Radical SAM Research Articles

Activation of Glycyl Radical Enzymes─Multiscale Modeling Insights into Catalysis and Radical Control in a Pyruvate Formate-Lyase-Activating Enzyme

Pyruvate formate-lyase (PFL) is a glycyl radical enzyme (GRE) playing a pivotal role in the metabolism of strict and facultative anaerobes. Its activation is carried out by a PFL-activating enzyme, a member of the radical S-adenosylmethionine (rSAM) superfamily of metalloenzymes, which introduces a glycyl radical into the Gly radical domain of PFL. The activation mechanism is still not fully understood and is structurally based on a complex with a short model peptide of PFL. Here, we present extensive molecular dynamics simulations in combination with quantum mechanics/molecular mechanics (QM/MM)-based kinetic and thermodynamic reaction evaluations of a more complete activation model comprising the 49 amino acid long C-terminus region of PFL. We reveal the benefits and pitfalls of the current activation model, providing evidence that the bound peptide conformation does not resemble the bound protein–protein complex conformation with PFL, with implications for the activation process. Substitution of the central glycine with (S)- and (R)-alanine showed excellent binding of (R)-alanine over unstable binding of (S)-alanine. Radical stabilization calculations indicate that a higher radical stability of the glycyl radical might not be the sole origin of the evolutionary development of GREs. QM/MM-derived radical formation kinetics further demonstrate feasible activation barriers for both peptide and C-terminus activation, demonstrating why the crystalized model peptide system is an excellent inhibitory system for natural activation. This new evidence supports the theory that GREs converged on glycyl radical formation due to the better conformational accessibility of the glycine radical loop, rather than the highest radical stability of the formed peptide radicals.

A Cobalamin-Dependent Radical SAM Enzyme Catalyzes the Unique Cα -Methylation of Glutamine in Methyl-Coenzyme M Reductase.

Methyl‐coenzyme M reductase, which is responsible for the production of the greenhouse gas methane during biological methane formation, carries several unique posttranslational amino acid modifications, including a 2‐(S)‐methylglutamine. The enzyme responsible for the Cα‐methylation of this glutamine is not known. Herein, we identify and characterize a cobalamin‐dependent radical SAM enzyme as the glutamine C‐methyltransferase. The recombinant protein from Methanoculleus thermophilus binds cobalamin in a base‐off, His‐off conformation and contains a single [4Fe‐4S] cluster. The cobalamin cofactor cycles between the methyl‐cob(III)alamin, cob(II)alamin and cob(I)alamin states during catalysis and produces methylated substrate, 5′‐deoxyadenosine and S‐adenosyl‐l‐homocysteine in a 1 : 1 : 1 ratio. The newly identified glutamine C‐methyltransferase belongs to the class B radical SAM methyltransferases known to catalyze challenging methylation reactions of sp3‐hybridized carbon atoms.

CircEZH2 inhibits opening of mitochondrial permeability transition pore via interacting with PiC and up-regulating RSAD2

Transmissible gastroenteritis virus (TGEV) infection can lead to mitochondrial damage in porcine intestinal epithelial cells-jejunum 2 (IPEC-J2) cell line. The abnormal opening of mitochondrial permeability transition pore (mPTP) is the most important factor for mitochondrial damage. We previously demonstrated that circEZH2 could inhibit the abnormal opening of mPTP by binding miR-22. However, circEZH2 binding to miR-22 cannot completely enable mPTP opening to recover to normal level compared with the control group. So, we assume that circEZH2 also regulates the mPTP opening in other ways. To prove it, we identified the differentially expressed proteins (DEPs) caused by circEZH2 and circEZH2-interacting proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). It turns out there are 123 DEPs (0.83 ≤ fold change ≥ 1.2) upon overexpression circEZH2 and 200 proteins interacted with circEZH2. The kyoto encyclopedia of genes and genomes (KEGG) analysis, gene ontology (GO) analysis, subcellular localization analysis, and protein interaction network results show that the DEPs and circEZH2-interacting proteins may involve in the regulation of mPTP opening. RNA immunoprecipitation (RIP) assay and flow cytometry (FCM) results indicate that circEZH2 can inhibit the opening of mPTP by interacting with Pi carrier (PiC, also named SLC25A3). Quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and FCM results reveal that circEZH2 can inhibit mPTP opening by promoting the expression of radical s-adenosyl methionine domain-containing protein 2 (RSAD2). In addition, PiC can promote RSAD2 expression. The data indicate that circEZH2 inhibits TGEV-induced mPTP opening by interacting with PiC and upregulating RSAD2.

