|
|
Original Paper
|
|
|||
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,
[email protected]
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 Halo�genases
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].
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.
References
1�� Gribble GW. Natural organohalogens: A new
frontier for medicinal agents? J Chem Educ 2004, 81: 1441-1449
2�� van P�e KH. Biosynthesis of halogenated
metabolites by bacteria. Annu Rev Microbiol 1996, 50: 375-399
3�� Marumo S, Hattori H, Abe H, Munakata K.
Isolation of 4-chloroindolyl-3-acetic acid from immature seeds of Pisum
sativum. Nature 1968, 219: 959-960
4�� Kanbe K, Naganawa H, Nakamura KT, Okami Y,
Takeuchi T. Thienodolin, a new plant growth-regulating substance produced by a Streptomycete
strain. II. Structure of thienodolin. Biosci Biotechnol Biochem 1993, 57: 636-637
5�� Xian-Ping C. Formation of novel
tryptophan-derived compounds by combinatorial biosynthesis using regioselective
tryptophan halogenases (Master�s thesis). Dresden: Dresden University of
Technology 2006
6�� Gribble GW. The diversity of naturally
occurring organobromine compounds. Chem Soc Rev 1999, 28: 335-346
7�� Itabashi T, Ogasawara N, Nozawa K, Kawai K.
Isolation and structures of new azaphilone derivatives, falconensins E-G., from
Emericella falconensis and absolute configurations of falconensins A-G.
Chem Pharm Bull 1996, 44: 2213-2217
8�� Falch BS, K�nig GM, Wright AD, Sticher O,
R�egger H, Bernardinelli G. Ambigol A and B: New biologically active
polychlorinated aromatic compounds from the terrestrial blue-green alga Fischeralla
ambigua. J Org Chem 1993, 58: 6570-6575
9�� Ballschmiter K. Pattern and sources of
naturally produced organohalogens in the marine environment: Biogenic formation
of organohalogens. Chemosphere 2003, 52: 313�324
10� de Boer E, van Kooyk Y, Tromp MGM, Plat H,
Wever R. Bromoperoxidase from Ascophyllum nodosum: A novel class of
enzymes containing vanadium as a prosthetic group? Biochim Biophys Acta 1986,
869: 48-53
11� Dunford HB. Heme peroxidases. New York: John
Wiley & Sons, 1999
12� Sundaramoorthy M, Terner J, Poulos TL.
Stereochemistry of the chloroperoxidase active site: crystallographic and
molecular-modeling studies. Chem Biol 1998, 5: 461-473
13� van P�e KH, Unversucht S. Biological
dehalogenation and halogenation reactions. Chemosphere 2003, 52: 299-312
14� Hofrichter M, Ullrich R. Heme-thiolate
haloperoxidases: versatile biocatalysts with biotechnological and environmental
significance. Appl Microbiol Biotechnol 2006, 71: 276-288
15� Farhangrazi ZS, Sinclair R, Yamazaki I, Powers
LS. Haloperoxidase activity of Phanerochaete chrysosporium lignin
peroxidases H2 and H8. Biochemistry 1992, 31: 10763-10768
16� Sheng D, Gold MH. Haloperoxidase activity of
manganese peroxidase from Phanerochaete chrysosporium. Arch Biochem
Biophys 1997, 345: 126-134
17� Munir IZ, Dordick JS. Soybean peroxidase as an
effective bromination catalyst. Enzyme Microb Technol 2000, 26: 337-341
18� Adak S, Bandyopadhyay D, Bandyopadhyay U,
Banerjee RK. An essential role of active site arginine residue in iodide
binding and histidine residue in electron transfer for iodide oxidation by
horseradish peroxidase. Mol Cell Biochem 2001, 218: 1-11
19� Taurog A, Dorris ML. Peroxidase-catalyzed
bromination of tyrosine, thyroglobulin, and bovine serum albumin: Comparison of
thyroid peroxidase and lactoperoxidase. Arch Biochem Biophys 1991, 287: 288-296
20� Harrison JE, Schultz J. Studies on the chlorinating
activity of myeloperoxidase. J Biol Chem 1976, 251: 1371-1374
21� Thomas EL, Bozeman PM, Jefferson MM, King CC.
Oxidation of bromide by the human leukocyte enzymes myeloperoxidase and
eosinophil peroxidase. J Biol Chem 1995, 270: 2906-2913
22� Thomas EL, Fishman M. Oxidation of chloride
and thiocyanate by isolated leukocytes. J Biol Chem 1986, 261: 9694-9702
23� Rawitch AB, Taurog A, Chernoff SB, Dorris ML.
Hog thyroid peroxidase: physical, chemical, and catalytic properties of the
highly purified enzyme. Arch Biochem Biophys 1979, 194: 244-257
24� Almeida M, Filipe S, Humanes M, Maia MF, Melo
R, Severino N, Da Silva JA et al. Vanadium haloperoxidases from brown
algae of the Laminariaceae family. Phytochemistry 2001, 57: 633-642
25� Messerschmidt A, Prade L, Wever R.
