Deoxyadenosyl Research Articles

Characterization of Radical SAM Adenosylhopane Synthase, HpnH, which Catalyzes the 5'-Deoxyadenosyl Radical Addition to Diploptene in the Biosynthesis of C35 Bacteriohopanepolyols.

Adenosylhopane is a crucial intermediate in the biosynthesis of bacteriohopanepolyols, which are widespread prokaryotic membrane lipids. Herein, it is demonstrated that reconstituted HpnH, a putative radical S-adenosyl-l-methionine (SAM) enzyme, commonly encoded in the hopanoid biosynthetic gene cluster, converts diploptene into adenosylhopane in the presence of SAM, flavodoxin, flavodoxin reductase, and NADPH. NMR spectra of the enzymatic reaction product were identical to those of synthetic (22R)-adenosylhopane, indicating that HpnH catalyzes stereoselective C-C formation between C29 of diploptene and C5' of 5'-deoxyadenosine. Further, the HpnH reaction in D2 O-containing buffer revealed that a D atom was incorporated at the C22 position of adenosylhopane. Based on these results, we propose a radical addition reaction mechanism catalyzed by HpnH for the formation of the C35 bacteriohopane skeleton.

Photoinduced Electron Transfer in a Radical SAM Enzyme Generates an S-Adenosylmethionine Derived Methyl Radical

Radical SAM (RS) enzymes use S-adenosyl-l-methionine (SAM) and a [4Fe-4S] cluster to initiate a broad spectrum of radical transformations throughout all kingdoms of life. We report here that low-temperature photoinduced electron transfer from the [4Fe-4S]1+ cluster to bound SAM in the active site of the hydrogenase maturase RS enzyme, HydG, results in specific homolytic cleavage of the S-CH3 bond of SAM, rather than the S-C5' bond as in the enzyme-catalyzed (thermal) HydG reaction. This result is in stark contrast to a recent report in which photoinduced ET in the RS enzyme pyruvate formate-lyase activating enzyme cleaved the S-C5' bond to generate a 5'-deoxyadenosyl radical, and provides the first direct evidence for homolytic S-CH3 bond cleavage in a RS enzyme. Photoinduced ET in HydG generates a trapped •CH3 radical, as well as a small population of an organometallic species with an Fe-CH3 bond, denoted ΩM. The •CH3 radical is surprisingly found to exhibit rotational diffusion in the HydG active site at temperatures as low as 40 K, and is rapidly quenched: whereas 5'-dAdo• is stable indefinitely at 77 K, •CH3 quenches with a half-time of ∼2 min at this temperature. The rapid quenching and rotational/translational freedom of •CH3 shows that enzymes would be unable to harness this radical as a regio- and stereospecific H atom abstractor during catalysis, in contrast to the exquisite control achieved with the enzymatically generated 5'-dAdo•.

Radical SAM enzymes: surprises along the path to understanding mechanism.

As the field of radical SAM enzymology has grown from a few examples in the 1990s to hundreds of thousands today, a fundamental question has remained: how does Nature use S-adenosyl-L-methionine to initiate radical reactions? This was a driving question when we first began studying pyruvate formate-lyase activating enzyme in 1993, and our journey for answers has brought us to many surprising discoveries, from the direct coordination of SAM to a unique iron in a [4Fe-4S] cluster, to our recent discovery of an organometallic intermediate and our ability to quantitatively produce and characterize the long-sought 5'-deoxyadenosyl radical intermediate. These adventures and what we have learned along the way about this fundamentally novel chemistry is described in this review.

The Elusive 5'-Deoxyadenosyl Radical: Captured and Characterized by Electron Paramagnetic Resonance and Electron Nuclear Double Resonance Spectroscopies.