Intratracheal instillation of graphene oxide decreases anti-virus responses and lipid contents via suppressing Toll-like receptor 3 in mouse livers.

Recent studies revealed a causal relationship between Toll-like receptors (TLRs) and lipid droplet biogenesis. Interestingly, it has been reported before that nanomaterials (NMs) were capable to modulate TLRs, but it remains unclear if NMs could affect lipid levels via TLR signaling pathways. In this study, we investigated the influences of airway exposure to graphene oxide (GO) on TLR3 signaling pathways and lipid levels in mouse livers. Intratracheal instillation of GO (0.1, 1, and 5mg/kg, once a day, totally 5days) induced inflammatory cell infiltrations as indicated by hematoxylin-eosin (H&E) staining and fibrosis as indicated by Masson staining in lungs, accompanying with decreased TLR3 proteins. Consistently, a TLR3-regulated anti-virus protein, namely interferon induced protein with tetratricopeptide repeats 1 (IFIT1), as well as two TLR3-regulated lipid proteins, namely radical S-adenosyl methionine domain containing 2 (RSAD2) and perilipin 2 (PLIN2), were decreased in lungs. The protein levels of interferon-β in serum were also decreased. In livers, GO exposure induced disorganization of liver cells but not fibrosis. In agreement with the trends observed in lungs, TLR3, IFIT1, RSAD2, and PLIN2 proteins were decreased in livers. As a possible consequence, GO exposure dose-dependently decreased lipid levels in livers as indicated by oil red O and BODIPY 493/503 staining. We concluded that airway exposure to GO decreased anti-virus responses and lipid levels in mouse livers via the suppression of TLR3.

The Biosynthetic Landscape of Triceptides Reveals Radical SAM Enzymes That Catalyze Cyclophane Formation on Tyr- and His-Containing Motifs.

Peptide-derived cyclophanes inhabit a unique niche in the chemical space of macrocyclic peptides with several examples of pharmaceutical importance. Although both synthetic and biocatalytic methods are available for constructing these macrocycles, versatile (bio)catalysts able to incorporate a variety of amino acids that compose the macrocycle would be useful for the creation of diverse peptide cyclophanes. In this report, we synergized the use of bioinformatic tools to map the biosynthetic landscape of radical SAM enzymes (3-CyFEs) that catalyze three-residue cyclophane formation in the biosynthesis of a new family of RiPP natural products, the triceptides. This analysis revealed 3940 (3113 unique) putative precursor sequences predicted to be modified by 3-CyFEs. Several uncharacterized maturase systems were identified that encode unique precursor types. Functional studies were carried out in vivo in Escherichia coli to identify modified precursors containing His and Tyr residues. NMR analysis of the products revealed that Tyr and His can also be incorporated into cyclophane macrocycles by 3-CyFEs. Collectively, all aromatic amino acids can be incorporated by 3-CyFEs, and the cyclophane formation strictly occurs via a C(sp2)-C(sp3) cross-link between the (hetero)aromatic ring to Cβ. In addition to 3-CyFEs, we functionally validated an Fe(II)/α-ketoglutarate-dependent hydroxylase, resulting in β-hydroxylated residues within the cyclophane rings. This study reveals the potential breadth of triceptide precursors and a systematic approach for studying these enzymes to broaden the diversity of peptide macrocycles.

In Vitro Demonstration of Human Lipoyl Synthase Catalytic Activity in the Presence of NFU1.