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 1997, 378: 309-315
26� Raugei S, Carloni P. Structure and function of
vanadium haloperoxidases. J Phys Chem B 2006, 110: 3747-3758
27� Keller S, Wage T, Hohaus K, H�lzer M, Eichhorn
E, van P�e KH. Purification and partial characterization of tryptophan
7-halogenase (PrnA) from Pseudomonas fluorescens. Angew Chem Int Ed Engl
2000, 39: 2300-2302
28� Dong C, Flecks S, Unversucht S, Haupt C, van
P�e KH, Naismith JH. Tryptophan 7-halogenase (PrnA) structure suggests a
mechanism for regioselective chlorination. Science 2005, 309: 2216-2219
29� Yeh E, Garneau S, Walsh CT. Robust in vitro
activity of RebF and RebH, a two-component reductase/halogenase, generating
7-chlorotryptophan during rebeccamycin biosynthesis. Proc Natl Acad Sci USA
2005, 102: 3960-3965
30� Zehner S, Kotzsch A, Bister B, S�ssmuth RD,
M�ndez C, Salas JA, van P�e KH. A regioselective tryptophan 5-halogenase is
involved in pyrroindomycin biosynthesis in Streptomyces rugosporus
LL-42D005. Chem Biol 2005, 12: 445-452
31� Seibold C, Schnerr H, Rumpf J, Kunzendorf A,
Hatscher C, Wage T, Ernyei AJ et al. A flavin-dependent tryptophan
6-halogenase and its use in modification of pyrrolnitrin biosynthesis. Biocatal
Biotransform 2006, 24: 401-408
32� Nowak-Thompson B, Chaney N, Wing JS, Gould SJ,
Loper JE. Characterization of the pyoluteorin biosynthetic gene cluster of Pseudomonas
fluorescens Pf-5. J Bacteriol 1999, 181: 2166-2174
33� Wynands I, van P�e KH. A novel halogenase gene
from the pentachloropseudilin producer Actinoplanes sp. ATCC 33002 and
detection of in vitro halogenase activity. FEMS Microbiol Lett 2004,
237: 363-367
34� van P�e KH, Patallo EP. Flavin-dependent
halogenases involved in secondary metabolism in bacteria. Appl Microbiol
Biotechnol 2006, 70: 631-641
35� Yeh E, Cole LJ, Barr EW, Bollinger JM, Ballou
DP, Walsh CT. Flavin redox chemistry precedes substrate chlorination in the
reaction of the flavin-dependent halogenase RebH. Biochemistry 2006, 45: 7904-7912
36� Unversucht S, Hollmann F, Schmid A, van P�e
KH. FADH2-dependence of tryptophan 7-halogenase. Advanced Synthesis Catalysis
2005, 347: 1163-1167
37� Yeh E, Blasiak LC, Koglin A, Drennan CL, Walsh
CT. Chlorination by a long-lived intermediate in the mechanism of
flavin-dependent halogenases. Biochemistry 2007, 46: 1284-1292
38� Nightingale ZD, Lancha AH, Handelman SK, Dolnikowski
GG, Busse SC, Dratz EA, Blumberg JB et al. Relative reactivity of lysine
and other peptide-bound amino acids to oxidation by hypochlorite. Free Radical
Biol Med 2000, 29: 425-433
39� Peskin AV, Winterbourn CC. Kinetics of the
reactions of hypochlorous acid and amino acid chloramines with thiols,
methionine, and ascorbate. Free Radical Biol Med 2001, 30: 572-579
40� Pattison, DI, Davies MJ. Absolute rate
constants for the reaction of hypochlorous acid with protein side chains and
peptide bonds. Chem Res Toxicol 2001, 14: 1453-1464
41� Hawkins CL, Pattison DI, Davies MJ.
Hypochlorite-induced oxidation of amino acids, peptides and proteins. Amino
Acids 2003, 25: 259-274
42� Vaillancourt FH, Yeh E, Vosburg DA,
Garneau-Tsodikova S, Walsh CT. Nature�s inventory of halogenation catalysts:
oxidative strategies predominate. Chem Rev 2006, 106: 3364-3378
43� Naismith JH. Inferring the chemical mechanism
from structures of enzymes. Chem Soc Rev 2006, 35: 763-770
44� Dorrestein PC, Yeh E, Garneau-Tsodikova S,
Kelleher NL, Walsh CT. Dichlorination of a pyrrolyl-S-carrier protein by FADH2-dependent
halogenase PltA during pyoluteorin biosynthesis. Proc Natl Acad Sci USA 2005,
102: 13843-13848
45� Bossemeyer D. The glycine-rich sequence of
protein kinases: A multifunctional element. Trends Biochem Sci 1994, 19: 201-205
46� Paulsen IT, Press CM, Ravel J, Kobayashi DY,
Myers GSA, Mavrodi DV, De Boy RT et al. Complete genome sequence of the
plant commensal Pseudomonas fluorescens Pf-5. Nat Biotechnol 2005, 23:
873-878
47� Galoni DP, Vaillancourt FH, Walsh CT.
