Radical SAM Superfamily Research Articles

Alternate routes to mnm5s2U synthesis in Gram-positive bacteria.

The wobble bases of tRNAs that decode split codons are often heavily modified. In bacteria, tRNAGlu, Gln, Asp contains a variety of xnm5s2U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negative Escherichia coli K12 model. Despite the ubiquitous presence of mnm5s2U modification, genomic analysis shows the absence of mnmC orthologous genes, suggesting the occurrence of alternate biosynthetic schemes for the conversion of cmnm5s2U to mnm5s2U. Using a combination of comparative genomics and genetic studies, a member of the YtqA subgroup of the radical Sam superfamily was found to be involved in the synthesis of mnm5s2U in both Bacillus subtilis and Streptococcus mutans. This protein, renamed MnmL, is encoded in an operon with the recently discovered MnmM methylase involved in the methylation of the pathway intermediate nm5s2U into mnm5s2U in B. subtilis. Analysis of tRNA modifications of both S. mutans and Streptococcus pneumoniae shows that growth conditions and genetic backgrounds influence the ratios of pathway intermediates owing to regulatory loops that are not yet understood. The MnmLM pathway is widespread along the bacterial tree, with some phyla, such as Bacilli, relying exclusively on these two enzymes. Although mechanistic details of these newly discovered components are not fully resolved, the occurrence of fusion proteins, alternate arrangements of biosynthetic components, and loss of biosynthetic branches provide examples of biosynthetic diversity to retain a conserved tRNA modification in Nature.IMPORTANCEThe xnm5s2U modifications found in several tRNAs at the wobble base position are widespread in bacteria where they have an important role in decoding efficiency and accuracy. This work identifies a novel enzyme (MnmL) that is a member of a subgroup of the very versatile radical SAM superfamily and is involved in the synthesis of mnm5s2U in several Gram-positive bacteria, including human pathogens. This is another novel example of a non-orthologous displacement in the field of tRNA modification synthesis, showing how different solutions evolve to retain U34 tRNA modifications.

Expression analysis and antiviral activity of koi carp (Cyprinus carpio) viperin against carp edema virus (CEV)

Viperin, also known as radical S-Adenosyl methionine domain containing 2 (RSAD2), is an IFN stimulated protein that plays crucial roles in innate immunity. Here, we identified a viperin gene from the koi carp (Cyprinus carpio) (kVip). The ORF of kVip is 1047 bp in length, encoding a polypeptide of 348 amino acids with neither signal peptide nor transmembrane protein. The predicted molecular weight is 40.37 kDa and the isoelectric point is 7.7. Multiple sequence alignment indicated that putative kVip contains a radical SAM superfamily domain and a conserved C-terminal region. kVip was highly expressed in the skin and spleen of healthy koi carps, and significantly stimulated in both natural and artificial CEV-infected koi carps. In vitro immune stimulation analysis showed that both extracellular and intracellular poly (I: C) or poly (dA: dT) caused a significant increase in kVip expression of spleen cells. Furthermore, intraperitoneal injection of recombinant kVip (rkVip) not only reduced the CEV load in the gills, but also improved the survival of koi carps following CEV challenge. Additionally, rkVip administration effectively regulated inflammatory and anti-inflammatory cytokines (IL-6, IL-1β, TNF-α, IL-10) and interferon-related molecules (cGAS, STING, MyD88, IFN-γ, IFN-α, IRF3 and IRF9). Collectively, kVip effectively responded to CEV infection and exerted antiviral function against CEV partially by regulation of inflammatory and interferon responses.

Discovery of a Biotin Synthase That Utilizes an Auxiliary 4Fe-5S Cluster for Sulfur Insertion.