The 5'-deoxyadenosyl radical (5'-dAdo·) abstracts a substrate H atom as the first step in radical-based transformations catalyzed by adenosylcobalamin-dependent and radical S-adenosyl-l-methionine (RS) enzymes. Notwithstanding its central biological role, 5'-dAdo· has eluded characterization despite efforts spanning more than a half-century. Here, we report generation of 5'-dAdo· in a RS enzyme active site at 12 K using a novel approach involving cryogenic photoinduced electron transfer from the [4Fe-4S]+ cluster to the coordinated S-adenosylmethionine (SAM) to induce homolytic S-C5' bond cleavage. We unequivocally reveal the structure of this long-sought radical species through the use of electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies with isotopic labeling, complemented by density-functional computations: a planar C5' (2pπ) radical (∼70% spin occupancy); the C5'(H)2 plane is rotated by ∼37° (experiment)/39° (DFT) relative to the C5'-C4'-(C4'-H) plane, placing a C5'-H antiperiplanar to the ribose-ring oxygen, which helps stabilize the radical against elimination of the 4'-H. The agreement between φ from experiment and in vacuo DFT indicates that the conformation is intrinsic to 5-dAdo· itself, and not determined by its environment.

Analysis of Electrochemical Properties of S-Adenosyl-l-methionine and Implications for Its Role in Radical SAM Enzymes.

S-Adenosyl-l-methionine (SAM) is the central cofactor in the radical SAM enzyme superfamily, responsible for a vast number of transformations in primary and secondary metabolism. In nearly all of these reactions, the reductive cleavage of SAM is proposed to produce a reactive species, 5′-deoxyadenosyl radical, which initiates catalysis. While the mechanistic details in many cases are well-understood, the reductive cleavage of SAM remains elusive. In this manuscript, we have measured the solution peak potential of SAM to be ∼−1.4 V (v SHE) and show that under controlled potential conditions, it undergoes irreversible fragmentation to the 5′-deoxyadenosyl radical. While the radical intermediate is not directly observed, its presence as an initial intermediate is inferred by the formation of 8,5′-cycloadenosine and by H atom incorporation into 5′-deoxyadenosine from solvent exchangeable site. Similarly, 2-aminobutyrate is also observed under electrolysis conditions. The implications of these results in the context of the reductive cleavage of SAM by radical SAM enzymes are discussed.

Revisiting the Mechanism of the Anaerobic Coproporphyrinogen III Oxidase HemN

AbstractHemN is a radical S‐adenosyl‐l‐methionine (SAM) enzyme that catalyzes the oxidative decarboxylation of coproporphyrinogen III to produce protoporphyrinogen IX, an intermediate in heme biosynthesis. HemN binds two SAM molecules in the active site, but how these two SAMs are utilized for the sequential decarboxylation of the two propionate groups of coproporphyrinogen III remains largely elusive. Provided here is evidence showing that in HemN catalysis a SAM serves as a hydrogen relay which mediates a radical‐based hydrogen transfer from the propionate to the 5′‐deoxyadenosyl (dAdo) radical generated from another SAM in the active site. Also observed was an unexpected shunt product resulting from trapping of the SAM‐based methylene radical by the vinyl moiety of the mono‐decarboxylated intermediate, harderoporphyrinogen. These results suggest a major revision of the HemN mechanism and reveal a new paradigm of the radical‐mediated hydrogen transfer in radical SAM enzymology.

Revisiting the Mechanism of the Anaerobic Coproporphyrinogen III Oxidase HemN.

HemN is a radical S-adenosyl-l-methionine (SAM) enzyme that catalyzes the oxidative decarboxylation of coproporphyrinogen III to produce protoporphyrinogen IX, an intermediate in heme biosynthesis. HemN binds two SAM molecules in the active site, but how these two SAMs are utilized for the sequential decarboxylation of the two propionate groups of coproporphyrinogen III remains largely elusive. Provided here is evidence showing that in HemN catalysis a SAM serves as a hydrogen relay which mediates a radical-based hydrogen transfer from the propionate to the 5'-deoxyadenosyl (dAdo) radical generated from another SAM in the active site. Also observed was an unexpected shunt product resulting from trapping of the SAM-based methylene radical by the vinyl moiety of the mono-decarboxylated intermediate, harderoporphyrinogen. These results suggest a major revision of the HemN mechanism and reveal a new paradigm of the radical-mediated hydrogen transfer in radical SAM enzymology.