Lipoyl synthase (LS) catalyzes the last step in the biosynthesis of the lipoyl cofactor, which is the attachment of sulfur atoms at C6 and C8 of an n-octanoyllysyl side chain of a lipoyl carrier protein (LCP). The protein is a member of the radical S-adenosylmethionine (SAM) superfamily of enzymes, which use SAM as a precursor to a 5′-deoxyadenosyl 5′-radical (5′-dA·). The role of the 5′-dA· in the LS reaction is to abstract hydrogen atoms from C6 and C8 of the octanoyl moiety of the substrate to initiate subsequent sulfur attachment. All radical SAM enzymes have at least one [4Fe–4S] cluster that is used in the reductive cleavage of SAM to generate the 5′-dA·; however, LSs contain an additional auxiliary [4Fe–4S] cluster from which sulfur atoms are extracted during turnover, leading to degradation of the cluster. Therefore, these enzymes catalyze only 1 turnover in the absence of a system that restores the auxiliary cluster. In Escherichia coli, the auxiliary cluster of LS can be regenerated by the iron–sulfur (Fe–S) cluster carrier protein NfuA as fast as catalysis takes place, and less efficiently by IscU. NFU1 is the human ortholog of E. coli NfuA and has been shown to interact directly with human LS (i.e., LIAS) in yeast two-hybrid analyses. Herein, we show that NFU1 and LIAS form a tight complex in vitro and that NFU1 can efficiently restore the auxiliary cluster of LIAS during turnover. We also show that BOLA3, previously identified as being critical in the biosynthesis of the lipoyl cofactor in humans and Saccharomyces cerevisiae, has no direct effect on Fe–S cluster transfer from NFU1 or GLRX5 to LIAS. Further, we show that ISCA1 and ISCA2 can enhance LIAS turnover, but only slightly.

Substrate specificity and reaction directionality of a three-residue cyclophane forming enzyme PauB

Three-residue cyclophane-forming enzymes (3-CyFEs) are a group of radical S-adenosylmethionine (SAM) enzymes involved in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs). 3-CyFE catalyzes the crosslinking between an aromatic residue (Ω1) and a non-aromatic residue (X3) in a Ω1-X2-X3 motif to produce a cyclophane ring, a key step in the biosynthesis of the RiPP natural product triceptide. In this study, we perform a genome-wide search for the Xye-type triceptides, showing these RiPPs are likely class-specific and only present in gamma-proteobacteria. The 3-CyFE PauB from Photorhabdus australis exhibits a relaxed substrate specificity on the X3 position, but glycine in this position is not suitable for cyclophane formation. We also reconstituted the activity of PauB in vitro, showing it produces the N-terminal cyclophane firstly, and then the C-terminal ring, whereas the middle cyclophane is produced in the last step.

Dioxane Bridge Formation during the Biosynthesis of Spectinomycin Involves a Twitch Radical S-Adenosyl Methionine Dehydrogenase That May Have Evolved from an Epimerase.

Spectinomycin is a dioxane-bridged, tricyclic aminoglycoside produced by Streptomyces spectabilis ATCC 27741. While the spe biosynthetic gene cluster for spectinomycin has been reported, the chemistry underlying construction of the dioxane ring is unknown. The twitch radical SAM enzyme SpeY from the spe cluster is shown here to catalyze dehydrogenation of the C2' alcohol of (2'R,3'S)-tetrahydrospectinomycin to yield (3'S)-dihydrospectinomycin as a likely biosynthetic intermediate. This reaction is radical-mediated and initiated via H atom abstraction from C2' of the substrate by the 5'-deoxyadenosyl radical equivalent generated upon reductive cleavage of SAM. Crystallographic analysis of the ternary Michaelis complex places serine-183 adjacent to C2' of the bound substrate opposite C5' of SAM. Mutation of this residue to cysteine converts SpeY to the corresponding C2' epimerase mirroring the opposite phenomenon observed in the homologous twitch radical SAM epimerase HygY from the hygromycin B biosynthetic pathway. Phylogenetic analysis suggests a relatively recent evolutionary branching of putative twitch radical SAM epimerases bearing homologous cysteine residues to generate the SpeY clade of enzymes.

