Reference Work Entry

Handbook of Marine Natural Products

pp 977-1024

Date:

Mechanisms of Halogenation of Marine Secondary Metabolites

  • Claudia WagnerAffiliated withInstitute for Pharmaceutical Biology, University of Bonn Email author 
  • , Gabriele M. KönigAffiliated withInstitute for Pharmaceutical Biology, University of Bonn Email author 

Abstract

Chemical halogenation often requires harsh reaction conditions and results in unwanted by-product formation. It is thus of great interest to investigate the biosynthesis of halogenated natural products and the biotechnological potential of halogenating enzymes. Most of the biogenic organohalogens known today are marine-derived and often proposed to serve as antifeedant and antibacterial defense agents; however, knowledge on biological halogenation in marine organisms still is very limited. Today, mainly vanadate-depending haloperoxidases (Va-HPO) and nonheme FeII/α-ketoglutarate/O2-dependent halogenases are described for secondary metabolite biosynthesis in marine organisms. Beyond that, also enzymes utilizing S-adenosyl-l-methionine in halogen transfer are found in marine environments. This review aims to give a comprehensive overview on the different strategies used by nature to incorporate halogens into secondary metabolites.

Abstract

Chemical halogenation often requires harsh reaction conditions and results in unwanted by-product formation. It is thus of great interest to investigate the biosynthesis of halogenated natural products and the biotechnological potential of halogenating enzymes. Most of the biogenic organohalogens known today are marine-derived and often proposed to serve as antifeedant and antibacterial defense agents; however, knowledge on biological halogenation in marine organisms still is very limited. Today, mainly vanadate-depending haloperoxidases (Va-HPO) and nonheme FeII/α-ketoglutarate/O2-dependent halogenases are described for secondary metabolite biosynthesis in marine organisms. Beyond that, also enzymes utilizing S-adenosyl-l-methionine in halogen transfer are found in marine environments. This review aims to give a comprehensive overview on the different strategies used by nature to incorporate halogens into secondary metabolites.

19.1 Introduction

Approximately 4,500 compounds known to be produced by living organisms are organohalogens [14]. Structural classes range from simple phenolic or aliphatic compounds to complex polyketides and oligopeptides (Figs. 19.4, 19.6, 19.9, 19.10, 19.11, 19.12, 19.13). Biological activities are equally diverse, e.g., antibiotic and antitumor activity as known for chloramphenicol (Fig. 19.12), glycopeptide antibiotics, and cryptophycin (Fig. 19.9), or specific proteasome inhibition as found for salinosporamide A (Fig. 19.16). While from terrestrial organisms a variety of chlorinated metabolites are known, marine halogenated secondary metabolites are more commonly brominated. In general, fluorinated metabolites are extremely rare, mainly due to the high desolvation energy required to activate F in aqueous solution [5].
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Fig. 19.4

Natural products to be halogenated by FeII/α-ketoglutarate/O2-dependent halogenases. Cyclopropyl ring formation of coronatine is executed via a γ-chlorination of l-allo-isoleucine followed by intramolecular cyclization

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Fig. 19.6

Compounds with chlorinated indole moieties formed from tryptophan. In pyrrolnitrin, rebeccamycin, and AT2433 biosynthesis, a tryptophan-7-halogenase; in thienodolin biosynthesis, a tryptophan-6-halogenase; and in pyrroindomycin biosynthesis, a tryptophan-5-halogenase were identified to catalyze the initial biosynthetic step. In the kutznerides biosynthesis, a tryptophan-7-halogenase and a tryptophan-6-halogenase work in a tandem action to produce 6,7-dichloro-l-tryptophan. Only the tryptophan halogenation at position C-2 in chondramide B and D biosynthesis occurs during NRPS assembly

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Fig. 19.9

Compounds with chlorinated phenolic structures where FADH2-dependent halogenation most likely occurs within multienzymatic biosynthesis assembly lines (e.g., PKS/NRPS)

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Fig. 19.10

Compounds with chlorinated pyrrole structural elements halogenated by FADH2-dependent halogenases

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Fig. 19.11

Further compounds with halogenated aromatic substructures for which an involvement of FADH2-dependent halogenases is discussed

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Fig. 19.12

Compounds with chlorinated aliphatic moieties for which an involvement of FADH2-dependent halogenases is discussed

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Fig. 19.13

Marine compounds potentially synthesized via corresponding FADH2-dependent halogenases, e.g., compounds with halogenated indole moieties as isolated from marine actinomycetes (lynamicins [162] and NPI 3114 [163]), algae (flustrabromine [164]), and sponges (bromofascaplysins [165], cyclocinamide A [166], as well as the chondramide analog jaspamide [167]). Secondary metabolites with halogenated phenol and pyrrole moieties are also known from various marine actinomycete (e.g., chinikomycins [168], sporolides [169], cyanosporasides [170], and marmycins B [171]) and sponges (psammaplin A [172], carteramine A[173], nagelamides [174], and mukanadins [174])

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Fig. 19.16

SAM in biohalogenation processes. (a) Enzymatic conversion of inorganic fluoride to organic fluoride by 5´-fluoro-5´-deoxyadenosine synthetase. (b) The chlorinated moiety of salinosporamide A could be shown to derive from tetrose, and SalL was identified as a 5’-FDAS analog halogenating enzyme. The intermediate chloroethylmalonyl-CoA has been identified to act as an unusual starter unit for the subsequent PKS/NRPS biosynthesis part. (c) Methyltransferases mediate the transfer of a methyl group from SAM to a halide (X: halide ion)

Overall, most of the biogenic organohalogens are marine-derived [1] and often proposed to serve as antifeedant and antibacterial defense agents. However, knowledge on biological halogenation in marine organisms still is very limited. Today, mainly vanadate-depending haloperoxidases (Va-HPO) and nonheme FeII/α-ketoglutarate/O2-dependent halogenases are described for secondary metabolite biosynthesis in marine organisms. More recently, the unique S-adenosyl-l-methionine-requiring enzyme, SalL, has been identified to be involved in the salinosporamide A biosynthesis by the marine actinomycete Salinispora tropica [6]. Beyond that, to complete the picture, this review will also include the important group of FADH2-depending halogenases, even though such enzymes were not yet identified from marine organisms.

19.1.1 Classification of Halogenating Enzymes According to their Mechanism

Halogenating enzymes mainly can be grouped into two classes:
  1. 1.

    Less specific haloperoxidases (HPO), utilizing hydrogen peroxide and having heme or vanadate involved as cofactor.

     
  2. 2.

    Highly substrate-specific halogenases requiring dioxygen for enzymatic activity.

    In dioxygen-depending halogenases, either α-ketoglutarate, in nonheme FeII/α-ketoglutarate/O2-dependent halogenases, or flavin, in FADH2-depending halogenases, is found to function as a cosubstrate.

     
Furthermore, methyltransferases are involved in the formation of the carbon–halogen bonds of CH3Cl, CH3Br, and CH3I, and other enzymes requiring S-adenosyl-l-methionine as catalyst have been identified to be involved in fluorination and chlorination reactions [6, 7].