Biotin synthase (BioB) is a member of the Radical SAM superfamily of enzymes that catalyzes the terminal step of biotin (vitamin B7) biosynthesis, in which it inserts a sulfur atom in desthiobiotin to form a thiolane ring. How BioB accomplishes this difficult reaction has been the subject of much controversy, mainly around the source of the sulfur atom. However, it is now widely accepted that the sulfur atom inserted to form biotin stems from the sacrifice of the auxiliary 2Fe-2S cluster of BioB. Here, we bioinformatically explore the diversity of BioBs available in sequence databases and find an unexpected variation in the coordination of the auxiliary iron-sulfur cluster. After in vitro characterization, including the determination of biotin formation and representative crystal structures, we report a new type of BioB utilized by virtually all obligate anaerobic organisms. Instead of a 2Fe-2S cluster, this novel type of BioB utilizes an auxiliary 4Fe-5S cluster. Interestingly, this auxiliary 4Fe-5S cluster contains a ligated sulfide that we propose is used for biotin formation. We have termed this novel type of BioB, Type II BioB, with the E. coli 2Fe-2S cluster sacrificial BioB representing Type I. This surprisingly ubiquitous Type II BioB has implications for our understanding of the function and evolution of Fe-S clusters in enzyme catalysis, highlighting the difference in strategies between the anaerobic and aerobic world.

Inside Cover Picture

This cover picture depicts an unprecedented chemistry of the radical SAM superfamily enzymes, which removes the chelated iron from heme, one of the most abundant cofactors in nature. Demetallation of heme is a rather difficult chemical reaction, which barely occurs in physiological conditions. The remarkable reaction of ChuW is reminiscent of a story from the Legend of Sword and Fairy, a Chinese fantasy game. Only the fairy Zhao Ling’er is able to pick the Sacred Deads from the cleric and call for a rainstorm for the Miao people, restoring peace to the land. More details are given in the article by zhang et al. on page 1053—1058.image

Broken-Symmetry Density Functional Theory Analysis of the Ω Intermediate in Radical S-Adenosyl-l-methionine Enzymes: Evidence for a Near-Attack Conformer over an Organometallic Species.

Radical S-adenosyl-l-methionine (SAM) enzymes are found in all domains of life and catalyze a wide range of biochemical reactions. Recently, an organometallic intermediate, Ω, has been experimentally implicated in the 5'-deoxyadenosyl radical generation mechanism of the radical SAM superfamily. In this work, we employ broken-symmetry density functional theory to evaluate several structural models of Ω. The results show that the calculated hyperfine coupling constants (HFCCs) for the proposed organometallic structure of Ω are inconsistent with the experiment. In contrast, a near-attack conformer of SAM bound to the catalytic [4Fe-4S] cluster, in which the distance between the unique iron and SAM sulfur is ∼3 Å, yields HFCCs that are all within 1 MHz of the experimental values. These results clarify the structure of the ubiquitous Ω intermediate and suggest a paradigm shift reversal regarding the mechanism of SAM cleavage by members of the radical SAM superfamily.

RadicalSAM.org: A Resource to Interpret Sequence-Function Space and Discover New Radical SAM Enzyme Chemistry.

The radical SAM superfamily (RSS), arguably the most functionally diverse enzyme superfamily, is also one of the largest with ~700K members currently in the UniProt database. The vast majority of the members have uncharacterized enzymatic activities and metabolic functions. In this Perspective, we describe RadicalSAM.org, a new web-based resource that enables a user-friendly genomic enzymology strategy to explore sequence-function space in the RSS. The resource attempts to enable identification of isofunctional groups of radical SAM enzymes using sequence similarity networks (SSNs) and the genome context of the bacterial, archaeal, and fungal members provided by genome neighborhood diagrams (GNDs). Enzymatic activities and in vivo functions frequently can be inferred from genome context given the tendency for genes of related function to be clustered. We invite the scientific community to use RadicalSAM.org to (i) guide their experimental studies to discover new enzymatic activities and metabolic functions, (ii) contribute experimentally verified annotations to RadicalSAM.org to enhance the ability to predict novel activities and functions, and (iii) provide suggestions for improving this resource.

Lignin induced iron reduction by novel sp., Tolumonas lignolytic BRL6-1.