Enzymatic Cascade Reactions in Biosynthesis.

Enzyme-mediated cascade reactions are widespread in biosynthesis. To facilitate comparison with the mechanistic categorizations of cascade reactions by synthetic chemists and delineate the common underlying chemistry, we discuss four types of enzymatic cascade reactions: those involving nucleophilic, electrophilic, pericyclic, and radical reactions. Two subtypes of enzymes that generate radical cascades exist at opposite ends of the oxygen abundance spectrum. Iron-based enzymes use O2 to generate high valent iron-oxo species to homolyze unactivated C-H bonds in substrates to initiate skeletal rearrangements. At anaerobic end, enzymes reversibly cleave S-adenosylmethionine (SAM) to generate the 5'-deoxyadenosyl radical as a powerful oxidant to initiate C-H bond homolysis in bound substrates. The latter enzymes are termed radical SAM enzymes. We categorize the former as "thwarted oxygenases".

Mechanism of Radical Initiation in the Radical S-Adenosyl-l-methionine Superfamily.

The seeds for recognition of the vast superfamily of radical S-adenosyl-l-methionine (SAM) enzymes were sown in the 1960s, when Joachim Knappe found that the dissimilation of pyruvate was dependent on SAM and Fe(II), and Barker and co-workers made similar observations for lysine 2,3-aminomutase. These intriguing observations, coupled with the evidence that SAM and Fe were cofactors in radical catalysis by these enzyme systems, drew us in the 1990s to explore how Fe(II) and SAM initiate radical reactions. Our early work focused on the same enzyme Knappe had originally characterized: the pyruvate formate-lyase activating enzyme (PFL-AE). Our discovery of an iron-sulfur cluster in this enzyme, together with similar findings for other SAM-dependent enzymes at the time, led to the recognition of an emerging class of enzymes that use iron-sulfur clusters to cleave SAM, liberating the 5'-deoxyadenosyl radical (5'-dAdo•) that initiates radical reactions. A major bioinformatics study by Heidi Sofia and co-workers identified the enzyme superfamily denoted Radical SAM, now known to span all kingdoms of life with more than 100,000 unique sequences encoding enzymes that catalyze remarkably diverse reactions. Despite the limited sequence similarity and vastly divergent reactions catalyzed, the radical SAM enzymes appear to employ a common mechanism for initiation of radical chemistry, a mechanism we have helped to clarify over the last 25 years. A reduced [4Fe-4S]+ cluster provides the electron needed for the reductive cleavage of SAM. The resulting [4Fe-4S]2+ cluster can be rereduced either by an external reductant, with SAM acting as a cosubstrate, or by an electron provided during the reformation of SAM in cases where SAM is used as a cofactor. The amino and carboxylate groups of SAM bind to the unique iron of the catalytic [4Fe-4S] cluster, placing the sulfonium of SAM in close proximity to the cluster. Surprising recent results have shown that the initiating enzymatic cleavage of SAM generates an organometallic intermediate prior to liberation of 5'-dAdo•, which initiates radical chemistry on the substrate. This organometallic intermediate, denoted Ω, has a 5'-deoxyadenosyl moiety directly bound to the unique iron of the [4Fe-4S] cluster via the 5'-C, giving a structure that is directly analogous to the Co-(5'-C) bond of the organometallic cofactor adenosylcobalamin. Our observation that this intermediate Ω is formed throughout the superfamily suggests that this is a key intermediate in initiating radical SAM reactions, and that organometallic chemistry is much more broadly relevant in biology than previously thought.