19.2 Haloperoxidases

Until the mid-1990s, the understanding of biological halogenation reactions was restricted to haloperoxidases (HPO). These enzymes generate hypohalous acid (HOX) or related halogenating intermediates, such as OX, X 3 , and X+, by the reaction of a metal-bound hydroperoxy species with a halide ion (Fig. 19.1). In most cases, the halogenating species is then released from the enzyme to act on organic substrates that are susceptible to an attack by electrophiles. The enzymes are found to contain either heme or vanadate as cofactor in their active site [810]. In heme-containing HPOs, the formation of hypohalous acid is driven by a redox mechanism, whereas vanadate-containing HPOs do not change their oxidation state during the enzymatic cycle but rather function as Lewis acids. HPOs are further classified according to the halide ion which they are predominantly able to utilize for halogenation [11]. Thus, the nomenclature is based on the most electronegative halide oxidized by the enzyme. Chloroperoxidases (CPOs) catalyze the oxidation of chloride, bromide, and iodide, while bromoperoxidases (BPOs) only catalyze the oxidation of bromide and iodide, and iodoperoxidases (IPOs) are specific for iodide oxidation [12]. However, hydrogen peroxide does not have the driving force to oxidize fluoride [8].
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Fig. 19.1

Both heme- and vanadium-dependent HPOs are thought to generate bound hypohalite intermediates and hypohalous acid which react as X+ equivalents with electron-rich substrates (X: halide, A: organic substrate)

The first halogenating enzyme was described from the caldariomycin-producing fungus Caldariomyces fumago as a heme-containing HPO [13]. Hager and coworkers developed a spectrophotometric assay, using monochlorodimedone as a mimic of the natural precursor of caldariomycin, to monitor for HPO activity (Fig. 19.2). Based on this assay, several heme HPOs were subsequently discovered from diverse fungi, algae, and microorganisms [9, 10, 14]. The detected HPOs were shown to have a broad substrate specificity, accepting organic compounds, which are generally susceptible to electrophilic attack. Thus, regioselectivity was the same as that seen in chemical halogenations.
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Fig. 19.2

2-Chloro-1,3-cyclopentanedione is a late intermediate in caldariomycin biosynthesis, the natural product from Caldariomyces fumago. Based on this reaction, the conversion of the synthetic analog monochlorodimedone is applied in HPO activity assays

In addition to halogenating activity, ferric heme HPOs have been shown to share catalytic properties with at least three further classes of heme-containing oxidoreductases, namely, classical peroxidases, cytochrome P450 monooxygenases, and catalases [10]. Intensive studies on structural and functional aspects of the heme HPO have focused mainly on the initially described CPO from C. fumago. Under investigation was the chlorination activity of this enzyme, as well as its epoxidation and sulfoxidation activity in the absence of halide [10, 1519]. A recent study on the chlorination mechanism confirmed that the final chlorine transfer to various substrates occurs outside the active site via a free diffusible species without any special mode of substrate recognition [20]. In contrast, epoxidation and sulfoxidation are performed regioselectively, providing C. fumago CPO as a useful tool in synthetic applications. Further investigations were conducted towards the dehalogenation of trihalophenols and p-halophenols [21]. C. fumago CPO proved to be capable of catalyzing such degradation processes and was significantly more robust than other peroxidases. Thus, this CPO might be applicable in biodegradation of polychlorinated phenols and further noxious haloaromatic compounds, under reaction conditions often too harsh for other biocatalysts.

Vanadate ion–containing bromoperoxidases (Va-BPOs) have been isolated mainly from marine algae. This group of enzymes most likely seems to play a role in the formation of marine natural products, e.g., the halogenation and cyclization of terpenes [22]. Va-HPOs bind hydrogen peroxide and halides leading to a putative enzyme-bound or active site–trapped brominating moiety [23]. X-ray crystal structures of the Va-BPO from Curvularia inaequalis [24], Ascophyllum nodosum [25], and Corallina officinalis [26] displayed a channel leading to the vanadium binding site, which is proposed to influence substrate specificity [27]. Competitive kinetic studies, comparing the bromination of indole substrates by Va-HPO with enzyme-free preparations under aqueous conditions, showed that these reactions were not congruent, since Va-HPO preferably brominated indole derivatives even in the presence of other substrates, such as monochlorodimedone or phenol red [8, 28]. In the absence of a suitable substrate, however, HOBr is released from the enzyme and carries out unselective bromination as observed for aqueous bromine. The role of Va-BrPO in biosynthesis has been further established by the observation that the ratio of formed diastereomers was also found to differ between purely chemical and enzymatic reactions. Accordingly, asymmetric bromination and cyclization of the terpenoid precursor (E)-(+)-nerolidol could be demonstrated for Va-BPO, producing single diastereomers of the marine natural product β-snyderol as isolated from Laurencia sp. [27] (Fig. 19.3).
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Fig. 19.3

Proposed mechanism of the stereospecific snyderol biosynthesis by Va-BrPO [27]

The first Va-BPO to be characterized was from the brown seaweed Ascophyllum nodosum [29]. However, with the isolation of a Va-BPO from the lichen Xanthoria parietina, it became obvious that this group of enzymes not only occurs in the marine environment but also can be found in terrestrial eukaryotic organisms [30]. Va-CPOs on the other hand have been mainly found in fungi even though, to date, halogenated natural products have not been identified from the corresponding strains [31]. The first indication of the potential involvement of a Va-CPO in the biosynthesis of a marine natural product has recently been discussed regarding the chlorination and cyclization of napyradiomycin (Fig. 19.12), a compound obtained from the marine sediment–derived Streptomyces sp. CNQ-525 [31, 32].

In contrast to the iron heme haloperoxidases, Va-HPOs do not catalyze the direct disproportion of hydrogen peroxide and, therefore, have no catalase activity [33]. However, their structural similarity to acid phosphatases is noticeable, particularly in the domains providing the vanadate and phosphate binding site. Therefore, a common evolutionary origin is proposed as being likely [23].

Recent studies aim to characterize the active site in Va-HPO more precisely, whereby the residues involved in the selection and binding of the specific halide are the main focus [31]. Heterologous expression and mutagenesis studies of Va-BPO from Curvularia inaequalis and Corallina officinalis explore the potential of this type of enzymes in biotransformations [34, 35].

Hypohalous acid is a bactericidal agent, and many halogenated compounds have antimicrobial activity, illustrating the possible involvement of HPO systems in natural defense mechanisms. This might prevent biofouling by microorganisms on the surface of marine algae as well as act as an antifeeding system. Experiments with the marine alga Laminaria digitata demonstrated that natural HPO systems are also capable of mediating the deactivation of acylated homoserine lactones, which are important as cell-to-cell signaling molecules for biofilm formation [36]. This suggests that oxidized halogens may control biofouling not only via a bactericidal mechanism but also by possibly interfering with bacterial cell signaling systems. As described above, an involvement of HPOs in the biosynthesis of natural products has only been suggested in a very few cases [22, 27, 32]. The physiological function of CPOs in fungi is mainly proposed as being important for the degradation of plant cell walls. The hypohalous acid produced thus oxidizes lignocellulose and facilitates penetration of the fungal hyphen into a host [23]. Furthermore, Va-IPOs seem to play a role in iodine accumulation of brown algae as a main vector of the iodine biogeochemical cycle [37]. Species like Laminaria digitata are thereby able to take up an average content of 1% of their dry weight, representing approximately a 30,000-fold accumulation of iodine from seawater. Iodine efflux and the production of volatile halocarbons, then again, are proposed to be part of an early defense response to various biotic and abiotic stresses by these organisms. This assumption was strengthened, since the transcription of members of the Va-IPO gene family was demonstrated to be rapidly and highly induced as a defense response [38].