Lignin is the second most abundant carbon polymer on earth and despite having more fuel value than cellulose, it currently is considered a waste byproduct in many industrial lignocellulose applications. Valorization of lignin relies on effective and green methods of de-lignification, with a growing interest in the use of microbes. Here we investigate the physiology and molecular response of the novel facultative anaerobic bacterium, Tolumonas lignolytica BRL6-1, to lignin under anoxic conditions. Physiological and biochemical changes were compared between cells grown anaerobically in either lignin-amended or unamended conditions. In the presence of lignin, BRL6-1 accumulates higher biomass and has a shorter lag phase compared to unamended conditions, and 14% of the proteins determined to be significantly higher in abundance by log2 fold-change of 2 or greater were related to Fe(II) transport in late logarithmic phase. Ferrozine assays of the supernatant confirmed that Fe(III) was bound to lignin and reduced to Fe(II) only in the presence of BRL6-1, suggesting redox activity by the cells. LC-MS/MS analysis of the secretome showed an extra band at 20 kDa in lignin-amended conditions. Protein sequencing of this band identified a protein of unknown function with homology to enzymes in the radical SAM superfamily. Expression of this protein in lignin-amended conditions suggests its role in radical formation. From our findings, we suggest that BRL6-1 is using a protein in the radical SAM superfamily to interact with the Fe(III) bound to lignin and reducing it to Fe(II) for cellular use, increasing BRL6-1 yield under lignin-amended conditions. This interaction potentially generates organic free radicals and causes a radical cascade which could modify and depolymerize lignin. Further research should clarify the extent to which this mechanism is similar to previously described aerobic chelator-mediated Fenton chemistry or radical producing lignolytic enzymes, such as lignin peroxidases, but under anoxic conditions.

Sulfonium-Based Homolytic Substitution Observed for the Radical SAM Enzyme HemN.

Sulfur-based homolytic substitution (SH reaction) plays an important role in synthetic chemistry, yet whether such a reaction could occur on the positively charged sulfonium compounds remains unknown. In the study of the anaerobic coproporphyrinogen III oxidase HemN, a radical S-adenosyl-l-methionine (SAM) enzyme involved in heme biosynthesis, we observed the production of di-(5'-deoxyadenosyl)methylsulfonium, which supports a deoxyadenosyl (dAdo) radical-mediated SH reaction on the sulfonium center of SAM. The sulfonium-based SH reactions were then investigated in detail by density functional theory calculations and model reactions, which showed that this type of reactions is thermodynamically favorable and kinetically competent. These findings represent the first report of sulfonium-based SH reactions, which could be useful in synthetic chemistry. Our study also demonstrates the remarkable catalytic promiscuity of the radical SAM superfamily enzymes.

Sulfonium‐Based Homolytic Substitution Observed for the Radical SAM Enzyme HemN

AbstractSulfur‐based homolytic substitution (SH reaction) plays an important role in synthetic chemistry, yet whether such a reaction could occur on the positively charged sulfonium compounds remains unknown. In the study of the anaerobic coproporphyrinogen III oxidase HemN, a radical S‐adenosyl‐l‐methionine (SAM) enzyme involved in heme biosynthesis, we observed the production of di‐(5′‐deoxyadenosyl)methylsulfonium, which supports a deoxyadenosyl (dAdo) radical‐mediated SH reaction on the sulfonium center of SAM. The sulfonium‐based SH reactions were then investigated in detail by density functional theory calculations and model reactions, which showed that this type of reactions is thermodynamically favorable and kinetically competent. These findings represent the first report of sulfonium‐based SH reactions, which could be useful in synthetic chemistry. Our study also demonstrates the remarkable catalytic promiscuity of the radical SAM superfamily enzymes.

Radical S-adenosylmethionine maquette chemistry: Cx3Cx2C peptide coordinated redox active [4Fe-4S] clusters.