Halogenation of unactivated carbon centers in natural product biosynthesis:
trichlorination of leucine during barbamide biosynthesis. J Am Chem Soc 2006,
128: 3900-3901
48� Blasiak LC, Vaillancourt FH, Walsh CT, Drennan
CL. Crystal structure of the non-haem iron halogenase SyrB2 in syringomycin
biosynthesis. Nature 2006, 440: 368-371
49� Vaillancourt FH, Yin J, Walsh CT. SyrB2 in
syringomycin E biosynthesis is a nonheme FeII
alpha-ketoglutarate- and O2-dependent halogenase. Proc
Natl Acad Sci USA 2005, 102: 10111-10116
50� Galonic DP, Barr EW, Walsh CT, Bollinger Jr
JM, Krebs C. Two interconverting Fe(IV) intermediates in aliphatic chlorination
by the halogenase CytC3. Nat Chem Biol 2007, 3: 113-116
51� Vaillancourt FH, Yeh E, Vosburg DA, O�Connor
SE, Walsh CT. Cryptic chlorination by a non-haem iron enzyme during cyclopropyl
amino acid biosynthesis. Nature 2005, 436: 1191-1194
52� Parry RJ, Lin MT, Walker AE, Mhaskar S. The
biosynthesis of coronatine: Investigations of the biosynthesis of coronamic
acid. J Am Chem Soc 1991, 113: 1849-1850
53� Bender C, Palmer D, Penaloza-Vazquez A,
Rangasswamy V, Ullrich M. Biosynthesis of coronatine, a thermoregulated
phytotoxin produced by the phytopathogen Pseudomonas syringae. Arch
Microbiol 1996, 166: 71-75
54� Krebs C, Galonić Fujimori D, Walsh CT,
Bollinger JM Jr. Non-heme Fe(IV)-oxo intermediates. Acc Chem Res 2007, 40: 484-492
55� Schaffrath C, Deng H, O�Hagan D. Isolation and
characterisation of 5�-fluorodeoxyadenosine synthase, a fluorination enzyme
from Streptomyces cattleya. FEBS Lett 2003, 547: 111-114
56� Huang FL, Haydock SF, Spiteller D, Mironenko
T, Li T, Leadlay PF, O�Hagan D et al. Characterisation of a locus
involved in fluorometabolite biosynthesis in Streptomyces cattleya. Chem
Biol 2006, 13: 475-484
57� Cobb SL, Deng H, Hamilton JT, McGlinchey RP,
O�Hagan D. Identification of 5-fluoro-5-deoxy-D-ribose-1-phosphate as an
intermediate in fluorometabolite biosynthesis in Streptomyces cattleya.
Chem Commun 2004, 592-593
58� O�Hagan D. Recent developments on the
fluorinase from Streptomyces cattleya. J Fluorine Chem 2006, 127: 1479-1483
59� Deng H, Cobb SL, McEwan A, McGlinchey RP,
Naismith JH, O�Hagan D, Robinson DA et al. The fluorinase from Streptomyces
cattleya is also a chlorinase. Angew Chem Int Ed Engl 2006, 45: 759-762
60� Cadicamo CD, Courtieu J, Deng H, Meddour A,
O�Hagan D. Enzymatic fluorination in Streptomyces cattleya takes place
with an inversion of configuration consistent with an SN2 reaction mechanism.
Chembiochem 2004, 5: 685-690
61� Ulber R, Protsch C, Solle D, Hitzmann B,Wilke
B, Faurie R, Scheper T. Use of bioanalytical systems for the improvement of
industrial tryptophan production. Chem Ing Technol 2001, 24: 15-17
62� Muffler K, Retzlaff M, van P�e KH, Ulber R.
Optimisation of halogenase enzyme activity application of a genetic algorithm.
J Biotechnol 2007, 127: 425-433
63� Vankeerberghen P, Smeyers-Verbeke R, Leardi R,
Karr CL, Massart DL. Robust regression and outlier detection for non-linear models
using genetic algorithms. Chemometr Intell Lab 1995, 28: 73-87
64� Sanchez C, Zhu L, Brana AF, Salas AP, Rohr J,
Mendez C, Salas JA. Combinatorial biosynthesis of antitumor indolocarbazole
compounds. Proc Natl Acad Sci USA 2005, 102: 461-466
65� Hasegawa M, Yamada K, Nagahama Y, Somei M. The
chemistry of indoles. Part 94. A novel methodology for preparing 5-chloro- and
5-bromotryptamines and tryptophans, and its application to the synthesis of
(+/-)-bromochelonin B. Heterocycles 1999, 51: 2815-2821
66� M�ller D, Schl�mann M, Reineke W.
Maleylacetate reductases in chloroaromatic-degrading bacteria using the
modified ortho pathway: Comparison of catalytic properties. J Bacteriol 1996,
178: 298-300