Halogenases
Although a number of flavin-dependent halogenases have been investigated
in some detail, halogenating activity in vitro has only been shown for
the flavin adenine dinucleotide (FAD)-dependent tryptophan 7-halogenase (PrnA) (EC 1.14.13.2) [27,28]
and PrnC [27] from pyrrolnitrin biosynthesis in Pseudomonas fluorescens
Bl915, RebH from rebeccamycin biosynthesis in Lechevalieria aerocolonigenes
[29], PyrH from pyrroindomycin biosynthesis in Streptomyces rugosporus
[30], Thal from the thienodolin producer S. albogriseolus [31], PltA
from pyoluteorin biosynthesis in P. fluorescens Pf-5 [32], and HalB from the pentachloropseudilin
producer Actinoplanes sp. ATCC 33002 [33]. All of the flavin-dependent
halogenases require reduced FADH2 (provided by a partner flavin
reductase), chloride ion, and oxygen as co-substrates for halogenation reaction
[34]. The reaction of FADH2 and O2 in the
halogenase active site was presumed to form a typical 4a-hydroperoxyflavin (FAD-4a-OOH)
intermediate [34,35]. Two reaction mechanisms have been proposed for the
flavin-dependent halogenases. One is the nucleophilic mechanism, suggesting the
initial formation of an epoxide [27] or the addition of a hydroxyl group [36]
from the substrate’s reaction with the FAD-4a-OOH intermediate. This
would then be followed by the nucleophilic attack of a halide ion (chloride or
bromide), leading to the formation of a halohydrin [34]. The other is the
electrophilic mechanism that proposed the reaction of the FAD-4a-OOH
intermediate with chloride ion to form FAD-4a-OCl [5,35]. Attack of the
aromatic p electrons on the FAD-4a-OCl intermediate would lead to formation of a
chlorinated substrate intermediate that, after deprotonation, would give the
chlorinated product [34,35]. The elucidation of the 3-D structure [28] of PrnA involved in
pyrrolnitrin biosynthesis suggests that neither the nucleophilic nor the
electrophilic mechanism is correct [34]. The crystal structure of PrnA has been
resolved and it indicates that the protein is composed of two modules, an FAD
binding module and a tryptophan binding module [5,28]. The structure reveals
that the initially formed FAD-4a-OOH cannot interact directly with the substrate tryptophan because
the bound tryptophan lays 10 ? from the FAD. On the basis of this finding, the
catalytic mechanism of PrnA was proposed, illustrated in Fig. 3. After
formation of a FAD-4a-OOH intermediate, hypochlorous acid (HOCl) will be produced by
nucleophilic attack of Cl– on FAD-4a-OOH (Fig. 3). K79 provides a hydrogen bond to the HOCl,
positioning it in the correct orientation to react with the tryptophan
7-position. Furthermore, the Wheland intermediate formed during the
electrophilic addition of chlorine to tryptophan is stabilized by a glutamate
residue in the substrate binding site (E346), that deprotonates the
intermediate yielding 7-chlorotryptophan (Fig. 3). Site-directed
mutagenesis experiments showed the importance of these residues to the activity
of PrnA: an E346®Q346 mutation significantly affects
turnover, and a K79®A79
mutation destroys activity completely [28]. RebH is another tryptophan 7-halogenase that catalyzes the formation
of 7-chlorotryptophan as the initial step in the biosynthesis of antitumor
agent rebeccamycin [29,35]. Both structural and kinetic evidence of PrnA and
RebH support the subsequent formation of HOCl in the active site when FAD-4a-OOH is captured
by Cl– [28,37]. However, two observations from the RebH reaction kinetics
and the RebH structure seemed to challenge the suggested mechanism of PrnA.
First, during stopped flow studies to monitor formation of flavin intermediates
in RebH, flavin reactions leading to HOCl production were observed with or
without L-Trp present, suggesting that this potent oxidant is formed in the
active site without available substrate for reaction [35]. Second, in the
crystal structure of RebH with bound flavin and tryptophan solved at 2.1 ?, Lys79
occupies a key position between the binding pockets for flavin and substrate
tryptophan (corresponding to the same residue in PrnA) [28,37]. Studies of
protein oxidation by HOCl show that the eNH2 of lysine reacts rapidly with HOCl to form a long-lived chloramine,
Lys-eNH-Cl (t1/2>25 h) [37,38].