19.3 Nonheme FeII/α-Ketoglutarate/O2-Dependent Halogenases

In the last years, evidence has accumulated that another class of halogenating enzymes, able to carry out chlorination of inactivated carbon centers, plays an important role in the biosynthesis of marine organohalogens. Using α-ketoglutarate (α-KG) as cosubstrate, these halogenases are involved in the chlorination of terminal methyl groups of amino acids linked to peptidyl carrier proteins (Table 19.1).
Table 19.1

FeII/α-ketoglutarate/O2-dependent halogenases

Halogenases

Natural product Organism

Halogenated structural element

GenBank accession number [reference]

SyrB2

Syringomycin E

l-threoninea

U25130, AF047828 [40, 127, 128]

Pseudomonas syringae

BarB1/BarB2

Barbamide

l-leucineb

AF516145 [48, 49, 129]

Lyngbya majuscula

DysB1/DysB2

Dysidenin

N.S.d

AY628171 AY628172 [50]

Oscillatoria spongeliae 35P1c

CytC3

Armentomycin

l-aminobutyratee

n.df [55]

Streptomyces spp.

HctB

Hectochlorin

N.S.d

AY974560 [130]

Lyngbya majuscula

CmaBg

Coronatine

l-allo-isoleucine

U14657 [57, 131]

Pseudomonas syringae

KtzDh

Kutznerides

N.S.d

EU074211 [132]

Kutzneria sp. 744

JamHali

Jamaicamides

N.S.d

AY522504 [51, 52]

Lyngbya majuscula JHB

CurHalj

Curacin A

(S)-3-hydroxy-3-methlyglutaryl

AY652953 [52, 133]

Lyngbya majuscula JHB

aOnly when bound to the peptidyl carrier protein SyrB1

bThe triple chlorination is catalyzed by a tandem action of the two enzymes

c Oscillatoria spongeliae 39P1: AY628173, AY628174 [50]; Oscillatoria spongeliae AY648943 [134]

dNot specified

eBound to the peptidyl carrier protein CytC2

fNo sequence data available in GenBank

gMediates the formation of the cyclopropane ring

hHypothesized to be involved in the formation of the cyclopropane ring

iEmbedded in the PKS module JamE

jEmbedded in the PKS module CurA

Initial insight into their catalytic mechanism is derived from investigations on SyrB2, which chlorinates the γ-methyl group of l-threonine in syringomycin E (Fig. 19.4) biosynthesis in Pseudomonas syringae [39, 40]. Halogenation of l-threonine occurs only when bound to the peptidyl carrier protein SyrB1, while free l-threonine is not accepted as a substrate. Reactions performed with an excess of sodium bromide demonstrated that SyrB2 was also capable of incorporating bromine into the threonyl-S-protein [41], however, with a clear preference to chlorine over bromine. From crystallographic data [42], it was confirmed that FeII of the active site is complexed by two histidine residues. A halide ligand (chloride or bromide) together with α-KG and water coordinates the iron in the resting FeII state (Fig. 19.5). Dioxygen attack results in decarboxylation of α-KG, and a highly reactive iron-oxo species (FeIV = O) is formed as a general key feature of catalysis by these α-oxoacid-dependent dioxygenases [43]. The FeIV = O abstracts a hydrogen radical from an aliphatic carbon center of the actual substrate, which, in turn, abstracts the halide from the coordination sphere. It has been shown that in vitro SyrB2 was rapidly deactivated during catalysis and no more than about seven turnovers of the enzyme were possible [43]. Further mechanistic studies, especially focusing on the preference of halogenation over hydroxylation during the catalytic cycle, are still ongoing, e.g., as recently published by de Visser and Latifi [44], Wong et al. [45], Matthews et al. [46, 47], and Kulik et al. [4].
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Fig. 19.5

Proposed mechanism of the catalytic cycle of FeII/α-ketoglutarate/O2-dependent halogenases. R-CH3 is the methyl group of the threonyl-S-protein substrate [2]

For several marine natural products that are halogenated at an unactivated carbon center, analogous halogenating enzymes were suggested to be involved in their biosynthesis, e.g., barbamide, dysidenin, dysideathiazole, jamaicamides, and herbamide B (Fig. 19.4) [41, 4854]. Indeed, in the barbamide gene cluster of Lyngbya majuscula, two genes, i.e., barB1 and barB2, homologous to syrB2 could be identified. The initial biosynthetic step is the chlorination of l-leucine to a trichloroleucine derivative, followed by a subsequent conversion to a unique trichloroisovaleryl starter unit in a mixed nonribosomal peptide (NRPS)/polyketide (PKS) pathway [48, 49]. Regarding dysidenin production in dictyoceratid sponges, similar genes (dysB1/dysB2) were found and could be ascribed to the cyanobacterial symbiont Oscillatoria spongeliae [50].

A FeII/α-ketoglutarate/O2-dependent halogenase homologous enzyme, CytC3, involved in the formation of the γ,γ-dichloroaminobutyrate armentomycin (Fig. 19.4) was identified from a soil Streptomyces sp. [55]. Interestingly, the γ,γ-dichloroaminobutyryl moiety is postulated to be further cyclized and to act as a precursor in cytotrienin A formation. CytC3 could be demonstrated to act on l-aminobutyrate bound to a peptidyl carrier protein, CytC2. Spectroscopic monitoring of the enzymatic reaction confirmed that the chlorination proceeds through an FeIV intermediate that cleaves the C–H bond of the amino acid substrate [56], as described above. By analogy, γ-chlorination of an l-allo-isoleucine by the FeII/α-ketoglutarate/O2-dependent halogenase CmaB mediates the formation of the cyclopropane ring in coronatine (Fig. 19.4), which is produced by several Pseudomonas syringae strains [57]. The intermediate γ-chloroaminoacyl β-thioester is then converted to a γ-carbanion, which, in turn, is accessible to intramolecular cyclization [58]. Evidence for a very similar biosynthetic mechanism was observed for the marine secondary metabolite curacin A (Fig. 19.4). From the respective gene cluster of Lyngbya majuscula, it was deduced that the formation of the β-branched cyclopropane moiety is initiated by an analogous γ-chlorination step [52].

