Naturally produced halogenated compounds are ubiquitous across all domains of life where they perform a multitude of biological functions and adopt a diversity of chemical structures. Accordingly, a diverse collection of enzyme catalysts to install and remove halogens from organic scaffolds has evolved in nature. Accounting for the different chemical properties of the four halogen atoms (fluorine, chlorine, bromine, and iodine) and the diversity and chemical reactivity of their organic substrates, enzymes performing biosynthetic and degradative halogenation chemistry utilize numerous mechanistic strategies involving oxidation, reduction, and substitution. Biosynthetic halogenation reactions range from simple aromatic substitutions to stereoselective C-H functionalizations on remote carbon centers and can initiate the formation of simple to complex ring structures. Dehalogenating enzymes, on the other hand, are best known for removing halogen atoms from man-made organohalogens, yet also function naturally, albeit rarely, in metabolic pathways. This review details the scope and mechanism of nature’s halogenation and dehalogenation enzymatic strategies, highlights gaps in our understanding, and posits where new advances in the field might arise in the near future.

Nature has developed an exquisite array of methods to introduce halogen atoms into organic compounds. Most of these enzymes are oxidative and require either hydrogen peroxide or molecular oxygen as a cosubstrate to generate a reactive halogen atom for catalysis. Vanadium-dependent haloperoxidases contain a vanadate prosthetic group and utilize hydrogen peroxide to oxidize a halide ion into a reactive electrophilic intermediate. These metalloenzymes have a large distribution in nature, where they are present in macroalgae, fungi, and bacteria, but have been exclusively characterized in eukaryotes. In this minireview, we highlight the chemistry and biology of vanadium-dependent haloperoxidases from fungi and marine algae and the emergence of new bacterial members that extend the biological function of these poorly understood halogenating enzymes. VanadiumVanadium is a trace element that is widely distributed in nature. After molybdenum, vanadium is the second most abundant transition metal in the ocean, with a concentration of 35-50 nM (1) and up to 100 mg/kg in carbon-containing sediments of marine origin. In fresh water, the concentration is reported as 1.3 g/liter (50 nM), and in the Earth's crust, vanadium is present at 100 ppm (1, 2). Vanadium exists in many oxidation states, with V(V) being the most common in sea water (1-3). Only the V(III), V(IV), and V(V) oxidation states are involved, however, in biological systems, where vanadium has limited distribution as an essential mineral in organisms such as sea squirts and mushrooms and as a cofactor in metalloenzymes. The most prevalent form of vanadium at neutral pH is the oxyanion vanadate, which is an oxidizing agent that is structurally and electronically similar to phosphate (1-3). Hence, vanadate and vanadate derivatives have been employed to interrogate a range of enzymes that interact with phosphorylated substrates (3). Interestingly, acid phosphatase enzymes have evolved to accommodate vanadate as a redox cofactor (4, 5). Vanadium-containing EnzymesTo date, two classes of vanadium-containing enzymes have been identified: vanadium nitrogenases and vanadium-dependent haloperoxidases (V-HPOs).2 Nitrogenases are utilized by nitrogen-fixing bacteria to reduce dinitrogen to ammonia. Although this metalloenzyme system commonly contains a molybdenum-iron cofactor, some bacteria produce additional nitrogenases that are genetically distinct and instead contain V-Fe or Fe-Fe central metals (6 -8). Vanadium nitrogenases have been identified from a diverse group of diazotrophic microorganisms and are synthesized under molybdenum-limiting conditions. On the other hand, V-HPOs have a larger distribution in nature, where they are present in macroalgae, fungi, and bacteria (1, 4 -5, 9). These enzymes, which contain a ligated vanadate ion, oxidize halide ions to their corresponding hypohalous acids at the expense of hydrogen peroxide and are classified by the most electronegative halide they oxidize. Thus, vanadium chloroperoxidases (V-ClPOs) oxidize chloride, bromide, ...