Deconvoluting the Reduction Potentials for the Three [4Fe-4S] Clusters in an AdoMet Radical SCIFF Maturase.

Enzymes in the S-adenosyl-l-methionine (AdoMet) radical enzyme superfamily are metalloenzymes that catalyze a wide variety of complex radical-mediated transformations with the aid of a [4Fe-4S] cluster, which is required for activation of AdoMet to generate the 5′-deoxyadenosyl radical to initiate the catalytic cycle. In addition to this cluster, some enzymes share an additional domain, the SPASM domain, that houses auxiliary FeS clusters whose functional significance is not clearly understood. The AdoMet radical enzyme Tte1186, which catalyzes a thioether cross-link in a cysteine rich peptide (SCIFF), has two auxiliary [4Fe-4S] clusters within a SPASM domain that are required for enzymatic activity but not for the generation of the 5′-deoxyadenosyl radical intermediate. Here we demonstrate the ability to measure independently the midpoint potentials of each of the three [4Fe-4S] clusters by employing Tte1186 variants for which only the first, second, or AdoMet binding cluster is bound. This allows, for the first time, assignment of reduction potentials for all clusters in an AdoMet radical enzyme with a SPASM domain. Our results show that the clusters have midpoint potentials that are within 100 mV of each other, suggesting that their electrochemical properties are not greatly influenced by the presence of the nearby clusters.

Reconstitution and substrate specificity for isopentenyl pyrophosphate of the antiviral radical SAM enzyme viperin

Viperin is a radical SAM enzyme that has been shown to possess antiviral activity against a broad spectrum of viruses; however, its molecular mechanism is unknown. We report here that recombinant fungal and archaeal viperin enzymes catalyze the addition of the 5'-deoxyadenosyl radical (5'-dA•) to the double bond of isopentenyl pyrophosphate (IPP), producing a new compound we named adenylated isopentyl pyrophosphate (AIPP). The reaction is specific for IPP, as other pyrophosphate compounds involved in the mevalonate biosynthetic pathway did not react with 5'-dA• Enzymatic reactions employing IPP derivatives as substrates revealed that any chemical change in IPP diminishes its ability to be an effective substrate of fungal viperin. Mutational studies disclosed that the hydroxyl group on the side chain of Tyr-245 in fungal viperin is the likely source of hydrogen in the last step of the radical addition, providing mechanistic insight into the radical reaction catalyzed by fungal viperin. Structure-based molecular dynamics (MD) simulations of viperin interacting with IPP revealed a good fit of the isopentenyl motif of IPP to the active site cavity of viperin, unraveling the molecular basis of substrate specificity of viperin for IPP. Collectively, our findings indicate that IPP is an effective substrate of fungal and archaeal viperin enzymes and provide critical insights into the reaction mechanism.

Probing the coordination and function of Fe4S4 modules in nitrogenase assembly protein NifB

NifB is an essential radical S-adenosylmethionine (SAM) enzyme for nitrogenase cofactor assembly. Previous studies show that NifB couples a putative pair of [Fe4S4] modules (designated K1 and K2) into an [Fe8S9C] cofactor precursor concomitant with radical SAM-dependent carbide insertion through the action of its SAM-binding [Fe4S4] module. However, the coordination and function of the NifB cluster modules remain unknown. Here, we use continuous wave and pulse electron paramagnetic resonance spectroscopy to show that K1- and K2-modules are 3-cysteine-coordinated [Fe4S4] clusters, with a histidine-derived nitrogen serving as the fourth ligand to K1 that is lost upon K1/K2-coupling. Further, we demonstrate that coexistence of SAM/K2-modules is a prerequisite for methyltransfer to K2 and hydrogen abstraction from the K2-associated methyl by a 5′-deoxyadenosyl radical. These results establish an important framework for mechanistic explorations of NifB while highlighting the utility of a synthetic-cluster-based reconstitution approach employed herein in functional analyses of iron–sulfur (FeS) enzymes.

