Mechanism of Radical Initiation in the Radical SAM Enzyme Superfamily.

Radical S-adenosylmethionine (SAM) enzymes use a site-differentiated [4Fe-4S] cluster and SAM to initiate radical reactions through liberation of the 5'-deoxyadenosyl (5'-dAdo•) radical. They form the largest enzyme superfamily, with more than 700,000 unique sequences currently, and their numbers continue to grow as a result of ongoing bioinformatics efforts. The range of extremely diverse, highly regio- and stereo-specific reactions known to be catalyzed by radical SAM superfamily members is remarkable. The common mechanism of radical initiation in the radical SAM superfamily is the focus of this review. Most surprising is the presence of an organometallic intermediate, Ω, exhibiting an Fe-C5'-adenosyl bond. Regioselective reductive cleavage of the SAM S-C5' bond produces 5'-dAdo• to form Ω, with the regioselectivity originating in the Jahn-Teller effect. Ω liberates the free 5'-dAdo• as the catalytically active intermediate through homolysis of the Fe-C5' bond, in analogy to Co-C5' bond homolysis in B12, which was once viewed as biology's choice of radical generator.

Macrocyclization and Backbone Modification in RiPP Biosynthesis.

The past decade has seen impressive advances in understanding the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs). One of the most common modifications found in these natural products is macrocyclization, a strategy also used by medicinal chemists to improve metabolic stability and target affinity and specificity. Another tool of the peptide chemist, modification of the amides in a peptide backbone, has also been observed in RiPPs. This review discusses the molecular mechanisms of biosynthesis of a subset of macrocyclic RiPP families, chosen because of the unusual biochemistry involved: the five classes of lanthipeptides (thioether cyclization by Michael-type addition), sactipeptides and ranthipeptides (thioether cyclization by radical chemistry), thiopeptides (cyclization by [4+2] cycloaddition), and streptide (cyclization by radical C-C bond formation). In addition, the mechanisms of backbone amide methylation, backbone epimerization, and backbone thioamide formation are discussed, as well as an unusual route to small molecules by posttranslational modification.

Biosynthesis of the Metalloclusters of Nitrogenases.

Nitrogenase is a versatile metalloenzyme that is capable of catalyzing two important reactions under ambient conditions: the reduction of nitrogen (N2) to ammonia (NH3), a key step in the global nitrogen cycle; and the reduction of carbon monoxide (CO) and carbon dioxide (CO2) to hydrocarbons, two reactions useful for recycling carbon waste into carbon fuel. The molybdenum (Mo)- and vanadium (V)-nitrogenases are two homologous members of this enzyme family. Each of them contains a P-cluster and a cofactor, two high-nuclearity metalloclusters that have crucial roles in catalysis. This review summarizes the progress that has been made in elucidating the biosynthetic mechanisms of the P-cluster and cofactor species of nitrogenase, focusing on what is known about the assembly mechanisms of the two metalloclusters in Mo-nitrogenase and giving a brief account of the possible assembly schemes of their counterparts in V-nitrogenase, which are derived from the homology between the two nitrogenases.

Identification of two archaeal GDGT lipid-modifying proteins reveals diverse microbes capable of GMGT biosynthesis and modification.

Archaea produce unique membrane-spanning lipids (MSLs), termed glycerol dialkyl glycerol tetraethers (GDGTs), which aid in adaptive responses to various environmental challenges. GDGTs can be modified through cyclization, cross-linking, methylation, hydroxylation, and desaturation, resulting in structurally distinct GDGT lipids. Here, we report the identification of radical SAM proteins responsible for two of these modifications-a glycerol monoalkyl glycerol tetraether (GMGT) synthase (Gms), responsible for covalently cross-linking the two hydrocarbon tails of a GDGT to produce GMGTs, and a GMGT methylase (Gmm), capable of methylating the core hydrocarbon tail. Heterologous expression of Gms proteins from various archaea in Thermococcus kodakarensis results in the production of GMGTs in two isomeric forms. Further, coexpression of Gms and Gmm produces mono- and dimethylated GMGTs and minor amounts of trimethylated GMGTs with only trace GDGT methylation. Phylogenetic analyses reveal the presence of Gms homologs in diverse archaeal genomes spanning all four archaeal superphyla and in multiple bacterial phyla with the genetic potential to synthesize fatty acid-based MSLs, demonstrating that GMGT production may be more widespread than previously appreciated. We demonstrate GMGT production in three Gms-encoding archaea, identifying an increase in GMGTs in response to elevated temperature in two Archaeoglobus species and the production of GMGTs with up to six rings in Vulcanisaeta distributa. The occurrence of such highly cyclized GMGTs has been limited to environmental samples and their detection in culture demonstrates the utility of combining genetic, bioinformatic, and lipid analyses to identify producers of distinct archaeal membrane lipids.