Chloramines can also carry out chlorination reactions [39–41], and might
play an important role in the flavin halogenase mechanism [42,43]. Lys-eNH-Cl was formed
in the RebH active site when the reaction of FADH2, Cl–, and O2 was catalyzed in the absence of substrate tryptophan, and the
chlorinating species is remarkably long-lived with t1/2 of 63 h at 4 ?C and 28 h at 25 ?C [37]. Based on these observations,
a challenge to the mechanism of PrnA was put forward by Yeh et al [37]
in a different mechanism that HOCl reacts with the active site Lys79
of RebH to form a lysine chloramine Lys-eNH-Cl before reaching
the substrate tryptophan (Fig. 4). This intermediate remained on the
enzyme after removal of FAD and transferred chlorine to tryptophan with
kinetically competent rates (Fig. 4). Furthermore, a similar
chlorinating species has also been detected in the halogenase PltA from
pyoluteorin producer P. fluorescens Pf-5 [37]. Three proteins (PltA,
PltD, and PltM) involved in pyoluteorin biosynthesis [34] are homologous to
FADH2-dependent halogenases found in other non-ribosomal peptide
synthetases (NRPS) and polyketide synthases (PKS) biosynthetic gene clusters [32].
Assay of halogenating activity with L-pyrrolyl-S-PltL as the substrate
in vitro revealed that only PltA catalysed the incorporation of both
chlorine atoms [44]. All flavin-dependent halogenases have two conserved motifs (Fig.
5). The first motif (GxGxxG), which is the FAD-binding site, is located
near the N-terminus [30], and is also known to be involved in the binding of
nucleotide co-factors of the large family of protein kinases [45]. However, in
PltD this motif is not absolutely conserved (GxSxxV), it is only a
halogenase-like protein of unknown function [34,46]. The second absolutely
conserved motif located near the middle of the enzymes contains two tryptophan
residues (WxWxIP) (Fig. 5). Again, this motif is not absolutely
conserved in PltD (WxGxIP), showing that this enzyme is not a halogenase [34].
The two tryptophan residues of this motif are located near the flavin, and they
are suggested to block the binding of a substrate close to the flavin and thus
prevent the enzyme from catalysing a monooxygenase reaction [28,30].
a-Ketoglutarate (aKG)-dependent Halogenases
A class of aKG-dependent halogenases
responsible for halogenation of unactivated carbon centers in the biosyntheses of
several compounds of non-ribosomal peptide origin has recently been
characterized [47–53]. Unlike haloperoxidases and
flavin-dependent halogenases, this novel type of halogenase does not require a
substrate with a double bond for introduction of halogen atoms [34]. Studies
showed that the in vitro reconstitution of the aliphatic halogenation
activity of these enzymes requires halogenase, FeII, and three
small-molecule co-substrates, aKG, oxygen, and chloride [48,49].
Halogen incorporation follows the consensus mechanism of non-ribosomal peptide
biosyntheses, an amino acid will be used as its initial substrate and initial
activation of the amino acid by an adenylation (A) domain is followed by its
loading on the phosphopantetheinyl arm of the thiolation (T) module. The
resultant aminoacyl-S-T protein is the substrate for the halogenase, which
chlorinates an unactivated methyl group of the tethered amino acid [50]. For
example, chlorination of the methyl group of L-threonine tethered to the A-T
didomain protein SyrB1 by the halogenase SyrB2 produces
4-chloro-L-threonine-S-SyrB1, an intermediate in the biosynthesis of the
antifungal agent syringomycin E [Fig. 6(A)] [49]. Similar chlorination
also occurs in the biosynthesis of the non-halogenated phytotoxin coronatine in
P. syringae pv. tomato DC3000 [Fig. 6(B)]
[51]. The aKG-dependent halogenase CmaB chlorinates the L-allo-isoleucine to
form the g-Cl-L-allo-isoleucine, an intermediate in the formation of the
cyclopropane ring of CMA, a substrate for coronatine biosynthesis [52,53].