A radical S-adenosyl-L-methionine enzyme and a methyltransferase catalyze cyclopropane formation in natural product biosynthesis

Cyclopropanation of unactivated olefinic bonds via addition of a reactive one-carbon species is well developed in synthetic chemistry, whereas natural cyclopropane biosynthesis employing this strategy is very limited. Here, we identify a two-component cyclopropanase system, composed of a HemN-like radical S-adenosyl-l-methionine (SAM) enzyme C10P and a methyltransferase C10Q, catalyzes chemically challenging cyclopropanation in the antitumor antibiotic CC-1065 biosynthesis. C10P uses its [4Fe-4S] cluster for reductive cleavage of the first SAM to yield a highly reactive 5′-deoxyadenosyl radical, which abstracts a hydrogen from the second SAM to produce a SAM methylene radical that adds to an sp2-hybridized carbon of substrate to form a SAM-substrate adduct. C10Q converts this adduct to CC-1065 via an intramolecular SN2 cyclization mechanism with elimination of S-adenosylhomocysteine. This cyclopropanation strategy not only expands the enzymatic reactions catalyzed by the radical SAM enzymes and methyltransferases, but also sheds light on previously unnoticed aspects of the versatile SAM-based biochemistry.

Stereospecific Radical-Mediated B12-Dependent Methyl Transfer by the Fosfomycin Biosynthesis Enzyme Fom3.

Fom3, the antepenultimate enzyme in the fosfomycin biosynthetic pathway in Streptomyces spp., is a class B cobalamin-dependent radical SAM methyltransferase that catalyzes methylation of (5'-cytidylyl)-2-hydroxyethylphosphonate (2-HEP-CMP) to form (5'-cytidylyl)-2-hydroxypropylphosphonate (2-HPP-CMP). Previously, the reaction of Fom3 with 2-HEP-CMP produced 2-HPP-CMP with mixed stereochemistry at C2. Mechanistic characterization has been challenging because of insoluble expression and poor cobalamin (B12) incorporation in Escherichia coli. Recently, soluble E. coli expression and incorporation of cobalamin into Fom3 were achieved by overexpression of the BtuCEDFB cobalamin uptake system. Herein, we use this new method to obtain Fom3 from Streptomyces wedmorensis. We show that the initiator 5'-deoxyadenosyl radical stereospecifically abstracts the pro- R hydrogen atom from the C2 position of 2-HEP-CMP and use the downstream enzymes FomD and Fom4 to demonstrate that our preparation of Fom3 produces only (2 S)-2-HPP-CMP. Additionally, we show that the added methyl group originates from SAM under multiple-turnover conditions, but the first turnover uses a methyl donor already present on the enzyme; furthermore, cobalamin isolated from Fom3 reaction mixtures contains methyl groups derived from SAM. These results are consistent with a model in which Fom3 catalyzes methyl transfer from SAM to cobalamin and the resulting methylcobalamin (MeCbl) is the ultimate methyl source for the reaction.

A Radical Clock Probe Uncouples H Atom Abstraction from Thioether Cross-Link Formation by the Radical S-Adenosyl-l-methionine Enzyme SkfB.

Sporulation killing factor (SKF) is a ribosomally synthesized and post-translationally modified peptide (RiPP) produced by Bacillus. SKF contains a thioether cross-link between the α-carbon at position 40 and the thiol of Cys32, introduced by a member of the radical S-adenosyl-l-methionine (SAM) superfamily, SkfB. Radical SAM enzymes employ a 4Fe–4S cluster to bind and reductively cleave SAM to generate a 5′-deoxyadenosyl radical. SkfB utilizes this radical intermediate to abstract the α-H atom at Met40 to initiate cross-linking. In addition to the cluster that binds SAM, SkfB also has an auxiliary cluster, the function of which is not known. We demonstrate that a substrate analogue with a cyclopropylglycine (CPG) moiety replacing the wild-type Met40 side chain forgoes thioether cross-linking for an alternative radical ring opening of the CPG side chain. The ring opening reaction also takes place with a catalytically inactive SkfB variant in which the auxiliary Fe–S cluster is absent. Therefore, the CPG-containing peptide uncouples H atom abstraction from thioether bond formation, limiting the role of the auxiliary cluster to promoting thioether cross-link formation. CPG proves to be a valuable tool for uncoupling H atom abstraction from peptide modification in RiPP maturases and demonstrates potential to leverage RS enzyme reactivity to create noncanonical amino acids.