Facile and dynamic cleavage of every iron-sulfide bond in cuboidal iron-sulfur clusters.

Nature employs weak-field metalloclusters to support a wide range of biological processes. The most ubiquitous metalloclusters are the cuboidal Fe-S clusters, which are comprised of Fe sites with locally high-spin electronic configurations. Such configurations enhance rates of ligand exchange and imbue the clusters with a degree of structural plasticity that is increasingly thought to be functionally relevant. Here, we examine this phenomenon using isotope tracing experiments. Specifically, we demonstrate that synthetic [Fe4S4] and [MoFe3S4] clusters exchange their Fe atoms with Fe2+ ions dissolved in solution, a process that involves the reversible cleavage and reformation of every Fe-S bond in the cluster core. This exchange is facile-in most cases occurring at room temperature on the timescale of minutes-and documented over a range of cluster core oxidation states and terminal ligation patterns. In addition to suggesting a highly dynamic picture of cluster structure, these results provide a method for isotopically labeling pre-formed clusters with spin-active nuclei, such as 57Fe. Such a protocol is demonstrated for the radical S-adenosyl-l-methionine enzyme, RlmN.

Biosynthesis of 3-thia-α-amino acids on a carrier peptide.

A subset of natural products, such as polyketides and nonribosomal peptides, is biosynthesized while tethered to a carrier peptide via a thioester linkage. Recently, we reported that the biosyntheses of 3-thiaglutamate and ammosamide, single amino acid-derived natural products, employ a very different type of carrier peptide to which the biosynthetic intermediates are bound via an amide linkage. During their biosyntheses, a peptide aminoacyl-transfer ribonucleic acid (tRNA) ligase (PEARL) first loads an amino acid to the C terminus of the carrier peptide for subsequent modification by other enzymes. Proteolytic removal of the modified C-terminal amino acid yields the mature product. We termed natural products that are biosynthesized using such pathways pearlins. To investigate the diversity of pearlins, in this study we experimentally characterized another PEARL-encoding biosynthetic gene cluster (BGC) from Tistrella mobilis (tmo). The enzymes encoded in the tmo BGC transformed cysteine into 3-thiahomoleucine both in vitro and in Escherichia coli. During this process, a cobalamin-dependent radical S-adenosylmethionine (SAM) enzyme catalyzes C-isopropylation. This work illustrates that the biosynthesis of amino acid-derived natural products on a carrier peptide is a widespread strategy in nature and expands the spectrum of thiahemiaminal analogs of amino acids that may serve a broader, currently unknown function.

ThnL, a B12-dependent radical S-adenosylmethionine enzyme, catalyzes thioether bond formation in carbapenem biosynthesis.

Complex carbapenems are important clinical antibiotics used to treat recalcitrant infections. Their biosynthetic gene clusters contain three essential B12-dependent radical S-adenosylmethionine (rSAM) enzymes. The majority of characterized enzymes in this subfamily catalyze methyl transfer, but only one is required to sequentially install all methionine-derived carbons in complex carbapenems. Therefore, it is probable that the other two rSAM enzymes have noncanonical functions. Through a series of fermentation and in vitro experiments, we show that ThnL uses radical SAM chemistry to catalyze thioether bond formation between C2 of a carbapenam precursor and pantetheine, uniting initial bicycle assembly common to all carbapenems with later tailoring events unique to complex carbapenems. ThnL also catalyzes reversible thiol/disulfide redox on pantetheine. Neither of these functions has been observed previously in a B12-dependent radical SAM enzyme. ThnL expands the known activity of this subclass of enzymes beyond carbon-carbon bond formation or rearrangement. It is also the only radical SAM enzyme currently known to catalyze carbon-sulfur bond formation with only an rSAM Fe-S cluster and no additional auxiliary clusters.