Consecutive Methylation Catalyzed by TsrM, an Atypical Class B Radical SAM Methylase

Comprehensive SummaryTsrM is a cobalamin‐dependent radical S‐adenosylmethionine (SAM) methyltransferase belonging to the Class B radical SAM methylase (RSM) family. This enzyme catalyzes the C‐2 methylation of L‐tryptophan to produce 2‐methyltrytophan (2‐MeTrp), an intermediate involved in the biosynthesis of thiostrepton A. In this work, we report characterization of an unexpected activity of TsrM, which carries out an additional methylation reaction on the product 2‐MeTrp. A series of isotopic labeling studies and assays with different Trp analogs revealed that TsrM is able to transfer a methyl group from SAM to the C4 of 2‐MeTrp to produce 2,4‐dimethyltryptophan. These results reveal the intriguing substrate specificity of TsrM, further expanding the reaction promiscuity of the radical SAM superfamily enzymes.

Lysine 2,3-Aminomutase and a Newly Discovered Glutamate 2,3-Aminomutase Produce β-Amino Acids Involved in Salt Tolerance in Methanogenic Archaea.

Many methanogenic archaea synthesize β-amino acids as osmolytes that allow survival in high salinity environments. Here, we investigated the radical S-adenosylmethionine (SAM) aminomutases involved in the biosynthesis of Nε-acetyl-β-lysine and β-glutamate in Methanococcus maripaludis C7. Lysine 2,3-aminomutase (KAM), encoded by MmarC7_0106, was overexpressed and purified from Escherichia coli, followed by biochemical characterization. In the presence of l-lysine, SAM, and dithionite, this archaeal KAM had a kcat = 14.3 s-1 and a Km = 19.2 mM. The product was shown to be 3(S)-β-lysine, which is like the well-characterized Clostridium KAM as opposed to the E. coli KAM that produces 3(R)-β-lysine. We further describe the function of MmarC7_1783, a putative radical SAM aminomutase with a ∼160 amino acid extension at its N-terminus. Bioinformatic analysis of the possible substrate-binding residues suggested a function as glutamate 2,3-aminomutase, which was confirmed here through heterologous expression in a methanogen followed by detection of β-glutamate in cell extracts. β-Glutamate has been known to serve as an osmolyte in select methanogens for a long time, but its biosynthetic origin remained unknown until now. Thus, this study defines the biosynthetic routes for β-lysine and β-glutamate in M. maripaludis and expands the importance and diversity of radical SAM enzymes in all domains of life.

Biochemical Characterization of the Radical SAM Methylase Involved in Tetrahydromethanopterin Biosynthesis in Methanogenic Archaea

Tetrahydromethanopterin (H4MPT) is a tetrahydrofolate (H4F) analog that serves as a one‐carbon carrier cofactor in the methanogenesis pathway in methanogenic microorganisms. One major structural feature that distinguishes H4MPT from H4F and other pterin‐containing biomolecules is the presence of methyl groups at the C7 and C9 positions. Although the biosynthesis of H4MPT has been relatively well‐defined, the enzyme responsible for the methylation reactions at the unreactive carbon centers has remained unclear. Our previous work identified the radical S‐adenosyl‐L‐methionine (SAM) methylase, MjMptM, from Methanocaldococcus jannaschii that likely catalyzes the addition of both methyl groups during H4MPT biosynthesis. Our current data indicate that MjMptM does not utilize SAM as the methyl group donor and instead uses methylenetetrahydrofolate for this purpose, thus making MjMptM the founding member of “Class D” radical SAM methylases. Here, we describe our recent progress towards the biochemical and spectroscopic characterization of MjMptM. We demonstrated that the enzyme catalyzes two methylation reactions to produce a dimethylated folate species using methylenetetrahydrofolate as the C1 source for the in vitro methylation reactions and dihydrofolate as the in vitrosubstrate. Site‐directed mutagenesis and UV‐Vis spectroscopic analysis indicate that MjMptM harbors at least two [4Fe‐4S] clusters which reside in the two canonical CX3CX2C radical SAM motifs in the N‐terminus. This result was further confirmed by EPR spectroscopy, which shows the binding of at least two [4Fe‐4S] clusters with unique signals. Only one of the [4Fe‐4S] clusters catalyzes the reductive cleavage of SAM to produce 5’deoxyadenosine, the key first step in radical SAM enzyme catalysis, and thus the most N‐terminal auxiliary cluster is likely involved in electron transfer. Current work is focused on elucidating the details of the novel methylation reaction mechanism.