C-Methylation Catalyzed by Fom3, a Cobalamin-Dependent Radical S-adenosyl-l-methionine Enzyme in Fosfomycin Biosynthesis, Proceeds with Inversion of Configuration.

Fom3, a cobalamin-dependent radical S-adenosyl-l-methionine (SAM) methyltransferase, catalyzes C-methylation at the C2 position of cytidylylated 2-hydroxyethylphosphonate (HEP-CMP) to afford cytidylylated 2-hydroxypropylphosphonate (HPP-CMP) in fosfomycin biosynthesis. In this study, the Fom3 reaction product HPP-CMP was reanalyzed by chiral ligand exchange chromatography to confirm its stereochemistry. The Fom3 methylation product was found to be ( S)-HPP-CMP only, indicating that the stereochemistry of the C-methylation catalyzed by Fom3 is ( S)-selective. In addition, Fom3 reaction was performed with ( S)-[2-2H1]HEP-CMP and ( R)-[2-2H1]HEP-CMP to elucidate the stereoselectivity during the abstraction of the hydrogen atom from C2 of HEP-CMP. Liquid chromatography-electrospray ionization mass spectrometry analysis of the 5'-deoxyadenosine produced showed that the 2H atom of ( R)-[2-2H1]HEP-CMP was incorporated into 5'-deoxyadenosine but that from ( S)-[2-2H1]HEP-CMP was not. Retention of the 2H atom of ( S)-[2-2H1]HEP-CMP in HPP-CMP was also observed. These results indicate that the 5'-deoxyadenosyl radical stereoselectively abstracts the pro-R hydrogen atom at the C2 position of HEP-CMP and the substrate radical intermediate reacts with the methyl group on cobalamin that is located on the opposite side of the substrate from SAM. Consequently, it was clarified that the C-methylation catalyzed by Fom3 proceeds with inversion of configuration.

Paradigm Shift for Radical S-Adenosyl-l-methionine Reactions: The Organometallic Intermediate Ω Is Central to Catalysis.

Radical S-adenosyl-l-methionine (SAM) enzymes comprise a vast superfamily catalyzing diverse reactions essential to all life through homolytic SAM cleavage to liberate the highly reactive 5′-deoxyadenosyl radical (5′-dAdo·). Our recent observation of a catalytically competent organometallic intermediate Ω that forms during reaction of the radical SAM (RS) enzyme pyruvate formate-lyase activating-enzyme (PFL-AE) was therefore quite surprising, and led to the question of its broad relevance in the superfamily. We now show that Ω in PFL-AE forms as an intermediate under a variety of mixing order conditions, suggesting it is central to catalysis in this enzyme. We further demonstrate that Ω forms in a suite of RS enzymes chosen to span the totality of superfamily reaction types, implicating Ω as essential in catalysis across the RS superfamily. Finally, EPR and electron nuclear double resonance spectroscopy establish that Ω involves an Fe–C5′ bond between 5′-dAdo· and the [4Fe–4S] cluster. An analogous organometallic bond is found in the well-known adenosylcobalamin (coenzyme B12) cofactor used to initiate radical reactions via a 5′-dAdo· intermediate. Liberation of a reactive 5′-dAdo· intermediate via homolytic metal–carbon bond cleavage thus appears to be similar for Ω and coenzyme B12. However, coenzyme B12 is involved in enzymes catalyzing only a small number (∼12) of distinct reactions, whereas the RS superfamily has more than 100 000 distinct sequences and over 80 reaction types characterized to date. The appearance of Ω across the RS superfamily therefore dramatically enlarges the sphere of bio-organometallic chemistry in Nature.