A new member of the flavodoxin superfamily from Fusobacterium nucleatum that functions in heme trafficking and reduction of anaerobilin.

Fusobacterium nucleatum is an opportunistic oral pathogen that is associated with various cancers. To fulfill its essential need for iron, this anaerobe will express heme uptake machinery encoded at a single genetic locus. The heme uptake operon includes HmuW, a class C radical SAM-dependent methyltransferase that degrades heme anaerobically to release Fe2+ and a linear tetrapyrrole called anaerobilin. The last gene in the operon, hmuF encodes a member of the flavodoxin superfamily of proteins. We discovered that HmuF and a paralog, FldH, bind tightly to both FMN and heme. The structure of Fe3+-heme-bound FldH (1.6 Å resolution) reveals a helical cap domain appended to the ⍺/ß core of the flavodoxin fold. The cap creates a hydrophobic binding cleft that positions the heme planar to the si-face of the FMN isoalloxazine ring. The ferric heme iron is hexacoordinated to His134 and a solvent molecule. In contrast to flavodoxins, FldH and HmuF do not stabilize the FMN semiquinone but instead cycle between the FMN oxidized and hydroquinone states. We show that heme-loaded HmuF and heme-loaded FldH traffic heme to HmuW for degradation of the protoporphyrin ring. Both FldH and HmuF then catalyze multiple reductions of anaerobilin through hydride transfer from the FMN hydroquinone. The latter activity eliminates the aromaticity of anaerobilin and the electrophilic methylene group that was installed through HmuW turnover. Hence, HmuF provides a protected path for anaerobic heme catabolism, offering F. nucleatum a competitive advantage in the colonization of anoxic sites of the human body.

Intermolecular electron transfer in radical SAM enzymes as a new paradigm for reductive activation.

Radical S-adenosyl-L-methionine (rSAM) enzymes bind one or more Fe-S clusters and catalyze transformations that produce complex and structurally diverse natural products. One of the clusters, a 4Fe-4S cluster, binds and reductively cleaves SAM to generate the 5'-deoxyadenosyl radical, which initiates the catalytic cycle by H-atom transfer from the substrate. The role(s) of the additional auxiliary Fe-S clusters (ACs) remains largely enigmatic. The rSAM enzyme PapB catalyzes the formation of thioether cross-links between the ß-carbon of an Asp and a Cys thiolate found in the PapA peptide. One of the two ACs in the protein binds to the substrate thiol where, upon formation of a thioether bond, one reducing equivalent is returned to the protein. However, for the next catalytic cycle to occur, the protein must undergo an electronic state isomerization, returning the electron to the SAM-binding cluster. Using a series of iron-sulfur cluster deletion mutants, our data support a model whereby the isomerization is an obligatorily intermolecular electron transfer event that can be mediated by redox active proteins or small molecules, likely via the second AC in PapB. Surprisingly, a mixture of FMN and NADPH is sufficient to support both the reductive and the isomerization steps. These findings lead to a new paradigm involving intermolecular electron transfer steps in the activation of rSAM enzymes that require multiple iron-sulfur clusters for turnover. The implications of these results for the biological activation of rSAM enzymes are discussed.

Computational engineering of previously crystallized pyruvate formate-lyase activating enzyme reveals insights into SAM binding and reductive cleavage.