19.4 FADH2-Dependent Halogenases

Even though not yet firmly linked to any biosynthetic pathway from organisms of the marine habitat, FADH2-dependent halogenases form another very important class of halogenating enzymes and thus are included in this overview. This group of enzymes was first discovered when investigating pyrrolnitrin (Fig. 19.6) and chlorotetracycline (Fig. 19.9) biosynthesis [59, 60]. The four enzymes (PrnA, B, C, D) involved in pyrrolnitrin production have been identified from a 6.2-kb genomic DNA region of Pseudomonas fluorescens BL915 [59], and their function was proved by construction of deletion mutants for all four genes of the cluster [61]. PrnA (Table 19.2) and the PrnC (Table 19.4) were shown to act as highly substrate-specific halogenases, i.e., the tryptophan halogenase (PrnA) was not able to chlorinate the PrnC substrate monodechloramino-pyrrolnitrin and vice versa [62]. Both enzymes also perform the respective chlorination reaction highly regioselectively, and van Pée and coworkers demonstrated that PrnA is able to halogenate l-tryptophan in a site-specific manner to yield 7-chlorotryptophan [63]. Even though other indole derivatives were also accessible to halogenation by PrnA, they were chlorinated at different sites, i.e., at positions C-2 or C-3. Thus, for regioselectivity, the exact positioning of the specific biosynthetic intermediate at the active site of the halogenating enzyme seems to be of major importance and allows chlorination at a site of the molecule not accessible to chemical halogenation.
Table 19.2

FADH2-dependent halogenases that halogenate tryptophan in the initial step of the respective natural product biosynthesis

Halogenases

Natural product Organism

Halogenated structural element

GenBank accession number [reference]

PrnA

Pyrrolnitrin

Tryptophan at position C-7

PFU74493 [59]

Pseudomonas fluorescens BL915

RebH

Rebeccamycin

Tryptophan at position C-7

AJ414559 [72]

Lechevalieria aerocolonigenes ATCC 39243

PyrH

Pyrroindomycin B

Tryptophan at position C-5

AY623051 [77]

Streptomyces rugosporus LL-42D005

Thal

Thienodolin

Tryptophan at position C-6

EF095207 [71]

Streptomyces albogriseolus

KtzQa

Kutznerides

Tryptophan at position C-7

EU074211[132]

Kutzneria sp. 744

KtzRa

Kutznerides

7-Cl-tryptophan at position C-6b

EU074211[132]

Kutzneria sp. 744

AtmH

AT2433

Tryptophan at position C-7

DQ297453 [135]

Actinomadura melliaura

aWorking in a tandem action, first KtzQ on unmodified tryptophan followed by KtzR on 7-Cl-l-tryptophan to form 6,7-dichlorotryptophan [136]

bWith an approximate 120-fold preference over unsubstituted tryptophan [136]

Table 19.4

FADH2-dependent halogenases involved in the halogenation of pyrrole moieties

Halogenases

Natural product Organism

Halogenated structural element

GenBank accession number [reference]

PltA

Pyoluteorin

Prolinea

AF081920 [82]

Pseudomonas fluorescens Pf-5

HrmQ

Hormaomycin

Pyrroleb

EU583477 [98]

Streptomyces griseoflavus W-384

PrnC

Pyrrolnitrin

Monodechloramino-pyrrolnitrin

PFU74493 [59]

Pseudomonas fluorescens BL915

HalB

Pentachloropseudilin

Pyrrole

AF450451 [148]

Actinoplanes sp. ATCC33002

Pyr29c

Pyrrolomycin A, B, C, D

Proline

EF140901 [149]

Actinosporangium vitaminophilum ATCC31673

aProline halogenation while bound to a peptidyl carrier protein

bMost likely halogenation via a peptidyl carrier protein–bound proline

65% similarity to PltA

It was further shown that the enzymes, PrnA and PrnC ascribed to the two halogenating steps of pyrrolnitrin biosynthesis, require FADH2 as cofactor [64, 65], which is produced from FAD and NADH by a flavin reductase. A direct contact between the halogenase and the flavin reductase is thereby not absolutely necessary since free diffusible FADH2 can also be used. Even chemically reduced flavin, regenerated using organometallic complexes, can be utilized [66]. However, free FADH2 is rapidly decomposed into FAD and H2O2 with molecular oxygen, which leads to a significant decrease in halogenase activity. Recent sequencing of the Pseudomonas fluorescens Pf-5 genome [67] has revealed that the complete gene clusters for pyrrolnitrin as well as pyoluteorin biosynthesis contain genes encoding for a flavin reductase. This gives rise to the assumption that a distinct flavin reductase specifically interacts with each halogenase to prevent loss of FADH2 through autooxidation [68].

The mechanism for regioselective chlorination has been further characterized using a purified protein from a P. fluorescens BL915 PrnA overexpression system. Cocrystallization of the enzyme with tryptophan and FAD yielded yellow diamond-shaped crystals, indicating that FAD was bound to the protein [69]. From further investigations, it could be demonstrated that FAD and Cl are bound at the same site of the enzyme, while the tryptophan-binding module is located at a 10-Ǻ distance [70]. FADH2 reacts with O2 and forms a peroxide-linked isoalloxazine ring, as generally known from flavin-dependent monooxygenases. Subsequently, HOCl is produced as chlorinating agent and channeled through an intraenzymatic tunnel to the substrate binding pocket. However, HOCl is not released from the enzyme to act in a freely diffusible form in solution. Dong et al. postulated that chlorine may be hydrogen-bound to the Lys-79 residue located at the end of the tunnel. In this manner, chlorine would be activated by increased electrophilicity, allowing it to react with tryptophan. In addition, the importance of Glu-346 to stabilize and then deprotonate the substrate intermediate during this process was confirmed by site-directed mutagenesis experiments. The basis for regioselective halogenation is thus the controlled presentation of the substrate to the bound Cl-species via particular amino acid residues in the substrate binding site (Fig. 19.7) [71].
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Fig. 19.7

Mechanism of the halogenation reaction of a FADH2-dependent halogenase (gray-shaded are the processes that proceed within the halogenase enzyme)

RebH (Table 19.2) is another tryptophan-7-halogenase which has been identified within the rebeccamycin (Fig. 19.6) biosynthetic gene cluster of Lechevalieria aerocolonigenes ATCC39243 [72, 73]. RebH was shown to catalyze the initial step of rebeccamycin biosynthesis converting l-tryptophan to 7-chlorotryptophan [74]. RebF catalyzes the NADH-dependent reduction of FAD to provide FADH2 and thus acts together with RebH in a robust two-component reductase/halogenase system. From spectroscopically performed kinetic analysis of RebH, a detailed reaction scheme was proposed (Fig. 19.8) [75]. Initially, FADH2 is transferred to a FAD(C4a)-oxygenated species, and from this FAD(C4a)–OOH intermediate (observed as an increase in absorbance at 390 nm), an OH+ equivalent reacts with a chlorine ion yielding HOCl and FAD(C4a)–OH. Finally, an increase in absorbance at 450 nm indicated the dehydration of FAD(C4a)–OH to fully oxidize FAD. The actual chlorination of tryptophan thus occurs independently after the oxidative reaction of flavin is completed. Protein dynamics leading to conformational changes are assumed to shield the oxygenated FAD(C4a) intermediates from solvent, thereby preventing their rapid breakdown and providing a site for generating the chlorinating agent. Recently, it has been demonstrated that a stable long-living enzyme-bound chlorinating species (t½ of 28 h at 25°C) is formed during the reaction of RebH with FADH2, Cl, and O2, which remains on the active site even after removal of FAD until a substrate becomes available for reaction [76]. This chlorinating intermediate is proposed to be a covalently bound lysine chloramine (Lys79-εNH-Cl), located ideally to deliver a Cl+ equivalent for the electrophilic aromatic substitution of the tryptophan indole ring at C-7.
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Fig. 19.8

Timing of flavin intermediates and product formation as spectroscopically monitored by Walsh and coworkers [75]

Further specific halogenases (Table 19.2) capable to chlorinate tryptophan at positions C-6 and C-5 were also found. A selective tryptophan-5-halogenase (PyrH) was shown to be involved in pyrroindomycin B (Fig. 19.6) biosynthesis of Streptomyces rugosporus LL-42D005 [77] as well as a tryptophan-6-halogenase (Thal) in the thienodolin (Fig. 19.6) production by Streptomyces albogriseolus [71], both ascribed to catalyze initial steps in the biosynthesis. From comparison of the crystal structure of the tryptophan-5-halogenase (PyrH) with that of the tryptophan-7-halogenases, significant differences in the tryptophan-binding module were observed. This, together with site-directed mutagenesis studies, further explained the regioselectively controlled chlorination of tryptophan, showing that the respective carbon atom to be halogenated is solely presented to the chlorine species, while other potential reactive sites are shielded [78].