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With an ever-increasing amount of (meta)genomic data being deposited in sequence databases, (meta)genome mining for natural product biosynthetic pathways occupies a critical role in the discovery of novel pharmaceutical drugs, crop protection agents and biomaterials. The genes that encode these pathways are often organised into biosynthetic gene clusters (BGCs). In 2015, we defined the Minimum Information about a Biosynthetic Gene cluster (MIBiG): a standardised data format that describes the minimally required information to uniquely characterise a BGC. We simultaneously constructed an accompanying online database of BGCs, which has since been widely used by the community as a reference dataset for BGCs and was expanded to 2021 entries in 2019 (MIBiG 2.0). Here, we describe MIBiG 3.0, a database update comprising large-scale validation and re-annotation of existing entries and 661 new entries. Particular attention was paid to the annotation of compound structures and biological activities, as well as protein domain selectivities. Together, these new features keep the database up-to-date, and will provide new opportunities for the scientific community to use its freely available data, e.g. for the training of new machine learning models to predict sequence-structure-function relationships for diverse natural products. MIBiG 3.0 is accessible online at https://mibig.secondarymetabolites.org/.

Structural inspection of the bacterial meroterpenoid antibiotics belonging to the napyradiomycin family of chlorinated dihydroquinones suggests that the biosynthetic cyclization of their terpenoid subunits is initiated via a chloronium ion. The vanadium-dependent haloperoxidases that catalyze such reactions are distributed in fungi and marine algae and have yet to be characterized from bacteria. The cloning and sequence analysis of the 43-kb napyradiomycin biosynthetic cluster (nap) from Streptomyces aculeolatus NRRL 18422 and from the undescribed marine sediment-derived Streptomyces sp. CNQ-525 revealed 33 open reading frames, three of which putatively encode vanadium-dependent chloroperoxidases. Heterologous expression of the CNQ-525-based nap biosynthetic cluster in Streptomyces albus produced at least seven napyradiomycins, including the new analog 2-deschloro-2-hydroxy-A80915C. These data not only revealed the molecular basis behind the biosynthesis of these novel meroterpenoid natural products but also resulted in the first in vivo verification of vanadium-dependent haloperoxidases.Nature has devised several mechanisms to polarize the terminal olefin of linear terpenes to facilitate the creation of new C-X bonds. For instance, cyclization of the C 30 hydrocarbon squalene to steroids and hopanoids is initiated, respectively, by epoxidation or protonation of the terminal olefin. Although these biosynthetic strategies are widely distributed, a third mechanism for terpene cyclization has been characterized in marine macroalgae involving bromonium ion-induced ring closure (1-3). Oxidation of the halide is catalyzed by vanadiumdependent bromoperoxidase in the presence of hydrogen peroxide to produce the corresponding hypohalous acid. This species then further reacts with electron-rich organic substrates in a regio-and stereoselective manner, giving rise to brominated terpenes and other halogenated natural products (1, 2, 4). Vanadium-dependent bromoperoxidases are widely distributed in marine algae, and the first enzyme was discovered in 1984 from the brown alga Ascophyllum nodosum (5).Vanadium chloroperoxidases (V-ClPOs), 3 on the other hand, have been isolated primarily from dematiaceous hyphomycete fungi (1). The first enzyme was characterized in 1993 from Curvularia inaequalis (6), and even though there are numerous chlorinated marine natural products, V-ClPOs have not been reported from marine organisms to date (1, 2, 7). Although the biological function of V-ClPOs has not yet been elucidated, marine algal vanadium-dependent bromoperoxidases have been shown through in vitro chemoenzymatic conversions to catalyze bromonium ion-initiated cyclization of terpenes and ethers (1,8). These studies not only demonstrated that the enzymes were able to initiate cyclization of a terpene by a bromonium ion but also proved that the halogenation reaction occurred with stereochemical control. To date, all known V-dependent haloperoxidases have been characterized in vitro from eukaryotic systems (9).Structural inspection of...