Mechanistic studies of the radical SAM enzyme spore photoproduct lyase (SPL)

Spore photoproduct lyase (SPL) repairs a special thymine dimer 5-thyminyl-5,6-dihydrothymine, which is commonly called spore photoproduct or SP at the bacterial early germination phase. SP is the exclusive DNA photo-damage product in bacterial endospores; its generation and swift repair by SPL are responsible for the spores' extremely high UV resistance. The early in vivo studies suggested that SPL utilizes a direct reversal strategy to repair the SP in the absence of light. The research in the past decade further established SPL as a radical SAM enzyme, which utilizes a tri-cysteine CXXXCXXC motif to harbor a [4Fe-4S] cluster. At the 1+ oxidation state, the cluster provides an electron to the S-adenosylmethionine (SAM), which binds to the cluster in a bidentate manner as the fourth and fifth ligands, to reductively cleave the CS bond associated with the sulfonium ion in SAM, generating a reactive 5'-deoxyadenosyl (5'-dA) radical. This 5'-dA radical abstracts the proR hydrogen atom from the C6 carbon of SP to initiate the repair process; the resulting SP radical subsequently fragments to generate a putative thymine methyl radical, which accepts a back-donated H atom to yield the repaired TpT. SAM is suggested to be regenerated at the end of each catalytic cycle; and only a catalytic amount of SAM is needed in the SPL reaction. The H atom source for the back donation step is suggested to be a cysteine residue (C141 in Bacillus subtilis SPL), and the H-atom transfer reaction leaves a thiyl radical behind on the protein. This thiyl radical thus must participate in the SAM regeneration process; however how the thiyl radical abstracts an H atom from the 5'-dA to regenerate SAM is unknown. This paper reviews and discusses the history and the latest progress in the mechanistic elucidation of SPL. Despite some recent breakthroughs, more questions are raised in the mechanistic understanding of this intriguing DNA repair enzyme. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

DNA repair and free radicals, new insights into the mechanism of spore photoproduct lyase revealed by single amino acid substitution

The major DNA photoproduct in UV-irradiated Bacillus subtilis spores is the thymine dimer named spore photoproduct (SP, 5-(alpha-thyminyl)-5,6-dihydrothymine). The SP lesion has been found to be efficiently repaired by SP lyase (SPL) a very specific enzyme that reverses the SP to two intact thymines, at the origin of the great resistance of the spores to UV irradiation. SPL belongs to a superfamily of [4Fe-4S] iron-sulfur enzymes, called "Radical-SAM." Here, we show that the single substitution of cysteine 141 into alanine, a residue fully conserved in Bacillus species and previously shown to be essential for spore DNA repair in vivo, has a major impact on the outcome of the SPL-dependent repair reaction in vitro. Indeed the modified enzyme catalyzes the almost quantitative conversion of the SP lesion into one thymine and one thymine sulfinic acid derivative. This compound results from the trapping of the allyl-type radical intermediate by dithionite, used as reducing agent in the reaction mixture. Implications of the data reported here regarding the repair mechanism and the role of Cys-141 are discussed.     

S-adenosylmethionine as an oxidant: the radical SAM superfamily

A recently discovered superfamily of enzymes function using chemically novel mechanisms, in which S-adenosylmethionine (SAM) serves as an oxidizing agent in DNA repair and the biosynthesis of vitamins, coenzymes and antibiotics. Members of this superfamily, the radical SAM enzymes, are related by the cysteine motif CxxxCxxC, which nucleates the [4Fe-4S] cluster found in each. A common thread in the novel chemistry of these proteins is the use of a strong reducing agent--a low-potential [4Fe-4S](1+) cluster--to generate a powerful oxidizing agent, the 5'-deoxyadenosyl radical, from SAM. Recent results are beginning to determine the unique biochemistry for some of the radical SAM enzymes, for example, lysine 2,3 aminomutase, pyruvate formate lyase activase and biotin synthase.     