In contrast to the above-mentioned halogenases, the tryptophan-2-halogenase, CmdE, which is associated with the formation of chondramides B and D (Fig. 19.6; Table 19.3) from the myxobacterium Chondromyces crocatus Cm c5, was demonstrated to be part of a biosynthetic assembly line [79]. Chondramides are of mixed NRPS/PKS origin, and, to date, CmdE is the only example of a tryptophan halogenase integrated in such a modular biosynthetic pathway. Consequently, it is not surprising that the highest homologies of CmdE can be found in halogenases like those involved in cryptophycin (CrpH), avilamycin A (AviH), and clorobiocin (Clo-Hal) biosynthesis (Table 19.3) rather than in the above-described typical tryptophan halogenases (Table 19.2), i.e., only 20% identity to PryH, while no significant similarity to the tryptophan-7-halogenases, PrnA and RebH, was found [79].
Table 19.3

FADH2-dependent halogenases that halogenate phenolic moieties as an integrated step in multienzymatic biosynthesis

Halogenases

Natural product Organism

Halogenated structural element

GenBank accession number [reference]

CmdE

Chondramides B/D

Tryptophan at position C-2

AM179409 [79]

Chondromyces crocatus Cm c5

CTC-chl (=cts4)

Chlorotetracycline

4-Ketoanhydro-tetracyclinea

D38214 [60, 122]

Streptomyces aureofaciens NRRL3203

CrpH

Cryptophycin 1

Tyrosine

EF159954 [94]

Nostoc sp. ATCC 53789

ComH

Complestatin

Hydroxyphenylglycine

AF386507 [80]

Steptomyces lavendulae

“Halogenase gene

Vancomycin

N.S.b

AF486630 [137]

Amycolatopsis orientalis

BhaA

Balhimycin

β-Hydroxy-tyrosine

Y16952 [81, 138]

Amycolatopsis balhimycina DMS5908

Tcp21

Teicoplanin

Tyrosine and β-hydroxy-tyrosine

AJ605139 [139]

Actinoplanes teichomyceticus ATCC31131

ORF10 (=dbv10)

A40926

Dihydroxyphenylglycine and β-hydroxy-tyrosine

AJ561198 [140]

Nonomuraea sp. ATCC39727

ApdC

Anabaenopeptilide 90B

Tyrosine

AJ269505 [141]

Anabaena circinalis 90

McnDc

Cyanopeptolin-984

Tyrosine

DQ075244 [142]

Microcystis sp. NIVA-CYA 172/5

AerJd

Aerugenosin peptides

3-(4-Hydroxyphenyl) lactic acid

AM773664 [143]

Microcystis sp. PCC 9812

ChlB4

Chlorothricin

6-Methylsalicylic acid

DQ116941 [144]

Streptomyces antibioticus DSM 40725

SgcC3

C-1027

β-Tyrosinee

n.d.f [145]

Streptomyces globisporus

CndH

Chondrochloren

Tyrosine

AM988861 [88, 146]

Chondromyces crocatus Cm c5

aIndicates that halogenation occurs while the elongating acyl chain is still tethered to carrier proteins during the PKS assembly line

bNot specified

cAlso identified from other Microcystis strains (AM773679, AM773678, AM773677, AM773676, AM773675, AM773674) [143]

dAlso identified from other Microcystis strains (AM773663, AM773662, AM773661, AM773660, AM773659, AM773658, AM773657, AM773656, AM773655, AM773654 [143], PCC7806, NIES-98 [147])

eIn vitro characterized to halogenate tyrosine tethered to a peptidyl carrier protein

fNo sequence data available in GenBank

In general, FADH2-dependent halogenases can be grouped into those accepting free small molecules as described above and those that act on carrier-bound substrates. Further examples of halogenases involved in multienzymatic NRPS/PKS assembly lines are summarized in Tables 19.3 and 19.4, subdivided into those suggested to chlorinate phenolic moieties (Table 19.3, Fig. 19.9) as in complestatin [80] and glycopeptide antibiotic formation [81], e.g., vancomycin, and those that halogenate peptidyl carrier–bound pyrrole moieties (Table 19.4; Fig. 19.10), e.g., PltA in pyoluteorin biosynthesis [82]. In the latter case, proline is bound as a thioester to the carrier protein PltL and enzymatically desaturated to a pyrrolyl-S-PltL intermediate. This is then chlorinated regiospecifically at position C-5 and subsequently at position C-4 of the heteroaromatic ring, as observed through temporal product formation. Both halogenations are catalyzed by the same enzyme, i.e., PltA. During this process, chlorination is specific for pyrrolyl-S-PltL, since prolyl-S-PltL or free pyrrole-2-carboxylate is not accepted as substrate for halogenation by PltA.

Even more halogenases involved in the chlorination of phenolic moieties, but not explicitly described to function as an integral part of enzymatic assembly lines, are shown in Table 19.5 (for structures, see Fig. 19.11).
Table 19.5

FADH2-dependent halogenases involved in the halogenation of phenolic moieties

Halogenases

Natural product

Halogenated structural element

GenBank accession number [reference]

Organism

Pyr16/17

Pyrrolomycin A, B, C, D

Phenola

EF140901 [149]

Actinosporangium vitaminophilum ATCC31673

Asm12

Ansamitocin P-3

Proansamitocinb

AF453501 [150, 151]

Actinosynnema pretiosum ATCC31565

n.d.c

Ochratoxin A

Mellein intermediated

n.d.c [152]

Penicillium nordicum

Clo-Hal

Clorobiocin

Aminocoumarin

AF329398 [95, 153]

Streptomyces roseochromogenes

AviH

Avilamycin A

Orsellinic acide

AF333038 [154]

Streptomyces viridochromogenes Tü57

CalO3

Calicheamicin γ1

Orsellinic acid

AF497482 [155]

Micromonospora echinospora spp. calichensis

SimD4

Simocyclinone D8

Aminocoumarin

AF324838 [156]

Streptomyces antibioticus Tü6040

RadHf

Radiciol

Phenola

EU980390 [157]

Chaetomium chiversii

ChlA

DIF-1 (differentiation-inducing factor 1)

Phenola

DDB_G0290825g [158]