Lysine‐2,3‐aminomutase and a newly discovered glutamate‐2,3‐aminomutase produce β‐amino acids to overcome salt stress in methanogenic archaea

Many methanogenic archaea synthesize N ε ‐acetyl‐β‐lysine and/or β‐glutamate as osmolytes that allow survival in high salinity environments. Methanococcus maripaludis C7 contains two genes, MmarC7_0106 and MmarC7_1783, encoding radical S‐adenosylmethionine(SAM) enzymes with sequence similarity to lysine‐2,3‐aminomutase (KAM). MmarC7_0106 is immediately upstream of an acetyltransferase and was expected to be the KAM involved in the biosynthesis of N ε ‐acetyl‐β‐lysine. To confirm this, the gene was cloned and expressed with a hexahistidine tag in E. coli, followed by anaerobic purification and biochemical characterization. Although the bacterial KAMs involved in lysine degradation in Clostridium subterminale and a post‐translational modification of elongation factor P in E. coli have been well‐characterized, the archaeal KAM involved in compatible solute biosynthesis had never been studied in vitro before. In the presence of L‐lysine, SAM, and dithionite at 37°C, this archaeal KAM had a k cat = 0.42 s ‐1 and a K m = 17.8 mM. The product was shown to be 3(S)‐β‐lysine, which is like the well‐characterized Clostridium KAM as opposed to the E. coli KAM that produces 3(R)‐β‐lysine. The archaeal KAM was highly specific for L‐lysine, with no activity observed with D‐lysine or most other amino acids. However, low aminomutase activity was observed with L‐arginine. We further report the function of MmarC7_1783, a putative radical SAM aminomutase with a ~160 amino acid extension at its N‐terminus. Bioinformatic analysis of possible substrate binding residues suggested a function as glutamate‐2,3‐aminomutase, which was confirmed here through heterologous expression in a methanogen followed by detection of β‐glutamate in cell extracts. Finally, we investigated the differences in amino acid compatible solute usage amongst M. maripaludis C7 (contains both aminomutase genes) and M. maripaludis S2 (contains only the canonical KAM gene) in different salt concentrations. Taken together, this work defines the biosynthetic routes for β‐amino acids in methanogenic archaea and expands the importance and diversity of radical SAM enzymes in all domains of life.

Unraveling the secrets of radical SAM mechanisms

Radical SAM enzymes catalyze a remarkable diversity of biological radical reactions using a common radical initiation mechanism involving a [4Fe‐4S] cluster and SAM. The initial reductive cleavage of SAM leads to the formation of a catalytically central organometallic species (Ω) in which a SAM‐derived 5′‐deoxyadenosyl radical (5′‐dAdo•) is covalently bound through the 5′‐C to an iron of the [4Fe‐4S] cluster. Homolytic Fe‐C5′ bond cleavage then forms the 5′‐dAdo• radical intermediate, which abstracts a hydrogen atom from substrate. Efforts directed towards generating, trapping, and characterizing key radical intermediates during this radical initiation process will be presented and discussed. Further, recently‐discovered photochemistry in radical SAM enzymes, which can promote radical initiation in the absence of substrate, will be presented. This photochemistry has led to fundamental new understanding of the regioselectivity of reductive S‐C bond cleavage that is central to radical SAM mechanisms.

Structural Investigation of AlsA, a Radical S‐adenosylmethionine Enzyme Involved in the Biosynthesis of the Oxetane‐containing Herbicide Albucidin