Essential Cysteine Residues in Bacillus subtilis Spore Photoproduct Lyase Identified by Alanine Scanning Mutagenesis

Endospore-forming bacteria (Bacillus and Clostridium spp.) are highly ultraviolet (UV) resistant and repair UV-induced DNA damage in part using the spore-specific DNA repair enzyme spore photoproduct (SP) lyase. SP lyase in all known sporeformers contains four conserved cysteine residues; three absolutely conserved residues are located at the “Radical SAM” consensus (C91xxxC95xxC98), which presumably participates in [4Fe-4S] cluster formation. A fourth conserved cysteine, the function of which is unknown, is located at C141 in SP lyase from all Bacillus spp. sequenced to date. To probe the function of the fourth cysteine, each conserved cysteine in the B. subtilis SP lyase was systematically altered to alanine by site-directed mutagenesis. UV-visible spectroscopy of wild-type and mutant SP lyases indicated that C141 does not participate in [4Fe-4S] formation and redox chemistry; however, in vivo SP lyase activity was abolished in all mutants, indicating an essential role for C141 in SP lyase activity.     

Characterization of an active spore photoproduct lyase, a DNA repair enzyme in the radical S-adenosylmethionine superfamily

The major photoproduct in UV-irradiated Bacillus spore DNA is a unique thymine dimer called spore photoproduct (SP, 5-thyminyl-5,6-dihydrothymine). The enzyme spore photoproduct lyase (SP lyase) has been found to catalyze the repair of SP dimers to thymine monomers in a reaction that requires S-adenosylmethionine. We present here the first detailed characterization of catalytically active SP lyase, which has been anaerobically purified from overexpressing Escherichia coli. Anaerobically purified SP lyase is monomeric and is red-brown in color. The purified enzyme contains approximately 3.1 iron and 3.0 acid-labile S(2-) per protein and has a UV-visible spectrum characteristic of iron-sulfur proteins (410 nm (11.9 mM(-1) cm(-1)) and 450 nm (10.5 mM(-1) cm(-1))). The X-band EPR spectrum of the purified enzyme shows a nearly isotropic signal (g = 2.02) characteristic of a [3Fe-4S]1+ cluster; reduction of SP lyase with dithionite results in the appearance of a new EPR signal (g = 2.03, 1.93, and 1.89) with temperature dependence and g values consistent with its assignment to a [4Fe-4S]1+ cluster. The reduced purified enzyme is active in SP repair, with a specific activity of 0.33 micromol/min/mg. Only a catalytic amount of S-adenosylmethionine is required for DNA repair, and no irreversible cleavage of S-adenosylmethionine into methionine and 5'-deoxyadenosine is observed during the reaction. Label transfer from [5'-3H]S-adenosylmethionine to repaired thymine is observed, providing evidence to support a mechanism in which a 5'-deoxyadenosyl radical intermediate directly abstracts a hydrogen from SP C-6 to generate a substrate radical, and subsequent to radical-mediated beta-scission, a product thymine radical abstracts a hydrogen from 5'-deoxyadenosine to regenerate the 5'-deoxyadenosyl radical. Together, our results support a mechanism in which S-adenosylmethionine acts as a catalytic cofactor, not a substrate, in the DNA repair reaction.     

The Radical SAM Superfamily

The radical S-adenosylmethionine (SAM) superfamily currently comprises more than 2800 proteins with the amino acid sequence motif CxxxCxxC unaccompanied by a fourth conserved cysteine. The charcteristic three-cysteine motif nucleates a [4Fe-4S] cluster, which binds SAM as a ligand to the unique Fe not ligated to a cysteine residue. The members participate in more than 40 distinct biochemical transformations, and most members have not been biochemically characterized. A handful of the members of this superfamily have been purified and at least partially characterized. Significant mechanistic and structural information is available for lysine 2,3-aminomutase, pyruvate formate-lyase, coproporphyrinogen III oxidase, and MoaA required for molybdopterin biosynthesis. Biochemical information is available for spore photoproduct lyase, anaerobic ribonucleotide reductase activation subunit, lipoyl synthase, and MiaB involved in methylthiolation of isopentenyladenine-37 in tRNA. The radical SAM enzymes biochemically characterized to date have in common the cleavage of the [4Fe-4S](1 +) -SAM complex to [4Fe-4S](2 +)-Met and the 5' -deoxyadenosyl radical, which abstracts a hydrogen atom from the substrate to initiate a radical mechanism.     