Dictyostelium strain Ax2

aPhenol moiety polyketide derived

bHalogenation of proansamitocin as first PKS-tailoring reaction

cNo sequence data available in GenBank

dPost-PKS

eDichloro-isoeverninic acid attached to the preformed heptasaccharide

fAlso identified from Pochonia chlamydosporia (EU520419, termed Rdc2 [159])

gAt dictyBase, http://​dictybase.​org/​

Besides the FADH2-dependent halogenases involved in halogenation of aromatic compounds, recognizing tryptophan/indole or phenol/pyrrole moieties, enzymes involved in the halogenation of aliphatic compounds are known (Table 19.6; Fig. 19.12). For example, for chloramphenicol biosynthesis in Streptomyces venezuelae ISP5230, the function of CmlS as a halogenating enzyme has been proved by gene disruption experiments, whereby corynecins were produced in place of chloramphenicol [83]. CmlS shows the highest homology to the FADH2-dependent halogenase PltA (Table 19.4). It has been suggested that a substrate derived from glucose is halogenated and that dichloroacetyl-CoA is then attached to the amino group of the side chain of an enzyme-tethered p-aminophenylalanine [84]. For napyradiomycin (Fig. 19.12) from the marine sediment–derived Streptomyces sp. CNQ-525, not only Va-CPOs, as described above, are discussed to be involved in its biosynthesis but also a FADH2-dependent halogenase, NapH2. The gene for the latter was found within the biosynthesis cluster. Thus, NapH2 is the first FADH2-dependent halogenase described to be potentially involved in a halogenation reaction in a marine organism. Considering structural similarities between terrestrial and marine natural products, it may be conducted, however, that FADH2-dependent halogenases very likely also contribute to the biosynthesis of many marine compounds (Fig. 19.13).
Table 19.6

FADH2-dependent halogenases discussed to be involved in the chlorination of aliphatic structural elements

Halogenases

Natural product

Halogenated structural element

GenBank accession number [reference]

Organism

CmlS

Chloramphenicol

Aliphatic moietya

AY026946 [83]

Streptomyces venezuelae ISP5230

ORF3b

Neocarzilin A

Aliphatic moiety

AB097904 [160]

Streptomyces carinostaticus

n.d.c

(R-(Z))-4-amino-3-chloro-2-pentenedioic acid

Aliphatic moietyd

DQ782376 [161]

Streptomyces viridogenes ATCC39387

NapH2

Napyradiomycin A80915A-D

Aliphatic moietye

EF397638 [32]

Streptomyces sp. CNQ-525

aLikely in an enzyme-bound assembly line (similarity to PltA)

bSimilarity to HalB

cOnly partial gene sequence

dHalogenation postulated via a pyrrole intermediate

eAlso Va-CPOs (NapH1,3,4) identified within the gene cluster

19.4.1 Sequence Specificities and Enzymatic Activity of FADH2-Dependent Halogenases

The in vitro enzymatic activity of halogenases has been investigated only in a very few cases until now, and the function of many biosynthetic genes as halogenases (Tables 19.2, 19.3, 19.4, 19.5, 19.6) was assigned only putatively. Nevertheless, halogenases are known to have an approximate size of 550 amino acids and thus to be encoded by a corresponding DNA consisting of 1,500–1,600 bp (Fig. 19.14). The flavin binding site is located at the amino terminal end and includes a GxGxxG motive, which is extremely conserved within this enzyme group [85]. Another conserved region, i.e., WxWxIP, is found in the middle of the sequence [85]. The tryptophan residues of the latter motif are assumed to prevent the enzyme from catalyzing monooxygenase-type reactions by sterically hampering substrate binding next to the isoalloxazine ring of FADH2 [71].
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Fig. 19.14

General amino acid (aa) sequence assembly of FADH2-depending halogenases, showing the conserved motives in all FADH2-depending halogenases

Further insight in the mechanism how FADH2-dependent halogenases can halogenate such a wide range of substrates derived from the crystal structure of CmlS [86, 87], the halogenase involved in the chloramphenicol biosynthesis, in comparison to those characterized for tryptophan halogenases (e.g., PrnA) and CndH, a tyrosyl chlorinating halogenase acting during chondrochloren biosynthesis [88]. Based on characteristic features of these crystal structures, two variants of FADH2-dependent halogenase have been distinguished [88]. Variant A enzymes (e.g., PrnA), acting on free small molecules, enclose their substrate completely in the active halogenation site. Thereby, the C-terminus of the enzymes seems to cover the active halogenation site and to somehow form a tunnel into which the substrate must enter. In variant B enzymes (CndH), the active center remains open, giving access to a sterically much larger carrier-bound substrate. From simple primary structure alignment, CmlS would rather be classified as a variant B halogenase; however, due to the occurrence of a bulky C-terminal lobe, it seems more likely that a free small molecule enters the active halogenation site (variant A enzyme). The structural diversity observed in the C-termini of FADH2-dependent halogenases appears to reflect the overall diversity of substrates that are halogenated, potentially by sterically restricted access to the active halogenation site. Together with this, specific features within the active halogenating site contribute to the substrate selectivity and regioselectivity. In the case of CmlS, an overall hydrophobic pocket with three phenylalanine residues (F87, F304, F357) is present in the active halogenation site [87], in contrast to a glutamic acid accompanied by two histidine residues (E346, H101, H395) in PrnA, which are thought to ensure tryptophan halogenation. From the crystal structure of the tryptophan-5-halogenase PryH, it became obvious that as a result of amino acid insertion and deletions, compared to PrnA, the substrate binding pocket is substantially rearranged. As a result, tryptophan is bound differently, exposing the respective C atom for chlorination to the key lysine and glutamate residues, while the other sites of potential reactivity will be protected by the enzyme structure [78]. Amino acid sequence alignment of Thal (tryptophan-6-halogenases) and PrnA showed only few differences lining the active halogenation site, i.e., I56/I82/T348 in PrnA and V56/V86/S364 in Thal, which are potentially involved in the directed substrate presentation for regioselective halogenation [71].

Early studies investigating the incorporation of bromine instead of chlorine in chloramphenicol, chlorotetracycline, and pyrrolnitrin resulted in the formation of the corresponding bromo analogs, giving the first indication that the involved enzymes are not exclusively specific for chloride [8991]. Thus, the ability of FADH2-dependent halogenases to alternatively incorporate bromide instead of chloride has been subjected to further detailed investigations [9294]. For PyrH (Table 19.2), it could be shown that in the presence of bromide, 5-bromotryptophan was obtained, although chlorination is favored. In any case, only the regioselective monohalogenation of tryptophan at the C-5 position occurred [77]. Similarly, Thal and RebH (Table 19.2) were demonstrated to catalyze monochlorination regioselectively, as well as monobromination of tryptophan in vitro resulting in 6- and 7-halotryptophan, respectively [71, 74]. Bromide ions were also able to compete with and replace chloride in the PltA (Table 19.4) reaction on pyrrolyl-S-PltL. However, the sterically larger bromide slowed down the halogenation reaction to 10–20% conversion in 1 h, as opposed to >90% when chloride was the only halide present [82].

19.4.2 Biotechnological Potential of FADH2-Dependent Halogenases

The considerable progress made in understanding biological halogenation mediated through FADH2-dependent halogenases gave rise to probing the biotechnological potential of these enzymes. The aim of such investigations was to allow halogenations at specific sites of substrates not accessible to direct chemical halogenation as well as to avoid the formation of unwanted by-products.

