The ␣-ketoglutarate-dependent hydroxylases and halogenases employ similar reaction mechanisms involving hydrogen-abstracting Fe(IV)-oxo (ferryl) intermediates. In the halogenases, the carboxylate residue from the His 2(Asp/Glu)1''facial triad'' of iron ligands found in the hydroxylases is replaced by alanine, and a halide ion (X ؊ ) coordinates at the vacated site. Halogenation is thought to result from ''rebound'' of the halogen radical from the X-Fe ( ␣-ketoglutarate ͉ ferryl ͉ hydroxylase ͉ nonheme iron ͉ radical rebound T he ␣-ketoglutarate-dependent oxygenases activate O 2 at mononuclear Fe(II) cofactors, cleave ␣-ketoglutarate (␣KG) to CO 2 and succinate, and oxidize their substrates by two electrons (1-4). The most extensively studied members of the family are hydroxylases. A mechanism for the (pro)collagen-modifying prolyl-4-hydroxylase (P4H) originally proposed by Hanauske-Abel and Günzler (5) has accounted well for ensuing experimental data on multiple enzymes in the family. Its central tenets are the abstraction of a hydrogen atom (H • ) from the substrate by an Fe(IV)-oxo (ferryl) complex (Scheme 1A, black arrows) and the subsequent ''rebound'' of the coordinated hydroxyl radical to the substrate radical (red arrows) (6). The most compelling evidence for this mechanism was provided by the detection and spectroscopic characterization of the ferryl intermediates in taurine:␣KG dioxygenase (TauD) from Escherichia coli and a P4H from Paramecium bursaria Chlorella virus 1 (7, 8). The small Mössbauer isomer shifts (Ϸ0.3 mm/s) and S ϭ 2 electron-spin ground states of the freeze-trapped intermediates marked them as high-spin Fe(IV) complexes; the large substrate deuterium (D) kinetic isotope effects (KIEs) on their decay (k H /k D Ϸ 50) showed that they abstract hydrogen from the substrates (8, 9); and resonance Raman and X-ray absorption spectroscopic data on the TauD complex proved that it contains the ferryl unit (10, 11).Before the recent discovery of the aliphatic halogenases (12), a group of ␣KG-dependent oxygenases that synthesize precursors for assembly of halogen-containing natural products by nonribosomal peptide synthetases (NRPSs), iron coordination by a conserved 2-histidine-1-carboxylate ''facial triad'' had been considered a defining feature of the family (13). Sequence analysis and the crystal structure of SyrB2 (14), the halogenase that supplies the 4-chloro-L-threonine fragment incorporated into syringomycin E (a phytotoxin produced by Pseudomonas syringae B301D) (12), revealed that it lacks the carboxylate ligand, having an Ala residue where the contributing Asp or Glu would normally be found (Scheme 1 A). The observation that a halide ion (X Ϫ ) coordinates to the Fe(II) cofactor at the vacated site suggested a related mechanism for the halogenase reaction involving abstraction of H • from the substrate by the oxo group of a haloferryl intermediate and rebound of the coordinated halogen radical from the resultant X-Fe(III)-OH complex to the substrate radical (green arrows). Detection and ...

Methylation of small molecules and macromolecules is crucial in metabolism, cell signaling, and epigenetic programming and is most often achieved by S-adenosylmethionine (SAM)-dependent methyltransferases. Most employ an S(N)2 mechanism to methylate nucleophilic sites on their substrates, but recently, radical SAM enzymes have been identified that methylate carbon atoms that are not inherently nucleophilic via the intermediacy of a 5'-deoxyadenosyl 5'-radical. We have determined the mechanisms of two such reactions targeting the sp(2)-hybridized carbons at positions 2 and 8 of adenosine 2503 in 23S ribosomal RNA, catalyzed by RlmN and Cfr, respectively. In neither case is a methyl group transferred directly from SAM to the RNA; rather, both reactions proceed by a ping-pong mechanism involving intermediate methylation of a conserved cysteine residue.

