Halogenases: structures and functions
Graphical abstract
Halogenases: diverse structures mediating distinctive chemistries.
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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Cited by (17)
Structural and functional insights into the self-sufficient flavin-dependent halogenase
2024, International Journal of Biological MacromoleculesExpression, purification and structure determination of the chlorinase ClA2
2022, Biochemical and Biophysical Research CommunicationsCitation Excerpt :In contrast to chemical synthesis, the biochemical halogenation, like halogenase, is environmental friendly and reacts with high efficiency under mild and aqueous conditions [3]. Based on the catalytic mechanism, the bio-halogenase enzyme could be classified into three groups: electrophilic halogenase, nucleophilic halogenase, and radical halogenase [4,5]. For example, several S-Adenosyl-l-methionine (SAM)-dependent nucleophilic halogenating enzymes have been identified, which can catalyze fluoride/chloride-dependent nucleophilic displacement of l-methionine from SAM to generate 5′-fluoro-5′-deoxyadenosine(5′-FAD)/5′-chloro-5′-deoxyadenosine (5′-ClDA) and l-methionine(L-Met) [6–9].
Recent development of biomimetic halogenation inspired by vanadium dependent haloperoxidase
2022, Coordination Chemistry ReviewsCitation Excerpt :During the catalytic cycle of VBPO, all of Br+, HOBr, Br3+, enzyme-Br+ and enzyme-OBr (V-OBr) could possibly involve [59] (Scheme 3B). Since the radical engages in reactions catalyzed by Heme-HPO [18,21–23], it was rational to hypothesize that similar mechanism might engage in halogenations catalyzed by VHPO. However, based on the electron paramagnetic resonance (EPR) experiments, unlike the Heme-HPO, the redox of vanadium in An-VBPO and Ci-VCPO [42] remained untouched [60].
Brevianthrones, bianthrones from a Chinese isolate of the endophytic fungus Colletotrichum brevisporum
2021, PhytochemistryCitation Excerpt :The fungus was grown in the presence of NaCl, NaBr and KI (2%), however, according to the HPLC analysis, no new peaks were detected compared to the controls, suggesting that this fungus lacked a suitable halogenase capable of producing halogenated bianthrones. Halogenase enzymes are common in microorganisms isolated from marine environments (Ludewig et al., 2020), where higher levels of halide salts are present. Thus, the difference in biosynthetic capability of the two fungi might be attributed to the different environments from which they were isolated.
- 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.