Combinatorial biosynthesis yielded new derivatives of the aminocoumarin antibiotics, novobiocin, clorobiocin, and coumermycin A1 (Fig. 19.15). Inactivation of the clo-hal gene in the clorobiocin biosynthetic gene cluster of Streptomyces roseochromogenes resulted in the formation of the hydrogen analog novclobiocin 101, and coexpression of the putative methlytransferase novO led to the production of novclobiocin 102, which is methylated at C-8 of the aminocoumarin ring [95]. More interestingly, coexpression of clo-hal in a novO mutant resulted in the chlorinated novobiocin derivative novclobiocin 114 [96]. An attempt to functionally replace clo-hal by the balhimycin halogenase (Table 19.3) bhaA both in the clo-hal and novO mutant, did not yield in the respective chlorinated aminocoumarin derivatives (clorobiocin or novclobiocin 114), thus underlining the different substrate specificity of Clo-Hal and BhaA [96]. However, introduction of clo-hal into the cluster of coumermycin A1 again resulted in the formation of the respective mono- and dichlorinated derivatives [97]. In a study investigating the halogenating potential of the hormaomycin halogenase HrmQ (Table 19.4) in combinatorial biosynthesis, coexpression experiments of hrmQ in a cloN6 clorobiocin mutant strain were conducted [98]. The results confirmed that HrmQ specifically chlorinates pyrrole moieties at the C-5 position and thus yielded the new clorobiocin derivatives, novclobiocin 124 and 125.
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Fig. 19.15

Novel aminocoumarin derivatives derived by combinatorial biosynthesis

As a result of their potential therapeutic applications as antitumor and neuroprotective agents [99, 100], great efforts have been made to generate derivatives of indolocarbazole alkaloids. Novel halogenated compounds were obtained by recombining genes found within the biosynthetic gene clusters of rebeccamycin and staurosporine with the halogenases RebH, PyrH, and Thal (Table 19.2). In this way, Salas and coworkers were able to generate “hybrid” indolocarbazoles by modifying the staurosporine aglycon using rebeccamycin chlorination, glycosylation, and sugar methylation activities. Besides nonhalogenated products, this approach yielded chloro- and dichloro-staurosporine derivatives [100]. The major bis-indole products obtained when coexpressing PyrH and Thal (Table 19.2) had a chloride substitution at positions C-5 and C-6 of the indole moiety, respectively.

In another approach, Thal was investigated in a recombinant pyrrolnitrin producer strain [71]. Overexpression of thal yielded the new phenylpyrrole derivative, 3-(2´-amino-4´-chlorophenyl)pyrrole, but no pyrrolnitrin or aminopyrrolnitrin was detected. The authors concluded that the overproduction of Thal resulted in the chlorination of all the available tryptophan to 6-chlorotryptophan, which was then accepted by PrnB, the second enzyme in pyrrolnitrin biosynthesis catalyzing the subsequent ring rearrangement. However, the phenylpyrrole compound produced was not a substrate to PrnC for further halogenation reaction.

The use of tryptophan-5-halogenases (PyrH) (Table 19.2) in producing halogenated serotonin precursors has been the focus of further research [101]. The aim of these efforts was to obtain 5-bromotryptophan, which is regarded as a suitable intermediate for a subsequent nucleophilic hydroxylation to yield 5-hydroxytryptophan (oxitriptan). The latter is a component of many antidepressant drugs and, to date, commonly obtained by extraction of the seeds of the African plant Griffonia simplicifolia. The main limitation at this point is the low activity of the purified enzyme as well as the unsatisfactory production of the recombinant enzyme. Optimization of the reaction conditions, however, has already led to a nearly 20-fold improvement of the 5-bromotryptophan yield, and ongoing efforts aim to enhance the recombinant enzyme production [101]. The temperature sensitivity of the enzymes and the high NADH consumption to restore the cofactor FADH2 for the halogenase reaction, however, hamper an economical application of these enzymes [102].

19.5 Halogenating Enzymes Utilizing S-Adenosyl-l-Methionine

Fluoride is the most nucleophilic halogen and does not allow any electrophilic or radical biological chemistry, as is known for Cl, Br, and I. Even though, thermodynamically, the formation of carbon–fluoride bonds would be favorable, due to the extremely high desolvation energy required to activate F in aqueous solution, biological reactions with fluoride are rather rare [5]. Unique enzymes are needed to overcome this kinetic barrier, and, to date, the only one to be characterized is the 5´-fluoro-5´-deoxyadenosine synthetase (5´-FDAS). This enzyme was isolated from Streptomyces cattleya and utilizes S-adenosyl-l-methionine (SAM) as cosubstrate [7]. Fluoride is thereby thought to be placed in a hydrophobic pocket where desolvation is achieved as protein residues coordinate to water. In turn, a SN2 substitution of methionine at SAM takes place [103]. In this way, 5´-fluoro-5´-deoxyadenosine is formed as an intermediate and subsequently converted to fluoroacetate and 4-fluorothreonine (Fig. 19.16a).

For the biosynthesis of the potent proteasome inhibitor salinosporamide A (Fig. 19.16b) produced by the marine actinomycete Salinispora tropica, the deschloro analog, salinosporamide B, was initially presumed to be the direct precursor, and thus, the halogenation of the unactivated C-13 methyl group was suggested to be catalyzed by a nonheme iron halogenase. However, from feeding experiments, a different origin of the four-carbon moiety was demonstrated for these two molecules [104]. Thus, for salinosporamide B, a butyrate-derived ethylmalonyl-CoA was identified as a building block, whereas in salinosporamide A, an unprecedented PKS extender unit was shown to originate from a sugar precursor. The enzyme involved in the chlorination step (SalL) has been characterized as a 5´-FDAS similar enzyme, which converts SAM to 5´-chloro-5´-deoxyadenosine [6]. This sugar is subsequently converted to chloroethylmalonyl-CoA to further act as unique PKS extender unit [105]. The 5´-halo-5´-deoxyadenosine synthetase was shown to also be capable of utilizing bromide as well as iodide, but not fluoride.

Fluorine substitution is a prevalent strategy to enhance pharmacodynamic and pharmacokinetic properties of biologically active lead compounds; thus, the usefulness of the fluorinase 5´-FDAS to initiate C–F bonds in organohalogen synthesis has been further investigated. By genetic engineering, 5´-FDAS was successfully utilized in a salL-knockout mutant of S. tropica revealing fluorosalinosporamide [106]. Earlier studies, where this fluorometabolite was produced via chemical compensation of a salL-knockout mutant with synthetic 5´-fluoro-5´-deoxyadenosine, already showed that the proteasome inhibition activity of this salinosporamide derivative was slightly lower than that of the chlorinated natural product, however, also with reduced cytotoxicity [107]. This is thought to be due to a reversible binding of fluorosalinosporamide versus an irreversible binding of salinosporamide A. In further bioengineering approaches, the unique chloroethylmalonyl-CoA [105] and probably also analogous fluoroethylmalonyl-CoA [106] produced by salinosporamide A and respective genetically engineered biosynthetic systems may provide new opportunities in drug development. They may be used, e.g., as PKS extender units. Function of the fluorinase in isolated systems has already also been proven; thus, further application of this enzyme in in vitro biotransformation processes is conceivable [108].