The Radical SAM Superfamily

ABSTRACT

The radical S-adenosylmethionine (SAM) superfamily currently comprises more than 2800 proteins with the amino acid sequence motif CxxxCxxC unaccompanied by a fourth conserved cysteine. The charcteristic three-cysteine motif nucleates a [4Fe–4S] cluster, which binds SAM as a ligand to the unique Fe not ligated to a cysteine residue. The members participate in more than 40 distinct biochemical transformations, and most members have not been biochemically characterized. A handful of the members of this superfamily have been purified and at least partially characterized. Significant mechanistic and structural information is available for lysine 2,3-aminomutase, pyruvate formate-lyase, coproporphyrinogen III oxidase, and MoaA required for molybdopterin biosynthesis. Biochemical information is available for spore photoproduct lyase, anaerobic ribonucleotide reductase activation subunit, lipoyl synthase, and MiaB involved in methylthiolation of isopentenyladenine-37 in tRNA. The radical SAM enzymes biochemically characterized to date have in common the cleavage of the [4Fe–4S]^{1 +} –SAM complex to [4Fe–4S]^{2 +}–Met and the 5′ -deoxyadenosyl radical, which abstracts a hydrogen atom from the substrate to initiate a radical mechanism.     

Solution phase dynamics of the DNA repair enzyme spore photoproduct lyase as probed by H/D exchange

Spore photoproduct lyase (SPL) catalyzes the repair of the UV lesion spore photoproduct (SP) in a reaction dependent on S-adenosyl-l-methionine (SAM). We have utilized H/D exchange to show that in the presence of SAM, a significant reduction in H/D exchange is observed upon binding SPTpT or undamaged oligonucleotide, indicating a shift of 20 or 10 amide protons, respectively, from a rapidly-exchangable state to a fully-protected conformation. In the absence of SAM, neither the oligonucleotide nor the SPTpT produce a significant perturbation in H/D exchange, indicating SAM is a requisite binding partner. Performing the same experiments in aerobic conditions reduced the magnitude of ligand-induced structural changes, consistent with the importance of the oxygen-sensitive iron–sulfur cluster for SAM and substrate binding.     

Catalytic Activity of the Anaerobic Tyrosine Lyase Required for Thiamine Biosynthesis in Escherichia coli

Thiazole synthase in Escherichia coli is an αβ heterodimer of ThiG and ThiH. ThiH is a tyrosine lyase that cleaves the Cα–Cβ bond of tyrosine, generating p-cresol as a by-product, to form dehydroglycine. This reactive intermediate acts as one of three substrates for the thiazole cyclization reaction catalyzed by ThiG. ThiH is a radical S-adenosylmethionine (AdoMet) enzyme that utilizes a [4Fe-4S]⁺ cluster to reductively cleave AdoMet, forming methionine and a 5′-deoxyadenosyl radical. Analysis of the time-dependent formation of the reaction products 5′-deoxyadenosine (DOA) and p-cresol has demonstrated catalytic behavior of the tyrosine lyase. The kinetics of product formation showed a pre-steady state burst phase, and the involvement of DOA in product inhibition was identified by the addition of 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase to activity assays. This hydrolyzed the DOA and changed the rate-determining step but, in addition, substantially increased the uncoupled turnover of AdoMet. Addition of glyoxylate and ammonium inhibited the tyrosine cleavage reaction, but the reductive cleavage of AdoMet continued in an uncoupled manner. Tyrosine analogues were incubated with ThiGH, which showed a strong preference for phenolic substrates. 4-Hydroxyphenylpropionic acid analogues allowed uncoupled AdoMet cleavage but did not result in further reaction (Cα–Cβ bond cleavage). The results of the substrate analogue studies and the product inhibition can be explained by a mechanistic hypothesis involving two reaction pathways, a product-forming pathway and a futile cycle.     