Herbicides are a class of molecules commonly employed to maintain high crop yields by targeting weeds, a particularly damaging class of pests in agriculture. Albucidin is a novel herbicide found in Streptomyces albus subsp. Chlorinus NRRL B‐24108 that shows a suspected new mode of action. Albucidin is a nucleoside analogue containing an oxetane moiety similar to the antiviral compound oxetanocin‐A (OXT‐A). OXT‐A is the only other known naturally occurring oxetane‐containing nucleoside analogue and has a biosynthetic pathway with two key enzymes: OxsA and OxsB. OsxB is a B12‐dependent radical S‐adenosylmethionine (RS) enzyme and catalyzes formation of the oxetane ring via an oxidative ring contraction. A similar reaction has been proposed to be catalyzed by the B12‐dependent RS enzyme AlsB in the biosynthetic pathway of albucidin; however, the two biosynthetic pathways appear to diverge at this point as well. While OXT‐A is formed upon subsequent reduction and OxsA catalyzed dephosphorylation of the OxsB reaction product, the chemistry leading to albucidin is much less clear. In particular, one carbon must be eliminated, and AlsA, which is a biotin synthase‐like RS enzyme also encoded in the albucidin biosynthetic cluster, has been proposed to participate in this reaction. AlsA is most closely related to BioB‐like enzymes and demonstrates high sequence similarity with the canonical TIM barrel domain; however, AlsA also contains an N‐terminal domain of about 100 amino acids that shares minimal sequence homology to other RS enzymes with known structures. Herein, recent developments in the structural analysis of AlsA using X‐ray crystallography are reported.

Viperin Catalyzes the Conversion of Cytidine Triphosphate (CTP) to 3′‐deoxy‐3′,4′‐didehydro‐CTP by Radical S‐adenosylmethionine

Virus inhibitory protein, endoplasmic reticulum‐associated, interferon‐inducible (viperin) is a member of the radical S‐adenosylmethionine (SAM) superfamily of enzymes. Its expression is induced by interferon, and it inhibits a variety of DNA and RNA viruses such as influenza, HIV, dengue virus, West Nile virus, hepatitis C virus, and rabies virus. For flaviviridae especially, it catalyzes the conversion of cytidine triphosphate (CTP) to 3′‐deoxy‐3′,4′‐didehydro‐CTP (ddhCTP). When ddhCTP is incorporated by the viral RNA‐dependent RNA polymerase, RNA polymerization stops because the growing RNA strand lacks a 3’‐hydroxyl group. For other virus families, viperin has been found to interfere with mitochondrial metabolism and interact with a variety of cellular proteins. The Summit Country Day School MSOE Center for BioMolecular Modeling MAPS Team used 3D modeling and printing technology to examine structure‐function relationships of Mus musculus viperin bound with CTP. Viperin is a globular protein that has been shown to be highly conserved and is made up of three regions: N‐terminal extension, central domain, and the C‐terminal extension. The N‐terminal extension localizes viperin to the endoplasmic reticulum and lipid droplets where flaviviruses assemble. The central binding domain contains four sequence motifs associated with the radical SAM superfamily, including the tricysteine motif (CX3CX2C), responsible for binding the catalytic [4Fe‐4S] cluster. The C‐terminal extension binds to CTP and is involved in the installation of the [4Fe‐4S] cluster by the iron‐sulfur cluster–installing protein CIAO1. Binding of CTP to viperin results in conformational changes such as ordering of the C‐terminal extension and formation of an eight residue β‐hairpin loop that binds the γ‐phosphate of CTP. When bound, the 4’‐hydrogen of CTP is ideally positioned for abstraction by the 5′‐deoxyadenosyl radical generated by the reductive cleavage of SAM in the central domain. This is followed by the loss of the 3’‐hydroxyl group leading to the formation of the chain terminator, ddhCTP. The structural insights of CTP‐bound viperin provides an understanding of the role of the C‐terminal extension and its importance as an anti‐viral protein.

How do aerobic organisms solve the oxygen sensitivity problem of [4Fe‐4S] in radical SAM enzymes?

All radical‐SAM enzymes require [4Fe‐4S] clusters for activity. It is well known the [4Fe‐4S] cluster is very air sensitive and requires strict anaerobic conditions to reconstitute activity in vitro. However, aerobic species that live under oxygen do use radical SAM enzymes. How such enzymes function in vivo in the presence of oxygen is an interesting question. My lab works on the yeast diphthamide biosynthetic noncanonical radical‐SAM enzyme Dph1‐Dph2. Consistent with the known oxygen sensitivity, we found that in vitro the [4Fe‐4S] cluster in Dph1‐Dph2 is easily degraded into a [3Fe‐4S] cluster. Remarkably, a small iron‐containing protein Dph3 can donate one Fe atom to convert the [3Fe‐4S] cluster in Dph1‐Dph2 to a functional [4Fe‐4S] cluster during the radical‐SAM enzyme catalytic cycle. We believe this is a general strategy to solve the oxygen sensitivity problem and Dph3‐like proteins may exist to keep other radical‐SAM enzymes functional in aerobic environments.