The synthesis and characterization of short peptide-based maquettes of metalloprotein active sites facilitate an inquiry into their structure/function relationships and evolution. The [4Fe-4S]-maquettes of bacterial ferredoxin metalloproteins (Fd) have been used in the past to engineer redox active centers into artificial metalloenzymes. 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). The radical SAM superfamily enzymes contain site-differentiated, redox active [4Fe-4S] clusters coordinated to Cx3Cx2C or related motifs, which is in contrast to the Cx2Cx2C motif found in bacterial ferredoxins (Fd). Under an optimized set of experimental conditions, a high degree of reconstitution (80-100%) was achieved for both radical SAM- and Fd-maquettes. Negligible chemical speciation was observed for all sequences, with predominantly [4Fe-4S]2+ for the 'as-reconstituted' state. However, the reduction of [4Fe-4S]2+-maquettes provides low conversion (7-17%) to the paramagnetic [4Fe-4S]+ state, independent of either the spacing of the cysteine residues (Cx3Cx2C vs. Cx2Cx2C), the nature of intervening amino acids, or the length of the cluster binding motif. In the absence of the stabilizing protein environment, the reduction process is proposed to proceed via [4Fe-4S]2+ cluster disassembly and reassembly in a more reduced state. UV-Vis and EPR spectroscopic techniques are employed as analytical tools to quantitate the as-reconstituted (or oxidized) and one-electron reduced states of the [4Fe-4S] clusters, respectively. We demonstrate that short Fd and radical SAM derived 7- to 9-mer peptides containing appropriate cysteine motifs function equally well in coordinating redox active [4Fe-4S] clusters.

The A-type domain in Escherichia coli NfuA is required for regenerating the auxiliary [4Fe–4S] cluster in Escherichia coli lipoyl synthase

The lipoyl cofactor plays an integral role in several essential biological processes. The last step in its de novo biosynthetic pathway, the attachment of two sulfur atoms at C6 and C8 of an n-octanoyllysyl chain, is catalyzed by lipoyl synthase (LipA), a member of the radical SAM superfamily. In addition to the [4Fe-4S] cluster common to all radical SAM enzymes, LipA contains a second [4Fe-4S] auxiliary cluster, which is sacrificed during catalysis to supply the requisite sulfur atoms, rendering the protein inactive for further turnovers. Recently, it was shown that the Fe-S cluster carrier protein NfuA from Escherichia coli can regenerate the auxiliary cluster of E. coli LipA after each turnover, but the molecular mechanism is incompletely understood. Herein, using protein-protein interaction and kinetic assays as well as site-directed mutagenesis, we provide further insight into the mechanism of NfuA-mediated cluster regeneration. In particular, we show that the N-terminal A-type domain of E. coli NfuA is essential for its tight interaction with LipA. Further, we demonstrate that NfuA from Mycobacterium tuberculosis can also regenerate the auxiliary cluster of E. coli LipA. However, an Nfu protein from Staphylococcus aureus, which lacks the A-type domain, was severely diminished in facilitating cluster regeneration. Of note, addition of the N-terminal domain of E. coli NfuA to S. aureus Nfu, fully restored cluster-regenerating activity. These results expand our understanding of the newly discovered mechanism by which the auxiliary cluster of LipA is restored after each turnover.

Parsing redox potentials of five ferredoxins found within Thermotoga maritima.

Most organisms contain multiple soluble protein-based redox carriers such as members of the ferredoxin (Fd) family, that contain one or more iron-sulfur clusters. 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 [Fe4 S4 ]1+ oxidation state. As it is still unclear whether a single Fd in a given organism is specific to individual redox partners, we have examined the five Fd proteins found within Thermotoga maritima via direct electrochemistry, to compare them in a side-by-side fashion for the first time. While a single [Fe4 S4 ]-cluster bearing Fd (TM0927) has a potential of -420 mV, the other four 2x[Fe4 S4 ]-bearing Fds (TM1175, TM1289, TM1533, and TM1815) have potentials that vary significantly, including cases where the two clusters of the same Fd are essentially coincident (e.g., TM1175) and those where the potentials are well separate (TM1815).

