Introduction to Radical Sam

NifB, a radical SAM enzyme, catalyzes the biosynthesis of the L cluster (Fe8S9C), a structural homolog and precursor to the nitrogenase active-site M cluster ([MoFe7S9C·R-homocitrate]).

HygY is a SPASM/twitch radical SAM enzyme hypothesized to catalyze the C2'-epimerization of galacamine during the biosynthesis of hygromycin B.

These transformations share conceptual similarities with radical SAM enzymes found in microbial carbohydrate biosynthesis and present opportunities for synthetic chemists to access microbial and unnatural carbohydrate building blocks without the need for protecting groups or lengthy synthetic sequences.

It is the only 1,2-diol dehydratase in the radical SAM enzyme superfamily that has been identified and characterized in vitro.

elongatus, 7dSh is exclusively synthesized by promiscuous enzyme activity from an inhibitory by-product of radical SAM enzymes, without a specific gene cluster being involved.

Viperin is a broadly conserved radical SAM enzyme that synthesizes the antiviral nucleotide ddhCTP.

The family of radical SAM RNA-methylating enzymes comprises a large group of proteins that contains only a few functionally characterized members.

Additionally, co-immunoprecipitation studies revealed that viperin associated with NSP4 through regions including both its radical SAM domain and its C-terminal domain.

Viperin is a broadly conserved radical SAM enzyme that synthesizes the antiviral nucleotide ddhCTP.

The potential involvement of a radical SAM mechanism remains to be determined.

These appended amino acids are further sculpted by various enzyme classes such as radical SAM isomerases, PLP-dependent decarboxylases, flavin-dependent lyases and acetyltransferases.

As a radical SAM protein, MiaB contains one [Fe4S4]RS cluster used in the reductive cleavage of SAM to form a 5'-deoxyadenosyl 5'-radical, which is responsible for removing the C2 hydrogen of the substrate5.

TsrM is annotated as a cobalamin-dependent radical S -adenosylmethionine (SAM) methylase; however, TsrM does not reductively cleave SAM to the universal 5ʹ-deoxyadenosyl 5ʹ-radical intermediate, a hallmark of radical SAM (RS) enzymes.

Aminofutalosine synthase (MqnE) is a radical SAM enzyme that catalyzes the conversion of 3-((1-carboxyvinyl)oxy)benzoic acid to aminofutalosine during the futalosine-dependent menaquinone biosynthesis.

Homologues of TYW1 are found in both archaea and eukarya; archaeal homologues consist of a single domain, while eukaryal homologues contain a flavin binding domain in addition to the radical SAM domain shared with archaeal homologues.

This study not only reveals a key step in arsenosugar biosynthesis but also a new paradigm in radical SAM chemistry, highlighting the remarkable catalytic diversity of this superfamily of enzymes.

The structures give the first visualization of a cobalamin-dependent radical SAM methylase that employs the radical mechanism shared by a vast majority of these enzymes.

In nature, the radical SAM enzyme family plays a fascinating role in the radical chemistry, and the majority of them catalyze the cleavage of the S-C5′ bond to initiate the radical-based catalysis.

In addition to previously proposed mechanisms, we propose that impaired activity of "radical SAM" enzymes will result in reduced endogenous lipoic acid synthesis, reduced molybdenum cofactor synthesis and impaired porphyrin metabolism leading to mitochondrial dysfunction, porphyrinuria and impaired sulfation capacity.

Most radical SAM enzymes work via a mechanism in which cleavage.

In this review, we report on recent developments in radical SAM enzymology and how these unique biocatalysts have been shown to install complex and sometimes unprecedented posttranslational modifications in RiPPs with a special focus on microbiome derived enzymes.

Despite the extensive studies of structure-function relationships of radical SAM (RS) enzymes, the electronic state-dependent reactivity of [4Fe-4S] cluster in these enzymes remains elusive.

1H NMR spectroscopy can therefore contribute to the description of the catalityc mechanism of radical SAM enzymes.

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.

In the first step of diphthamide biosynthesis, a [4Fe–4S] cluster-containing radical SAM enzyme, Dph1–Dph2 heterodimer in eukaryotes or Dph2 homodimer in archaea, cleaves S-adenosylmethionine and transfers the 3-amino-3-carboxypropyl group to EF2.

Although TsrM is a member of the radical SAM superfamily, unlike all other annotated members, it does not catalyze a reductive cleavage of SAM to a 5′-deoxyadenosyl 5′-radical intermediate.

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.

When NS5A was overexpressed, this inhibited the replication of CSFV, and we determined that the radical SAM domain and N‐terminal domain of viperin was critical for its ability to bind to NS5A, with the latter being most important for this interaction.

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.

Results from SAM cleavage reactions support the supposition that PT is a radical SAM reaction.

The potential redundancy of Fd proteins is poorly understood, particularly in connection to the ability of Fd proteins to deliver reducing equivalents to members of the “radical SAM,” or S‐adenosylmethionine radical enzyme (ARE) superfamily, where the activity of all known AREs requires that an essential iron–sulfur cluster bound by the enzyme be reduced to the catalytically relevant [Fe4S4]1+ oxidation state.

Analysis of viperins functional domains required for anti-bacterial activity revealed the importance of both viperin’s N-terminal, and its radical SAM enzymatic function.

This reaction is catalyzed by the radical SAM enzyme PqqE.

