Non-haem Fe(II)/alpha-ketoglutarate (alphaKG)-dependent enzymes harness the reducing power of alphaKG to catalyse oxidative reactions, usually the hydroxylation of unactivated carbons, and are involved in processes such as natural product biosynthesis, the mammalian hypoxic response, and DNA repair. These enzymes couple the decarboxylation of alphaKG with the formation of a high-energy ferryl-oxo intermediate that acts as a hydrogen-abstracting species. All previously structurally characterized mononuclear iron enzymes contain a 2-His, 1-carboxylate motif that coordinates the iron. The two histidines and one carboxylate, known as the 'facial triad', form one triangular side of an octahedral iron coordination geometry. A subclass of mononuclear iron enzymes has been shown to catalyse halogenation reactions, rather than the more typical hydroxylation reaction. SyrB2, a member of this subclass, is a non-haem Fe(II)/alphaKG-dependent halogenase that catalyses the chlorination of threonine in syringomycin E biosynthesis. Here we report the structure of SyrB2 with both a chloride ion and alphaKG coordinated to the iron ion at 1.6 A resolution. This structure reveals a previously unknown coordination of iron, in which the carboxylate ligand of the facial triad is replaced by a chloride ion.

The flavin-dependent halogenase RebH catalyzes the formation of 7-chlorotryptophan as the initial step in the biosynthesis of antitumor agent rebeccamycin. The reaction of FADH2, Cl-, and O2 in the active site generates the powerful oxidant HOCl, which was presumed to carry out the chlorination reaction. Herein, we demonstrate the formation of a long-lived chlorinating intermediate (t1/2 = 63 h at 4 degrees C) when RebH, FADH2, Cl-, and O2 react in the absence of substrate tryptophan. This intermediate remained on the enzyme after removal of FAD and transferred chlorine to tryptophan with kinetically competent rates. The identity of this intermediate is suggested by the X-ray crystal structure of RebH, which revealed an active site Lys79 located in a central position between flavin and tryptophan binding sites and just 4.1 A above C7 of tryptophan. The chlorinating species is proposed to be a Lys-epsilonNH-Cl (lysine chloramine) from reaction of enzyme-generated HOCl with the active site Lys79. This covalent enzyme chloramine likely plays a key role in directing regiospecific chlorination of substrate in this important class of biosynthetic enzymes.

A fascinating feature of some bifunctional enzymes is the presence of an internal channel or tunnel to connect the multiple active sites. A channel can allow for a reaction intermediate generated at one active site to be used as a substrate at a second active site, without the need for the intermediate to leave the safety of the protein matrix. One such bifunctional enzyme is carbon monoxide dehydrogenase/acetyl-CoA synthase from Moorella thermoacetica (mtCODH/ACS). A key player in the global carbon cycle, CODH/ACS uses a Ni-Fe-S center called the C-cluster to reduce carbon dioxide to carbon monoxide and uses a second Ni-Fe-S center, called the A-cluster, to assemble acetyl-CoA from a methyl group, coenzyme A, and C-cluster-generated CO. mtCODH/ACS has been proposed to contain one of the longest enzyme channels (138 A long) to allow for intermolecular CO transport. Here, we report a 2.5 A resolution structure of xenon-pressurized mtCODH/ACS and examine the nature of gaseous cavities within this enzyme. We find that the cavity calculation program CAVENV accurately predicts the channels connecting the C- and A-clusters, with 17 of 19 xenon binding sites within the predicted regions. Using this X-ray data, we analyze the amino acid composition surrounding the 19 Xe sites and consider how the protein fold is utilized to carve out such an impressive interior passageway. Finally, structural comparisons of Xe-pressurized mtCODH/ACS with related enzyme structures allow us to study channel design principles, as well as consider the conformational flexibility of an enzyme that contains a cavity through its center.

Simple halogen substituents frequently afford key structural features that account for the potency and selectivity of natural products, including antibiotics and hormones. For example, when a single chlorine atom on the antibiotic vancomycin is replaced by hydrogen, the resulting antibacterial activity decreases by up to 70% (HarrisC. M.KannanR.KopeckaH.HarrisT. M.HarrisC. M.KannanR.KopeckaH.HarrisT. M.J. Am. Chem. Soc.198510766526658). This Account analyzes how structure underlies mechanism in halogenases, the molecular machines designed by nature to incorporate halogens into diverse substrates.Traditional synthetic methods of integrating halogens into complex molecules are often complicated by a lack of specificity and regioselectivity. Nature, however, has developed a variety of elegant mechanisms for halogenating specific substrates with both regio- and stereoselectivity. An improved understanding of the biological routes toward halogenation could lead to the development of novel synthetic methods for the creation of new compounds with enhanced functions. Already, researchers have co-opted a fluorinase from the microorganism Streptomyces cattleya to produce 18F-labeled molecules for use in positron emission tomography (PET) (DengH.CobbS. L.GeeA. D.LockhartA.MartarelloL.McGlincheyR. P.O’HaganD.OnegaM.DengH.CobbS. L.GeeA. D.LockhartA.MartarelloL.McGlincheyR. P.O’HaganD.OnegaM.Chem. Commun.2006652654). Therefore, the discovery and characterization of naturally occurring enzymatic halogenation mechanisms has become an active area of research.The catalogue of known halogenating enzymes has expanded from the familiar haloperoxidases to include oxygen-dependent enzymes and fluorinases. Recently, the discovery of a nucleophilic halogenase that catalyzes chlorinations has expanded the repertoire of biological halogenation chemistry (DongC.HuangF.DengH.SchaffrathC.SpencerJ. B.O’HaganD.NaismithJ. H.DongC.HuangF.DengH.SchaffrathC.SpencerJ. B.O’HaganD.NaismithJ. H.14765200Nature2004427561565). Structural characterization has provided a basis toward a mechanistic understanding of the specificity and chemistry of these enzymes. In particular, the latest crystallographic snapshots of active site architecture and halide binding sites have provided key insights into enzyme catalysis.Herein is a summary of the five classes of halogenases, focusing on the three most recently discovered: flavin-dependent halogenases, non-heme iron-dependent halogenases, and nucleophilic halogenases. Further, the potential roles of halide-binding sites in determining halide selectivity are discussed, as well as whether or not binding-site composition is always a seminal factor for selectivity. Expanding our understanding of the basic chemical principles that dictate the activity of the halogenases will advance both biology and chemistry. A thorough mechanistic analysis will elucidate the biological principles that dictate specificity, and the application of those principles to new synthetic techniques will expand the utility of halogenations in s...