Radical S-adenosyl-l-methionine (SAM) enzymes are ubiquitous in nature and carry out a broad variety of difficult chemical transformations initiated by hydrogen atom abstraction. Although numerous radical SAM (RS) enzymes have been structurally characterized, many prove recalcitrant to crystallization needed for atomic-level structure determination using X-ray crystallography, and even those that have been crystallized for an initial study can be difficult to recrystallize for further structural work. We present here a method for computationally engineering previously observed crystallographic contacts and employ it to obtain more reproducible crystallization of the RS enzyme pyruvate formate-lyase activating enzyme (PFL-AE). We show that the computationally engineered variant binds a typical RS [4Fe-4S]2+/+ cluster that binds SAM, with electron paramagnetic resonance properties indistinguishable from the native PFL-AE. The variant also retains the typical PFL-AE catalytic activity, as evidenced by the characteristic glycyl radical electron paramagnetic resonance signal observed upon incubation of the PFL-AE variant with reducing agent, SAM, and PFL. The PFL-AE variant was also crystallized in the [4Fe-4S]2+ state with SAM bound, providing a new high-resolution structure of the SAM complex in the absence of substrate. Finally, by incubating such a crystal in a solution of sodium dithionite, the reductive cleavage of SAM is triggered, providing us with a structure in which the SAM cleavage products 5'-deoxyadenosine and methionine are bound in the active site. We propose that the methods described herein may be useful in the structural characterization of other difficult-to-resolve proteins.

Structural features and substrate engagement in peptide-modifying radical SAM enzymes.

In recent years, the biological significance of ribosomally synthesized, post-translationally modified peptides (RiPPs) and the intriguing chemistry catalyzed by their tailoring enzymes has garnered significant attention. A subgroup of bacterial radical S-adenosylmethionine (rSAM) enzymes can activate C-H bonds in peptides, which leads to the production of a diverse range of RiPPs. The remarkable ability of these enzymes to facilitate various chemical processes, to generate and harbor high-energy radical species, and to accommodate large substrates with a high degree of flexibility is truly intriguing. The wide substrate scope and diversity of the chemistry performed by rSAM enzymes raise one question: how does the protein environment facilitate these distinct chemical conversions while sharing a similar structural fold? In this review, we discuss recent advances in the field of RiPP-rSAM enzymes, with a particular emphasis on domain architectures and substrate engagements identified by biophysical and structural characterizations. We provide readers with a comparative analysis of six examples of RiPP-rSAM enzymes with experimentally characterized structures. Linking the structural elements and the nature of rSAM-catalyzed RiPP production will provide insight into the functional engineering of enzyme activity to harness their catalytic power in broader applications.

Multiple Profile Models Extract Features from Protein Sequence Data and Resolve Functional Diversity of Very Different Protein Families.

Functional classification of proteins from sequences alone has become a critical bottleneck in understanding the myriad of protein sequences that accumulate in our databases. The great diversity of homologous sequences hides, in many cases, a variety of functional activities that cannot be anticipated. Their identification appears critical for a fundamental understanding of the evolution of living organisms and for biotechnological applications. ProfileView is a sequence-based computational method, designed to functionally classify sets of homologous sequences. It relies on two main ideas: the use of multiple profile models whose construction explores evolutionary information in available databases, and a novel definition of a representation space in which to analyze sequences with multiple profile models combined together. ProfileView classifies protein families by enriching known functional groups with new sequences and discovering new groups and subgroups. We validate ProfileView on seven classes of widespread proteins involved in the interaction with nucleic acids, amino acids and small molecules, and in a large variety of functions and enzymatic reactions. ProfileView agrees with the large set of functional data collected for these proteins from the literature regarding the organization into functional subgroups and residues that characterize the functions. In addition, ProfileView resolves undefined functional classifications and extracts the molecular determinants underlying protein functional diversity, showing its potential to select sequences towards accurate experimental design and discovery of novel biological functions. On protein families with complex domain architecture, ProfileView functional classification reconciles domain combinations, unlike phylogenetic reconstruction. ProfileView proves to outperform the functional classification approach PANTHER, the two k-mer-based methods CUPP and eCAMI and a neural network approach based on Restricted Boltzmann Machines. It overcomes time complexity limitations of the latter.

Biomimetic S-Adenosylmethionine Regeneration Starting from Multiple Byproducts Enables Biocatalytic Alkylation with Radical SAM Enzymes.

S-Adenosylmethionine (SAM) is an enzyme cofactor involved in methylation, aminopropyl transfer, and radical reactions. This versatility renders SAM-dependent enzymes of great interest in biocatalysis. The usage of SAM analogues adds to this diversity. However, high cost and instability of the cofactor impedes the investigation and usage of these enzymes. While SAM regeneration protocols from the methyltransferase (MT) byproduct S-adenosylhomocysteine are available, aminopropyl transferases and radical SAM enzymes are not covered. Here, we report a set of efficient one-pot systems to supply or regenerate SAM and SAM analogues for all three enzyme classes. The systems' flexibility is showcased by the transfer of an ethyl group with a cobalamin-dependent radical SAM MT using S-adenosylethionine as a cofactor. This shows the potential of SAM (analogue) supply and regeneration for the application of diverse chemistry, as well as for mechanistic studies using cofactor analogues.