CytC3, the halogenase isolated from soil Streptomyces sp., chlorinates
the methyl group of L-2-aminobutyric acid (L-Aba) or L-valine tethered to the
carrier protein CytC2 in [Fig. 6(C)] [54]. BarB1/BarB2 and DysB1/DysB2
have been suggested to be the halogenases catalysing the chlorinating reactions
in barbamide and disidenin/dysideathiazole biosynthesis, respectively [47,50].However, the mechanism of such aliphatic halogenations has not been
elucidated. Insight into the catalytic strategy of FeII/aKG-dependent halogenases came from the crystal structure of the
syringomycin halogenase SyrB2 [48]. In contrast to the aKG-dependent
dioxygenases, the Fe center of SyrB2 is coordinated by two protein-derived
histidines, bidentate aKG, water, and chloride. The
carboxylate of the “facial triad” that normally coordinates the FeII center is replaced with an alanine in the protein primary
structure, presenting a coordination site for the chloride ligand [54]. On the
basis of this observation, the mechanism of the FeII/aKG-dependent halogenases shown in Fig. 7 was proposed
[47,48,50]. The early steps of the mechanism leading to the ClFeIV-oxo complex are likely conserved among the dioxygenases and
halogenases [50]. The key postulated intermediate ClFeIV-oxo
complex activates the substrate by hydrogen atom abstraction to yield a ClFeIII-OH complex and a substrate radical (Fig. 7). Substrate
chlorination was proposed to proceed through “rebound” of a chloride
radical, rather than the hydroxyl radical rebound postulated for hydroxylases
[50,54]. Exclusive halogenation (rather than hydroxylation) reflects the lower
reduction potential of chlorine radical (Cl+e–®Cl–, 1.36 V) relative to hydroxyl radical (HO+e–®HO–, 2.02 V) [54]. The proposed mechanism of FeII/aKG-dependent halogenases was tested experimentally by direct
characterization of the intermediates (ClFeIV-oxo
complex) using a combination of kinetic and spectroscopic methods [54] in the
aliphatic halogenase CytC3 from soil Streptomyces sp. [50]. However, the mechanism of such aliphatic halogenations has not been
elucidated. Insight into the catalytic strategy of FeII/aKG-dependent halogenases came from the crystal structure of the
syringomycin halogenase SyrB2 [48]. In contrast to the aKG-dependent
dioxygenases, the Fe center of SyrB2 is coordinated by two protein-derived
histidines, bidentate aKG, water, and chloride. The
carboxylate of the “facial triad” that normally coordinates the FeII center is replaced with an alanine in the protein primary
structure, presenting a coordination site for the chloride ligand [54]. On the
basis of this observation, the mechanism of the FeII/aKG-dependent halogenases shown in Fig. 7 was proposed
[47,48,50]. The early steps of the mechanism leading to the ClFeIV-oxo complex are likely conserved among the dioxygenases and
halogenases [50]. The key postulated intermediate ClFeIV-oxo
complex activates the substrate by hydrogen atom abstraction to yield a ClFeIII-OH complex and a substrate radical (Fig. 7). Substrate
chlorination was proposed to proceed through “rebound” of a chloride
radical, rather than the hydroxyl radical rebound postulated for hydroxylases
[50,54]. Exclusive halogenation (rather than hydroxylation) reflects the lower
reduction potential of chlorine radical (Cl+e–®Cl–, 1.36 V) relative to hydroxyl radical (HO+e–®HO–, 2.02 V) [54]. The proposed mechanism of FeII/aKG-dependent halogenases was tested experimentally by direct
characterization of the intermediates (ClFeIV-oxo
complex) using a combination of kinetic and spectroscopic methods [54] in the
aliphatic halogenase CytC3 from soil Streptomyces sp. [50].