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.

Atlas of the Radical SAM Superfamily: Divergent Evolution of Function Using a "Plug and Play" Domain.

The radical SAM superfamily contains over 100,000 homologous enzymes that catalyze a remarkably broad range of reactions required for life, including metabolism, nucleic acid modification, and biogenesis of cofactors. While the highly conserved SAM-binding motif responsible for formation of the key 5'-deoxyadenosyl radical intermediate is a key structural feature that simplifies identification of superfamily members, our understanding of their structure-function relationships is complicated by the modular nature of their structures, which exhibit varied and complex domain architectures. To gain new insight about these relationships, we classified the entire set of sequences into similarity-based subgroups that could be visualized using sequence similarity networks. This superfamily-wide analysis reveals important features that had not previously been appreciated from studies focused on one or a few members. Functional information mapped to the networks indicates which members have been experimentally or structurally characterized, their known reaction types, and their phylogenetic distribution. Despite the biological importance of radical SAM chemistry, the vast majority of superfamily members have never been experimentally characterized in any way, suggesting that many new reactions remain to be discovered. In addition to 20 subgroups with at least one known function, we identified additional subgroups made up entirely of sequences of unknown function. Importantly, our results indicate that even general reaction types fail to track well with our sequence similarity-based subgroupings, raising major challenges for function prediction for currently identified and new members that continue to be discovered. Interactive similarity networks and other data from this analysis are available from the Structure-Function Linkage Database.

Purification, Characterization, and Biochemical Assays of Biotin Synthase From Escherichia coli.

Biotin synthase (BioB) catalyzes the oxidative insertion of a sulfur atom between the C6 methylene and the C9 methyl positions in dethiobiotin. The enzyme couples oxidation of each carbon position to reduction of the S-adenosyl-l-methionine (SAM) sulfonium center, generating 5'-deoxyadenosine and l-methionine, products that are characteristic of enzymes from the radical SAM superfamily. In bacteria, biotin biosynthesis is tightly regulated by the dual-function BirA repressor/holocarboxylase synthetase, resulting in very low levels of all biotin biosynthetic enzymes such that activity-based purification of BioB from the native organism is virtually impossible. However, overexpression and purification of recombinant BioB from E. coli are straight forward and, in contrast with many radical SAM enzymes, can be carried out under aerobic conditions. The active enzyme contains two iron-sulfur clusters, and the characterization and manipulation of these clusters are essential for a thorough understanding of enzyme catalysis and stability. An optimized in vitro assay for BioB is described herein that requires use of an auxiliary protein reducing system and must be carried out under anaerobic conditions to prevent oxidative damage to the reduced iron-sulfur clusters. Three methods for detection of biotin are described, with discussion of the advantages and limitations of each method. Challenges that may be encountered in adapting these assays to other organisms are also discussed.

Aminofutalosine Synthase (MqnE): A New Catalytic Motif in Radical SAM Enzymology.

Aminofutalosine synthase (MqnE) is a radical SAM enzyme involved in the futalosine-dependent menaquinone biosynthetic pathway. Its ability to add the 5'-deoxyadenosyl radical to the substrate-rather than abstract a hydrogen atom-and to catalyze radical addition to a stable benzene ring gives it a unique place in the radical SAM superfamily and required the development of new strategies for trapping radical intermediates. This chapter describes the methodologies used for enzyme overexpression, purification, and in vitro reconstitution. We also describe the development of fast, radical triggered, carbon-halogen bond fragmentation reactions for the trapping of intermediates. We anticipate that these methods will be of general use in the study of other transient enzymatic radicals.

Radical Breakthroughs in Natural Product and Cofactor Biosynthesis.