Noncanonical Radical SAM Enzyme Chemistry Learned from Diphthamide Biosynthesis.

Radical S-adenosylmethionine (SAM) enzymes are a superfamily of enzymes that use SAM and reduced [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical to catalyze numerous challenging reactions. We have reported a type of noncanonical radical SAM enzymes in the diphthamide biosynthesis pathway. These enzymes also use SAM and reduced [4Fe-4S] clusters, but generate a 3-amino-3-carboxypropyl (ACP) radical to modify the substrate protein, translation elongation factor 2. The regioselective cleavage of a different C-S bond of the sulfonium center of SAM in these enzymes comparing to canonical radical SAM enzymes is intriguing. Here, we highlight some recent findings in the mechanism of these types of enzymes, showing that the diphthamide biosynthetic radial SAM enzymes bound SAM with a distinct geometry. In this way, the unique iron of the [4Fe-4S] cluster in the enzyme can only attack the carbon on the ACP group to form an organometallic intermediate. The homolysis of the organometallic intermediate releases the ACP radical and generates the EF2 radial.

Expanding the Chemistry of the Class C Radical SAM Methyltransferase NosN by Using an Allyl Analogue of SAM

The radical S-adenosylmethionine (SAM) superfamily enzymes cleave SAM reductively to generate a highly reactive 5'-deoxyadenosyl (dAdo) radical, which initiates remarkably diverse reactions. Unlike most radical SAM enzymes, the class C radical SAM methyltransferase NosN binds two SAMs in the active site, using one SAM to produce a dAdo radical and the second as a methyl donor. Here, we report a mechanistic investigation of NosN in which an allyl analogue of SAM (allyl-SAM) was used. We show that NosN cleaves allyl-SAM efficiently and the resulting dAdo radical can be captured by the olefin moieties of allyl-SAM or 5'-allylthioadenosine (ATA), the latter being a derivative of allyl-SAM. Remarkably, we found that NosN produced two distinct sets of products in the presence and absence of the methyl acceptor substrate, thus suggesting substrate-triggered production of ATA from allyl-SAM. We also show that NosN produces S-adenosylhomocysteine from 5'-thioadenosine and homoserine lactone. These results support the idea that 5'-methylthioadenosine is the direct methyl donor in NosN reactions, and demonstrate great potential to modulate radical SAM enzymes for novel catalytic activities.

Expanding the Chemistry of the Class C Radical SAM Methyltransferase NosN by Using an Allyl Analogue of SAM

AbstractThe radical S‐adenosylmethionine (SAM) superfamily enzymes cleave SAM reductively to generate a highly reactive 5′‐deoxyadenosyl (dAdo) radical, which initiates remarkably diverse reactions. Unlike most radical SAM enzymes, the class C radical SAM methyltransferase NosN binds two SAMs in the active site, using one SAM to produce a dAdo radical and the second as a methyl donor. Here, we report a mechanistic investigation of NosN in which an allyl analogue of SAM (allyl‐SAM) was used. We show that NosN cleaves allyl‐SAM efficiently and the resulting dAdo radical can be captured by the olefin moieties of allyl‐SAM or 5′‐allylthioadenosine (ATA), the latter being a derivative of allyl‐SAM. Remarkably, we found that NosN produced two distinct sets of products in the presence and absence of the methyl acceptor substrate, thus suggesting substrate‐triggered production of ATA from allyl‐SAM. We also show that NosN produces S‐adenosylhomocysteine from 5′‐thioadenosine and homoserine lactone. These results support the idea that 5′‐methylthioadenosine is the direct methyl donor in NosN reactions, and demonstrate great potential to modulate radical SAM enzymes for novel catalytic activities.