Fluorinase
5‘-Fluoro-5‘-deoxyadenosine (5‘-FDA) synthase (EC 2.5.1.63) isolated from Streptomyces
cattleya is the first fluorinating enzyme [5,55]. The fluorinase gene (flA)
has been characterized recently, and 11 other putative open reading frames have
been identified [56]. Three of the proteins encoded by these genes have also
been characterized. FlB was the second enzyme in the biosynthetic pathway of
fluorometabolites, catalyzing the phosphorolytic cleavage of 5‘-FDA to produce the next intermediate
5-fluoro-5-deoxy-D-ribose-1-phosphate [57]. Fluoroacetaldehyde combines
with the amino acid L-threonine in a pyridoxal phosphate-dependent transaldol
reaction to generate the antibiotic 4-fluorothreonine. In a separate reaction
fluoroacetaldehyde is oxidised to fluoroacetate by the action of an
NADH-dependent aldehyde dehydrogenase. A summary of the fluorometabolite
pathway is shown in Fig. 8 [58]. The enzyme FlI is an
S-adenosylhomocysteine hydrolase that might act to relieve S-adenosylhomocysteine
inhibition of the fluorinase. Finally, FlK was proposed for the specific
degradation of fluoroacetyl-co-enzyme A into fluoroacetate and co-enzyme A (Fig.
8) [56]. The fluorinase from S. cattleya is also a chlorinase, and
can also use Cl– as a substrate generating 5‘-chloro-5‘-deoxyinosine (Fig. 8) [59]. The
reactions with both fluoride and chloride are reversible (Fig. 8)
[55,58,59]. A mechanism study that used stereospecifically-labeled S-adenosyl
methionine carrying deuterium at the 5‘-pro-S
site revealed that 5‘-FDA synthase
catalyses the synthesis of 5‘-FDA
from S-adenosyl methionine and fluoride by an SN2 substitution reaction that also occurs
in the biosynthesis of 5‘-chloro-5‘-deoxyinosine [60].
Optimisation of Halogenase
Enzyme Activity by Genetic Algorithm
5-Hydroxytryptophan (5-HTP) is a component of many antidepressant
drugs. Commonly it is obtained by seed extraction of the African plant Griffonia
simplicifolia. The bioanalytical system shown in Fig. 9 could also
generate 5-HTP for pharmaceutical and fine chemical applications [61]. However,
the production rate of the enzyme-producing bacteria and the activity of the
purified enzyme are too low for efficient application in the production of
5-HTP. To overcome the supply problem, a genetic algorithm (GA) was applied for
a tryptophan-5-halogenase activity assay formulation for enzyme activity
optimization that, in this special case, is influenced by six different
factors/parameters [62]. The GA makes an optimization step within a cycle of
four stages: creation of a population of individuals (experiments); evaluation
of these experiments; selection of best experiments and breeding; and, aided by
genetic manipulation, creation of a new population. Real variables are
generally encoded in the form of binary character strings. For a better
performance, all parameters were encoded according to the Gray code application
for the binary bit code [63]. The concentrations of six different medium
components were optimized and the maximum yield of the halogenated tryptophan
could be increased from 3.5% up to 65% [62]. This experiment showed that the
application of the GA for optimization of the enzyme assay composition led to
an improved enzyme assay within a few steps, using only a couple of experiments.
The GA can be saliently applied on a complex 6-D problem to obtain optimized
results within a minimum of experiments.
