Halogenases: structures and functions

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Over 5000 halogenated natural products have been reported so far, many of these arising from the marine environment. The introduction of a halogen into a molecule can significantly impact its bioavailability and bioactivity. More recently enzymatic halogenation has been used to enable late stage functionalisation through site-selective halogenation and cross-coupling. Halogenases are becoming increasingly valued tools. This review outlines the various classes of halogenases that have been discovered, and examines these from both a structural and a mechanistic perspective, reflecting upon the many recent advances in halogenase discovery.

Graphical abstract

Halogenases: diverse structures mediating distinctive chemistries.

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Introduction

Halogenation of molecules can significantly impact their activity and bioavailability [1,2] and is a strategy employed by both nature, and in bioactive molecule design, with 27% of small molecule drugs and more than 80% of agrochemicals bearing a halogen [3,4]. Additionally, incorporation of Cl, Br, or I provides chemically reactive and orthogonal handles for selective modification through cross-coupling chemistry [5]. Today, over 5000 halometabolites, predominantly chlorinated and brominated metabolites, have been identified [6]. Though more rare, approximately 100 iodinated natural metabolites and 6–13 natural, fluorinated metabolites have been found [6]. This plethora of halometabolites show exciting structural diversity indicating the operation of a large variety of powerful enzymes [7].

Though initially biosynthetic halogenation was considered an artefact, the first enzyme capable of halogenation was discovered by Lowell Hager et al. in 1959 [8••]. This enzyme was named chloroperoxidase (CPO), a haloperoxidase, from the fungus Calariomyces fumago [9]. For several decades the haloperoxidases remained the only halogenases known [9]. However, the turn of the millennium corresponded to a significant acceleration in this research field, largely fuelled by investigations into the biogenesis of natural products from bacteria, fungi and plants. In the last 5 years, bioinformatics led approaches have enabled the identification of halogenases, even from viruses [10••,11••].

Halogenases may be broadly classified by their mechanisms: electrophilic, nucleophilic, or radical, and may be further subdivided according to their catalytic species. The vast majority of halogenases studied so far employ electrophilic halogenation, enabling incorporation of Cl, Br, and I [7]; in recent years increasing numbers of radical halogenases have been identified [12,13,14]. Nucleophilic halogenases remain the most rare [15,16].

Here we overview the enzymatic mechanisms for each class of halogenase, reflecting upon the structures of the enzymes that mediate these chemistries.

Section snippets

Nucleophilic halogenases

A small handful of nucleophilic halogenases are known so far, the first of these, and the most well studied is the fluorinase or 5′-fluoro-5′-deoxyadenosine synthase (5′-FDAS) from Streptomyces cattleya [17]. Though fluorine is the most abundant halogen in the earth’s crust, and the 13th most abundant element, few natural organofluorine compounds exist. This is perhaps dominated by three factors: the poor solubility of fluoride salts, the high enthalpy of hydration of fluoride (490 kJ mol−1) and

Radical halogenases

All known enzymatic radical halogenation reactions are catalysed by non-heme-iron α-ketoglutarate (KG)-dependent enzymes; proteins which are able to selectively halogenate an unactivated aliphatic carbon centre. All Fe(II)/α-KG halogenation proceeds by the same general mechanism. The enzyme resting state (Figure 2a. I) generally consists of two active site histidine residues, a bidentate α-KG, a halide anion, and a weakly bound water (which is displaced by substrate (Figure 2a. II))

Haloperoxidases

Haloperoxidases (HPOs) are often considered as two main classes, in accordance with the prosthetic groups utilised in the active site: haem iron haloperoxidases (such as CPO), and vanadium-dependent haloperoxidases (V-HPOs). The mechanism of halogenation employed by these enzymes is similar; both (generally) are considered to produce free hypohalous acid, HOX, which reacts with the substrate. In the case of the haem-dependent haloperoxidases, resting state haem (Figure 3a. I) reacts with H2O2, (

Concluding thoughts

As indicated by compound structural diversity, so far only a very small proportion of existing halogenases have been explored. In silico approaches for the discovery of novel halogenases enable the identification of wildly different enzymes. Using such approaches, major advances have been seen in recent years including discovery of radical and FDH halogenases with biotechnologically interesting substrate scope.

Though the field is progressing rapidly, numerous exciting central questions remain

Conflict of interest statement

Nothing declared.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We thank ERC GenoChemetics (FP7/2007-2013/ERC consolidator grant GCGXC grant agreement no 614779 RJMG) for funding, CSC and EPSRC CRITICAT EP/L016419/1 for studentship support (KG & SM respectively).

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    1

    Present address: Department of Biochemistry, Brandeis University, Waltham, MA 02454, USA.

    2

    Present address: Howard Huges Medical Institute, Brandeis University, Waltham, MA 02454, USA.

    3

    Present Address: School of Molecular and Cellular Biology, University of Leeds, Leeds, Yorkshire, LS3 9JT, UK.

    4

    These authors contributed equally.

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