Sequential C-H Methylation Catalyzed by the B12 -Dependent SAM Enzyme TokK: Comprehensive Theoretical Study of Selectivities.

TokK is a B12 -dependent radical SAM enzyme involved in the biosynthesis of the ß-lactam antibiotic asparenomycin A. It can catalyze three methylations on different sp3 -hybridized carbon positions to introduce an isopropyl side chain at the ß-lactam ring of pantetheinylated carbapenem. Herein, we report a quantum chemical study of the reaction mechanism of TokK. A stepwise ''push-pull'' radical relay mechanism is proposed for each methylation: a 5'-deoxyadenosine radical first abstracts a hydrogen atom from the substrate in the active site, then methylcobalamin directionally donates a methyl group to the substrate. More importantly, calculations were able to uncover the origin of observed chemoselectivity and stereoselectivity for the first methylation and regioselectivity for the following two methylations. Further detailed distortion/interaction analysis can help to unravel the main factors controlling the selectivities. Our findings of sequential methylations by TokK could have profound implications for studying other B12 -dependent radical SAM enzymes.

Essentiality of the Escherichia coli YgfZ Protein for the In Vivo Thiomethylation of Ribosomal Protein S12 by the RimO Enzyme.

Enzymes carrying Iron-Sulfur (Fe-S) clusters perform many important cellular functions and their biogenesis require complex protein machinery. In mitochondria, the IBA57 protein is essential and promotes assembly of [4Fe-4S] clusters and their insertion into acceptor proteins. YgfZ is the bacterial homologue of IBA57 but its precise role in Fe-S cluster metabolism is uncharacterized. YgfZ is needed for activity of the radical S-adenosyl methionine [4Fe-4S] cluster enzyme MiaB which thiomethylates some tRNAs. The growth of cells lacking YgfZ is compromised especially at low temperature. The RimO enzyme is homologous to MiaB and thiomethylates a conserved aspartic acid in ribosomal protein S12. To quantitate thiomethylation by RimO, we developed a bottom-up LC-MS2 analysis of total cell extracts. We show here that the in vivo activity of RimO is very low in the absence of YgfZ and independent of growth temperature. We discuss these results in relation to the hypotheses relating to the role of the auxiliary 4Fe-4S cluster in the Radical SAM enzymes that make Carbon-Sulfur bonds.

Maturation of the [FeFe]-Hydrogenase: Direct Transfer of the (κ3 -cysteinate)FeII (CN)(CO)2 Complex B from HydG to HydE.

[FeFe]-hydrogenases efficiently catalyze the reversible oxidation of molecular hydrogen. Their prowess stems from the intricate H-cluster, combining a [Fe4 S4 ] center with a binuclear iron center ([2Fe]H ). In the latter, each iron atom is coordinated by a CO and CN ligand, connected by a CO and an azadithiolate ligand. The synthesis of this active site involves a unique multiprotein assembly, featuring radical SAM proteins HydG and HydE. HydG initiates the transformation of L-tyrosine into cyanide and carbon monoxide to generate complex B, which is subsequently transferred to HydE to continue the biosynthesis of the [2Fe]H -subcluster. Due to its instability, complex B isolation for structural or spectroscopic characterization has been elusive thus far. Nevertheless, the use of a biomimetic analogue of complex B allowed circumvention of the need for the HydG protein during in vitro functional investigations, implying a similar structure for complex B. Herein, we used the HydE protein as a nanocage to encapsulate and stabilize the complex B product generated by HydG. Using X-ray crystallography, we successfully determined its structure at 1.3 Šresolution. Furthermore, we demonstrated that complex B is directly transferred from HydG to HydE, thus not being released into the solution post-synthesis, highlighting a transient interaction between the two proteins.

Highlighting the Unique Roles of Radical S-Adenosylmethionine Enzymes in Methanogenic Archaea.