Viral infections continue to represent major challenges to public health, and an enhanced mechanistic understanding of the processes that contribute to viral life cycles is necessary for the development of new therapeutic strategies . Viperin, a member of the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes, is an interferon-inducible protein implicated in the inhibition of replication of a broad range of RNA and DNA viruses, including dengue virus, West Nile virus, hepatitis C virus, influenza A virus, rabies virus and HIV. Viperin has been suggested to elicit these broad antiviral activities through interactions with a large number of functionally unrelated host and viral proteins. Here we demonstrate that viperin catalyses the conversion of cytidine triphosphate (CTP) to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), a previously undescribed biologically relevant molecule, via a SAM-dependent radical mechanism. We show that mammalian cells expressing viperin and macrophages stimulated with IFNα produce substantial quantities of ddhCTP. We also establish that ddhCTP acts as a chain terminator for the RNA-dependent RNA polymerases from multiple members of the Flavivirus genus, and show that ddhCTP directly inhibits replication of Zika virus in vivo. These findings suggest a partially unifying mechanism for the broad antiviral effects of viperin that is based on the intrinsic enzymatic properties of the protein and involves the generation of a naturally occurring replication-chain terminator encoded by mammalian genomes.

The gut microbiota synthesize hundreds of molecules, many of which are known to impact host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), which accumulate at ~500 µM and are known to block C. difficile growth 1 , promote hepatocellular carcinoma 2 , and modulate host metabolism via the GPCR TGR5 3 . More broadly, DCA, LCA and their derivatives are a major component of the recirculating bile acid pool 4 ; the size and composition of this pool are a target of therapies for primary biliary cholangitis and nonalcoholic steatohepatitis. Despite the clear impact of DCA and LCA on host physiology, incomplete knowledge of their biosynthetic genes and a lack of genetic tools in their native producer limit our ability to modulate secondary bile acid levels in the host. Here, we complete the pathway to DCA/LCA by assigning and characterizing enzymes for each of the steps in its reductive arm, revealing a strategy in which the A-B rings of the steroid core are transiently converted into an electron acceptor for two reductive steps carried out by Fe-S flavoenzymes. Using anaerobic in vitro reconstitution, we establish that a set of six enzymes is necessary and sufficient for the 8-step conversion of cholic acid to DCA. We then engineer the pathway into Clostridium sporogenes, conferring production of DCA and LCA on a non-producing commensal and demonstrating that a microbiome-derived pathway can be expressed and controlled heterologously. These data establish a complete pathway to two central components of the bile acid pool, and provide a road map for deorphaning and engineering pathways from the microbiome as a critical step toward controlling the metabolic output of the gut microbiota.

Arylsulfatases require a maturating enzyme to perform a co-or posttranslational modification to form a catalytically essential formylglycine (FGly) residue. In organisms that live aerobically, molecular oxygen is used enzymatically to oxidize cysteine to FGly. Under anaerobic conditions, S-adenosylmethionine (AdoMet) radical chemistry is used. Here we present the structures of an anaerobic sulfatase maturating enzyme (anSME), both with and without peptidyl-substrates, at 1.6-1.8 Å resolution. We find that anSMEs differ from their aerobic counterparts in using backbone-based hydrogen-bonding patterns to interact with their peptidylsubstrates, leading to decreased sequence specificity. These anSME structures from Clostridium perfringens are also the first of an AdoMet radical enzyme that performs dehydrogenase chemistry. Together with accompanying mutagenesis data, a mechanistic proposal is put forth for how AdoMet radical chemistry is coopted to perform a dehydrogenation reaction. In the oxidation of cysteine or serine to FGly by anSME, we identify D277 and an auxiliary [4Fe-4S] cluster as the likely acceptor of the final proton and electron, respectively. D277 and both auxiliary clusters are housed in a cysteinerich C-terminal domain, termed SPASM domain, that contains homology to ∼1,400 other unique AdoMet radical enzymes proposed to use [4Fe-4S] clusters to ligate peptidyl-substrates for subsequent modification. In contrast to this proposal, we find that neither auxiliary cluster in anSME bind substrate, and both are fully ligated by cysteine residues. Instead, our structural data suggest that the placement of these auxiliary clusters creates a conduit for electrons to travel from the buried substrate to the protein surface.iron-sulfur cluster fold | radical SAM dehydrogenase