The gene product, ArsS, contains a characteristic CX3CX2C motif which is typical for the radical SAM superfamily.

In vitro biochemical investigations reveal that SAM is converted to AZE in the presence of VioH while MAZ is generated by coexpression of VioH and the radical SAM enzyme VioG in Myxococcus xanthus or by combination of VioH and the cell lysate of M.

However, the genome of this organism encodes five ferredoxins (TM0927, TM1175, TM1289, TM1533, and TM1815), each of which might donate the requisite electron to MiaB and perhaps to other radical SAM enzymes.

The radical SAM enzyme HydG generates CO- and CN--containing Fe complexes that are involved in the bioassembly of the [FeFe] hydrogenase active cofactor, the H-cluster.

Viperin (also known as radical SAM domain–containing 2 (RSAD2)) is an interferon-inducible and evolutionary conserved protein that participates in the cell's innate immune response against a number of viruses.

Fourteen of these clusters originated from microbes that were not increased in the patients, and the most significantly enriched one encoded a putative efflux protein and a radical SAM protein, indicating a potential role in PD.

The radical SAM enzyme viperin exerts a wide range of antiviral effects through both the synthesis of the antiviral nucleotide 3’-deoxy-3’, 4’-didehydro-CTP (ddhCTP) and through its interactions with various cellular and viral proteins.

Here we report investigation of the adenosylation reactions catalyzed by four radical SAM l-Tyr lyases (RSTLs), including HydG, FbiC, and two ThiH enzymes from different organisms.

Specifically, Broderick’s Bader Award recognizes her for making “seminal contributions to understanding the role of the 4Fe4S cluster in the Radical SAM Enzyme Superfamily: SAM binding and a novel organometallic intermediate.

The latter enzymes are termed radical SAM enzymes.

The interest in methyl group C-H bond activation near or bound to iron-containing clusters is of key biological importance, due to the broad relevance of radical SAM reactions.

The last step of archaeosine biosynthesis was found to involve two enzymes, the previously known ArcS with lysine transfer activity and a novel identified radical SAM enzyme named RaSEA.

Virus-inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) is a radical SAM enzyme that plays a multifaceted role in the cellular antiviral response.

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 this study, we identify 2 radical SAM proteins in Sulfolobus acidocaldarius that are required to produce these molecules.

We have applied this new screening method to the radical SAM enzyme CPH4 synthase (QueE), following a detailed molecular dynamics (MD) analysis that clarifies the role of both specific enzyme residues and bound Mg2+, Ca2+ or Na+.

The past decade has experienced an emergence of radical SAM enzymes that catalyze C-C bond formation reactions in a variety of pathways, including natural product and cofactor biosynthesis and RNA modifications.

These four proteins are now fairly well-characterized and span radical SAM activity (PqqE), aided by a peptide chaperone (PqqD), a dual hydroxylase (PqqB), and an eight-electron, eight-proton oxidase (PqqC).

This gene cluster which encodes notably for peptide precursors and putative radical SAM enzymes, has been proposed to be responsible for the biosynthesis of ruminococcin C (RumC), a ribosomally synthesized and posttranslationally modified peptide (RiPP) with potent activity against the human pathogen Clostridium perfringens.

Complex carbapenem β-lactam antibiotics contain diverse C6 alkyl substituents constructed by cobalamin-dependent radical SAM enzymes.

We show two radical SAM enzymes, tryptophan lyase NosL and the class C radical SAM methyltransferase NosN, are able to act on a sulfoxide SAHO and a sulfone SAHO2, both of which are structurally similar to SAM.

In addition, the radical SAM domain and C-terminal sequences of the predicted Viperin protein are highly conserved among various species.

S-Adenosyl methionine (SAM) is employed as a [4Fe-4S]-bound cofactor in the superfamily of radical SAM (rSAM) enzymes, in which one-electron reduction of the [4Fe-4S]-SAM moiety leads to homolytic cleavage of the S-adenosyl methionine to generate the 5′-deoxyadenosyl radical (5′dAdo•), a potent H-atom abstractor.

We also provide evidence for viperin’s radical SAM enzymatic activity to self-limit its immunomodulatory functions.

SCIFFs feature a different paradigm of non-α carbon thioether linkages, and they are exclusively formed by radical SAM enzymes, as opposed to the polar chemistry employed during lanthipeptide biosynthesis.

The cytosolic domain contains a radical SAM domain with a [4Fe-4S] cluster coordinated by three cysteine residues in the active site.

In an unusual twist, human cytomegalovirus (HCMV) co-opts the antiviral radical SAM enzyme, viperin (Virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible), to enhance viral infectivity.

Given that Mmp10 has not been annotated as a cobalamin-binding protein, these findings suggest that cobalamin-dependent radical SAM proteins are more prevalent than previously thought.

The novelty of our study is the application of maquettes to the superfamily of [4Fe–4S] cluster and S-adenosylmethionine-dependent radical metalloenzymes (radical SAM).

Callistemonol A (1) represents an example of a natural dibenzofuran with two phenyl moieties, and a plausible biogenetic pathway to generate this novel dibenzofuran through a C-C bond-forming radical SAM enzyme is proposed.

Interestingly, the hysteresis loop of the radical SAM is notably reduced when irradiated with NIR light, which we attribute to the bistable nature of this SAM in which neutral radical dyad molecules are excited into a zwitterionic state following a light driven intramolecular charge transfer (ICT) from the Fc unit to the PTM radical unit.