Radical S-adenosylmethionine (SAM) enzymes catalyze an impressive variety of difficult biochemical reactions in various pathways across all domains of life. These metalloenzymes employ a reduced [4Fe-4S] cluster and SAM to generate a highly reactive 5'-deoxyadenosyl radical that is capable of initiating catalysis on otherwise unreactive substrates. Interestingly, the genomes of methanogenic archaea encode many unique radical SAM enzymes with underexplored or completely unknown functions. These organisms are responsible for the yearly production of nearly 1 billion tons of methane, a potent greenhouse gas as well as a valuable energy source. Thus, understanding the details of methanogenic metabolism and elucidating the functions of essential enzymes in these organisms can provide insights into strategies to decrease greenhouse gas emissions as well as inform advances in bioenergy production processes. This minireview provides an overview of the current state of the field regarding the functions of radical SAM enzymes in methanogens and discusses gaps in knowledge that should be addressed.

The antiviral enzyme viperin inhibits cholesterol biosynthesis.

Many enveloped viruses bud from cholesterol-rich lipid rafts on the cell membrane. Depleting cellular cholesterol impedes this process and results in viral particles with reduced viability. Viperin (Virus Inhibitory Protein, Endoplasmic Reticulum-associated, Interferon iNducible) is an endoplasmic reticulum membrane-associated enzyme that exerts broad-ranging antiviral effects, including inhibiting the budding of some enveloped viruses. However, the relationship between viperin expression and the retarded budding of virus particles from lipid rafts on the cell membrane is unclear. Here, we investigated the effect of viperin expression on cholesterol biosynthesis using transiently expressed genes in the human cell line human embryonic kidney 293T (HEK293T). We found that viperin expression reduces cholesterol levels by 20% to 30% in these cells. Following this observation, a proteomic screen of the viperin interactome identified several cholesterol biosynthetic enzymes among the top hits, including lanosterol synthase (LS) and squalene monooxygenase (SM), which are enzymes that catalyze key steps in establishing the sterol carbon skeleton. Coimmunoprecipitation experiments confirmed that viperin, LS, and SM form a complex at the endoplasmic reticulum membrane. While coexpression of viperin was found to significantly inhibit the specific activity of LS in HEK293T cell lysates, coexpression of viperin had no effect on the specific activity of SM, although did reduce SM protein levels by approximately 30%. Despite these inhibitory effects, the coexpression of neither LS nor SM was able to reverse the viperin-induced depletion of cellular cholesterol levels, possibly because viperin is highly expressed in transfected HEK293T cells. Our results establish a link between viperin expression and downregulation of cholesterol biosynthesis that helps explain viperin's antiviral effects against enveloped viruses.

A bioactive molecule made by unusual salvage of radical SAM enzyme byproduct 5-deoxyadenosine blurs the boundary of primary and secondary metabolism.

5-Deoxyadenosine (5dAdo) is the byproduct of many radical S-adenosyl-l-methionine enzyme reactions in all domains of life. 5dAdo is also an inhibitor of the radical S-adenosyl-l-methionine enzymes themselves, making it necessary for cells to construct pathways to recycle or dispose of this toxic metabolite. However, the specific pathways involved have long remained unexplored. Recent research demonstrated a growth advantage in certain organisms by using 5dAdo or intermediates as a sole carbon source and elucidated the corresponding salvage pathway. We now provide evidence using supernatant analysis by GC-MS for another 5dAdo recycling route. Specifically, in the unicellular cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus), the activity of promiscuous enzymes leads to the synthesis and excretion first of 5-deoxyribose and subsequently of 7-deoxysedoheptulose. 7-Deoxysedoheptulose is an unusual deoxy-sugar, which acts as an antimetabolite of the shikimate pathway, thereby exhibiting antimicrobial and herbicidal activity. This strategy enables organisms with small genomes and lacking canonical gene clusters for the synthesis of secondary metabolites, like S. elongatus, to produce antimicrobial compounds from primary metabolism and enzymatic promiscuity. Our findings challenge the view of bioactive molecules as sole products of secondary metabolite gene clusters and expand the range of compounds that microorganisms can deploy to compete for their ecological niche.

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.