Radical SAM-dependent ether crosslink in daropeptide biosynthesis

Darobactin is a ribosomally synthesized and post-translationally modified peptide (RiPP), which possesses potent activity against various Gram-negative bacteria. Darobactin features a highly unique bicyclic scaffold, consisting of an ether crosslink between two Trp residues and a C–C crosslink between a Lys and a Trp. Here we report in vivo and in vitro activity of darobactin synthase DarE. We show DarE is a radical S-adenosylmethionine (rSAM) enzyme and is solely responsible for forming the bicyclic scaffold of darobactin. DarE mainly produced the ether-crosslinked product in vitro, and when the assay was performed in H218O, apparent 18O incorporation was observed into the ether-crosslinked product. These observations suggested an rSAM-dependent process in darobactin biosynthesis, involving a highly unusual oxygen insertion step from a water molecule and subsequent O–H and C–H activations. Genome mining analysis demonstrates the diversity of darobactin-like biosynthetic gene clusters, a subclade of which likely encode monocyclic products with only an ether linkage. We propose the name daropeptide for this growing family of ether-containing RiPPs produced by DarE enzymes.

L-tyrosine-bound ThiH structure reveals C\u2013C bond break differences within radical SAM aromatic amino acid lyases

2-iminoacetate synthase ThiH is a radical S-adenosyl-L-methionine (SAM) L-tyrosine lyase and catalyzes the L-tyrosine Cα–Cβ bond break to produce dehydroglycine and p-cresol while the radical SAM L-tryptophan lyase NosL cleaves the L-tryptophan Cα–C bond to produce 3-methylindole-2-carboxylic acid. It has been difficult to understand the features that condition one C–C bond break over the other one because the two enzymes display significant primary structure similarities and presumably similar substrate-binding modes. Here, we report the crystal structure of L-tyrosine bound ThiH from Thermosinus carboxydivorans revealing an unusual protonation state of L-tyrosine upon binding. Structural comparison of ThiH with NosL and computational studies of the respective reactions they catalyze show that substrate activation is eased by tunneling effect and that subtle structural changes between the two enzymes affect, in particular, the hydrogen-atom abstraction by the 5´-deoxyadenosyl radical species, driving the difference in reaction specificity.

Radical SAM-dependent formation of a nitrogenase cofactor core on NifB

Nitrogenase is a versatile metalloenzyme that reduces N2, CO and CO2 at its cofactor site. Designated the M-cluster, this complex cofactor has a composition of [(R-homocitrate)MoFe7S9C], and it is assembled through the generation of a unique [Fe8S9C] core prior to the insertion of Mo and homocitrate. NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that is essential for nitrogenase cofactor assembly. This review focuses on the recent work that sheds light on the role of NifB in the formation of the [Fe8S9C] core of the nitrogenase cofactor, highlighting the structure, function and mechanism of this unique radical SAM methyltransferase.

Exploring the Biosynthetic Potential of TsrM, a B12 -dependent Radical SAM Methyltransferase Catalyzing Non-radical Reactions.

B12 -dependent radical SAM enzymes are an emerging enzyme family with approximately 200,000 proteins. These enzymes have been shown to catalyze chemically challenging reactions such as methyl transfer to sp2- and sp3-hybridized carbon atoms. However, to date we have little information regarding their complex mechanisms and their biosynthetic potential. Here we show, using X-ray absorption spectroscopy, mutagenesis and synthetic probes that the vitamin B12 -dependent radical SAM enzyme TsrM catalyzes not only C- but also N-methyl transfer reactions further expanding its synthetic versatility. We also demonstrate that TsrM has the unique ability to directly transfer a methyl group to the benzyl core of tryptophan, including the least reactive position C4. Collectively, our study supports that TsrM catalyzes non-radical reactions and establishes the usefulness of radical SAM enzymes for novel biosynthetic schemes including serial alkylation reactions at particularly inert C-H bonds.