The radical SAM (S-adenosyl-l-methionine) superfamily is one of the largest group of enzymes with >113000 annotated sequences [Landgraf, B. J., et al. (2016) Annu. Rev. Biochem. 85, 485-514]. Members of this superfamily catalyze the reductive cleavage of SAM using an oxygen sensitive 4Fe-4S cluster to transiently generate 5'-deoxyadenosyl radical that is subsequently used to initiate diverse free radical-mediated reactions. Because of the unique reactivity of free radicals, radical SAM enzymes frequently catalyze chemically challenging reactions critical for the biosynthesis of unique structures of cofactors and natural products. In this Perspective, I will discuss the impact of characterizing novel functions in radical SAM enzymes on our understanding of biosynthetic pathways and use two recent examples from my own group with a particular emphasis on two radical SAM enzymes that are responsible for carbon skeleton formation during the biosynthesis of a cofactor and natural products.

Characterization of auxiliary iron-sulfur clusters in a radical S-adenosylmethionine enzyme PqqE from Methylobacterium extorquens AM1.

PqqE is a radical S‐adenosyl‐l‐methionine (SAM) enzyme that catalyzes the initial reaction of pyrroloquinoline quinone (PQQ) biosynthesis. PqqE belongs to the SPASM (subtilosin/PQQ/anaerobic sulfatase/mycofactocin maturating enzymes) subfamily of the radical SAM superfamily and contains multiple Fe–S clusters. To characterize the Fe–S clusters in PqqE from Methylobacterium extorquens AM1, Cys residues conserved in the N‐terminal signature motif (CX 3 CX 2C) and the C‐terminal seven‐cysteine motif (CX 9–15 GX 4 CX n CX 2 CX 5 CX 3 CX nC; n = an unspecified number) were individually or simultaneously mutated into Ser. Biochemical and Mössbauer spectral analyses of as‐purified and reconstituted mutant enzymes confirmed the presence of three Fe–S clusters in PqqE: one [4Fe–4S]2+ cluster at the N‐terminal region that is essential for the reductive homolytic cleavage of SAM into methionine and 5′‐deoxyadenosyl radical, and one each [4Fe–4S]2+ and [2Fe–2S]2+ auxiliary clusters in the C‐terminal SPASM domain, which are assumed to serve for electron transfer between the buried active site and the protein surface. The presence of [2Fe–2S]2+ cluster is a novel finding for radical SAM enzyme belonging to the SPASM subfamily. Moreover, we found uncommon ligation of the auxiliary [4Fe–4S]2+ cluster with sulfur atoms of three Cys residues and a carboxyl oxygen atom of a conserved Asp residue.

On the Role of Additional [4Fe-4S] Clusters with a Free Coordination Site in Radical-SAM Enzymes.

The canonical CysXXXCysXXCys motif is the hallmark of the Radical-SAM superfamily. This motif is responsible for the ligation of a [4Fe-4S] cluster containing a free coordination site available for SAM binding. The five enzymes MoaA, TYW1, MiaB, RimO and LipA contain in addition a second [4Fe-4S] cluster itself bound to three other cysteines and thus also displaying a potentially free coordination site. This review article summarizes recent important achievements obtained on these five enzymes with the main focus to delineate the role of this additional [4Fe-4S] cluster in catalysis.

DNA Repair by the Radical SAM Enzyme Spore Photoproduct Lyase: From Biochemistry to Structural Investigations

Radical S-adenosyl-L-methionine (SAM) enzymes have emerged as one of the last superfamilies of enzymes discovered to date. Arguably, it is the most versatile group of enzymes involved in at least 85 biochemical transformations. One of the founding members of this enzyme superfamily is the spore photoproduct (SP) lyase, a DNA repair enzyme catalyzing the direct reversal repair of a unique DNA lesion, the so-called spore photoproduct, back into two thymidine residues. Discovered more than 20years ago in the bacterium Bacillus subtilis, SP lyase has been shown to be widespread in the endospore-forming Firmicutes from the Bacilli and Clostridia classes and to use radical-based chemistry to perform C-C bond breakage, a chemically challenging reaction. This review describes how the work on SP lyase has illuminated a unique strategy for DNA repair and provided major advances in our understanding of the emerging radical SAM superfamily of enzymes, from a biochemical and structural perspective.