Biotechnological and
Environmental Significance
AaP and related fungal peroxidases could become promising
biocatalysts in biotechnological applications because they seemingly fill the
gap between CPO and P450 enzymes and act as “self-sufficient”
peroxygenases. From the environmental point of view, the existence of a
halogenating mushroom enzyme is interesting because it could be linked to the
multitude of halogenated compounds known from these organisms. Following the
discovery of the haloperoxidase of A. aegerita, the specific
search for similar enzymes among the huge number of basidiomycetous fungi
colonizing litter or lignocelluloses will surely result in the discovery of
further haloperoxidases and could help better understanding of the natural
occurrence of organohalogens in terrestrial ecosystems [14]. The new findings
from the quantum mechanics/molecular mechanics study of the rest state of the
vanadium-dependent chloroperoxidase might help in understanding the action
mechanism of enzymes and give precious new insights for the design of
biomimetic compounds to be used in industrial catalytic conversions. Synthetic
haloperoxidases have been prepared by metal substitution incorporating Co, Ni,
Zn, and Cu [9].The genes of flavin-dependent halogenases have been identified in
the biosynthetic gene clusters of structurally very different compounds. In theory,
halogenating a range of organic substrates and a sensible program of
mutagenesis could lead to a diverse range of halogenated product. A series of
novel chloro-indolocarbazole compounds has been produced by co-expression of
rebeccamycin
genes with selected tryptophan halogenase genes rebH,
pyrH, and thal from other microorganisms in the Streptomyces
albus expression system [Fig. 10(A,B)] [5,64]. Transformation of the
pyrrolnitrin producer P. chlororaphis ACN with a plasmid containing the thal
gene led to the formation of the new aminopyrrolnitrin derivative 3-(2‘-amino-4‘-chlorophenyl) pyrrole [Fig. 10(C)] [5,31]. These
investigations have shown that such an approach to generating halogenated
analogs of biologically active compounds is feasible, and as more halogenases
are discovered, the range of applications will increase. Detection of the
long-lived chlorinating intermediate in the flavin-dependent halogenase
mechanism suggests nature’s ingenious solution to the chemical problem of
controlling a reactive and potentially destructive oxidant, HOCl, for C-Cl bond
construction [37]. aKG-dependent halogenases have no problem with
reactive power, but the system is complicated by the requirement of the
adenylation/thioesterase component for turnover [48–51,54]. If
aKG-dependent halogenases could be engineered to accept the
untethered substrate, a whole range of chemistry would be opened up. In contrast to haloperoxidases, halogenases (e.g. flavin-dependent
halogenases, aKG-dependent halogenases) are capable of catalysing the
regioselective formation of carbon halogen bonds and are therefore of
particular interest for applications in white biotechnology, as toxic
halogenating agents could be substituted through the less harmful halides [27],
and fewer by-products are produced [65]. Due to their application as
intermediates in palladium (Pd)-catalysed carbon coupling reactions they are
also of tremendous interest for the production of organic fine chemicals [62].
With the fluorinase gene (flA) characterized, there are clear
opportunities to clone it into other micro-organisms to “kick start”
organo-fluorine metabolite production in other organisms [55]. Huang et al.
have identified a cluster of approximately 10 genes that most likely express
proteins involved in the biosynthesis of the fluorometabolites of S.
cattleya [56]. It is attractive to consider inserting all of these genes as
a cassette into candidate alternative organisms to assess if that initiates the
biosynthesis of novel organo-fluorine compounds from such engineered
microorganisms. The application of stochastic search strategies (e.g. GAs) is
well suited to fast determination of the global optimum in multidimensional
search spaces, where statistical approaches or even the popular classical
one-factor-at-a-time method often fails by misleading to local optima.
Biotransformations to halogenated starting materials and building blocks from
inorganic halogen represent novel territory in organo-halogen chemistry and
merit investigation.A large number of halogenated compounds are 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 [13]. The
NADH-dependent enzyme maleylacetate reductase, which catalyzes a different type
of reductive dehalogenation reaction, has been isolated from several Pseudomonas
strains and Ralstonia eutropha [66]. The question whether there is any
connection between biological halogenation and dehalogenation, still can not be
answered. There are no data available showing that biological halogenation led
to the development of dehalogenating enzymes [13]. Additionally, similarities
between halogenating and dehalogenating enzymes have not yet been found. The
search for a halogenating enzyme also led to the development of dehalogenating
enzymes (e.g. CmaC from P. syringae) [53].
Acknowledgements
I would like to thank Prof. Dr. Erwin
Stoschek (The Department of Computational Engineering, Dresden University
of Technology) and Dr. Wenyong Li (Guilin University of Electronic Technology)
for critical readings of the manuscript.
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