SAM is also involved in the formation of monohalomethanes, albeit in a different way, namely, as methyl donor (Fig. 19.16c). At an early stage, Harper predicted that most likely other enzymes than haloperoxidases would be involved in the formation of halomethanes [109], and Wuosmaa and Hager were the first to detect and partially purify corresponding methyltransferases responsible for the methyl chloride production, e.g., from the red algae Endocladia muricata [110]. Several studies dealing with the emission of gaseous iodine and chlorine from the biosphere to the atmosphere resulted in the identification of respective enzymes in marine as well as terrestrial bacteria, macro- and microalgae, wood-rotting fungi, and terrestrial higher plants [111113].

19.6 Concluding Remarks

In the last years, our understanding of biohalogenation processes has extended tremendously. Several groups of halogenating enzymes have been discovered and investigated on the biochemical and genetic level. Each group of these enzymes performs halogenation reactions on chemically distinct and different substructures, i.e., indole, phenol, pyrrole, and aliphatic moieties, using a specific reaction mechanism. The halogenation of marine natural products, however, is still not as well investigated as that of terrestrial metabolites.

A detailed insight to natural biological halogenation processes will not only further our understanding of natural biological halogenation processes but will also give rise to alternative methods for generating halogenated analogs of biologically active metabolites [10, 85, 114, 115]. By means of such enzymatic reactions, modified compounds may be generated with potentially improved biological properties [95, 116118].

Investigations concerning the biotechnological potential of halogenating enzymes, however, are still in their infancy. Results obtained in the latter area so far prove that some halogenases can be expressed recombinantly and subsequently applied to directly halogenate suitable substrates, e.g., tryptophan halogenases [63, 71, 72, 100, 101]. Other halogenases only function in a multienzymatic biosynthetic environment, and combinatorial biosynthetic strategies need to be implemented to use their enzymatic capabilities.

For further details regarding biohalogenation beyond the scope of this book chapter, we recommend the comprehensive reviews based on the pioneering work of the research groups of van Pée and Walsh in the field of dioxygen-depending halogenases [64, 68, 85, 114, 118123]. The recently published comparison of the halide-binding sites of different halogenating enzymes and their corresponding selectivities are discussed by Blasiak and Drennan [117]. Further insights concerning the mechanisms of enzymatic halogenation were described by Dolfing [124], Anderson and Chapman [2], and Murphy [115], as well as Naismith [5]. In addition, reviews focusing on haloperoxidases have recently been published by Hofrichter and Ullrich [10], Butler and Carter-Franklin [8], and Raugei and Carloni [125], as well as Bortolini and Conte [126] and Winter and Moore [31].

19.7 Study Questions

  1. 1.

    Name the different halogenating enzymes known for natural product biosynthesis.

     
  2. 2.

    What are the cofactors utilized within haloperoxidases?

     
  3. 3.

    Which is the actual halogenation agent of haloperoxidases?

     
  4. 4.

    How do chloroperoxidases, bromoperoxidases, and iodoperoxidases differ?

     
  5. 5.

    Where are iodoperoxidases mainly found?

     
  6. 6.

    What is the halogenation strategy within nonheme FeII/α-ketoglutarate/O2-dependent halogenases?

     
  7. 7.

    Which are the structural elements halogenated by FeII/α-ketoglutarate/O2-dependent halogenases?

     
  8. 8.

    Which is the actual halogenation agent within FADH2 halogenases, and how is regioselective halogenation secured?

     
  9. 9.

    How are FADH2 halogenases further classified?

     
  10. 10.

    What is the function of SAM within the respective halogenating enzymes?

     
  11. 11.

    Why are fluorinated metabolites so rare in nature?

     

19.7.1 Answer–Keywords

  1. 1.

    Haloperoxidases, halogenases, S-adenosyl-l-methionine-utilizing enzymes

     
  2. 2.

    Heme or vanadate as cofactors

     
  3. 3.
    Hypohalous acid (HOX. or related halogenating intermediates, such as OX, X3−, and X+:
    • In heme HPO, the final chlorine transfer to various substrates occurs outside the active site via a free diffusible species without any special mode of substrate recognition.

    • Va-HPOs bind hydrogen peroxide and halides leading to a putative enzyme-bound or active site–trapped brominating moiety. In the absence of a suitable substrate, however, HOBr is released from the enzyme and carries out unselective bromination as observed for aqueous bromine.

     
  4. 4.

    Nomenclature is based on the most electronegative halide oxidized by the enzyme.

     
  5. 5.

    Va-IPOs seem to play a role in iodine accumulation of brown algae as a main vector of the iodine biogeochemical cycle.

     
  6. 6.

    A highly reactive iron-oxo species (FeIV = O) is formed within the catalytic cycle. This abstracts a hydrogen radical from an aliphatic carbon center of the actual substrate, which, in turn, abstracts the halide from the coordination sphere of the enzyme.

     
  7. 7.

    FeII/α-ketoglutarate/O2-dependent halogenase are involved in the chlorination of terminal methyl groups of amino acids linked to peptidyl carrier proteins and, occasionally, in the formation of cyclopropane moieties.

     
  8. 8.

    Enzyme-trapped HOCl is produced as chlorinating agent and channeled through an intraenzymatic tunnel to the substrate binding pocket. Here this chlorinating intermediate is proposed to be a covalently bound lysine chloramine (Lys79-εNH-Cl). Regioselective halogenation is thereby secured by controlled presentation of the substrate to the bound Cl-species via particular amino acid residues in the substrate binding site.

     
  9. 9.
    Halogenases can be grouped into:
    • Those accepting free small molecules and those that act on carrier-bound substrates, e.g., involved in multienzymatic NRPS/PKS assembly lines

    • Subdivided by the moieties to be chlorinated, i.e., aromatic compounds, recognizing tryptophan/indole or phenol/pyrrole moieties and enzymes involved in the halogenation of aliphatic compounds

     
  10. 10.
    Enzymes utilizing S-adenosyl-l-methionine (SAM) as cosubstrate:
    • Classically act in methyltransferases and, in this manner, are involved in the production of monohalomethanes.

    • Furthermore, in the unique enzyme 5´-FDA synthetase, substitution of methionine at SAM by fluoride takes place, and in turn, 5´-fluoro-5´-deoxyadenosine is formed as an intermediate (which is subsequently converted to fluoroacetate and 4-fluorothreonine). The chloro analog enzyme SalL converts SAM to 5´-chloro-5´-deoxyadenosine, which is subsequently converted to chloroethylmalonyl-CoA to further act as unique PKS extender unit.

     
  11. 11.
    Difficulties in the formation of fluorinated metabolites:
    • Extremely high desolvation energy required to activate F in aqueous solution makes biological reactions with fluoride rather rare.

    • Fluoride, as most nucleophilic halogens, does not allow any electrophilic or radical biological chemistry.

    • Unique enzymes are needed to overcome this kinetic barrier (e.g., hydrogen peroxide does not have the driving force to oxidize fluoride).

     

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© Springer Science+Business Media B.V. 2012
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