Spore photoproduct within DNA is a surprisingly poor substrate for its designated repair enzyme—The spore photoproduct lyase

DNA repair enzymes typically recognize their substrate lesions with high affinity to ensure efficient lesion repair. In UV irradiated endospores, a special thymine dimer, 5-thyminyl-5,6-dihydrothymine, termed the spore photoproduct (SP), is the dominant DNA photolesion, which is rapidly repaired during spore outgrowth mainly by spore photoproduct lyase (SPL) using an unprecedented protein-harbored radical transfer process. Surprisingly, our in vitro studies using SP-containing short oligonucleotides, pUC 18 plasmid DNA, and E. coli genomic DNA found that they are all poor substrates for SPL in general, exhibiting turnover numbers of 0.01–0.2 min⁻¹. The faster turnover numbers are reached under single turnover conditions, and SPL activity is low with oligonucleotide substrates at higher concentrations. Moreover, SP-containing oligonucleotides do not go past one turnover. In contrast, the dinucleotide SP TpT exhibits a turnover number of 0.3–0.4 min⁻¹, and the reaction may reach up to 10 turnovers. These observations distinguish SPL from other specialized DNA repair enzymes. To the best of our knowledge, SPL represents an unprecedented example of a major DNA repair enzyme that cannot effectively repair its substrate lesion within the normal DNA conformation adopted in growing cells. Factors such as other DNA binding proteins, helicases or an altered DNA conformation may cooperate with SPL to enable efficient SP repair in germinating spores. Therefore, both SP formation and SP repair are likely to be tightly controlled by the unique cellular environment in dormant and outgrowing spore-forming bacteria, and thus SP repair may be extremely slow in non-spore-forming organisms.     

Dinucleotide spore photoproduct, a minimal substrate of the DNA repair spore photoproduct lyase enzyme from Bacillus subtilis

The overwhelming majority of DNA photoproducts in UV-irradiated spores is a unique thymine dimer called spore photoproduct (SP, 5-thymine-5,6-dihydrothymine). This lesion is repaired by the spore photoproduct lyase (SP lyase) enzyme that directly reverts SP to two unmodified thymines. The SP lyase is an S-adenosylmethionine-dependent iron-sulfur protein that belongs to the radical S-adenosylmethionine superfamily. In this study, by using a well characterized preparation of the SP lyase enzyme from Bacillus subtilis, we show that SP in the form of a dinucleoside monophosphate (spore photoproduct of thymidilyl-(3'-5')-thymidine) is efficiently repaired, allowing a kinetic characterization of the enzyme. The preparation of this new substrate is described, and its identity is confirmed by mass spectrometry and comparison with authentic spore photoproduct. The fact that the spore photoproduct of thymidilyl-(3'-5')-thymidine dimer is repaired by SP lyase may indicate that the SP lesion does not absolutely need to be contained within a single- or double-stranded DNA for recognition and repaired by the SP lyase enzyme.     

Structural and functional comparison of HemN to other radical SAM enzymes

Radical SAM enzymes have only recently been recognized as an ancient family sharing an unusual radical-based reaction mechanism. This late appreciation is due to the extreme oxygen sensitivity of most radical SAM enzymes, making their characterization particularly arduous. Nevertheless, realization that the novel apposition of the established cofactors S-adenosylmethionine and [4Fe-4S] cluster creates an explosive source of catalytic radicals, the appreciation of the sheer size of this previously neglected family, and the rapid succession of three successfully solved crystal structures within a year have ensured that this family has belatedly been noted. In this review, we report the characterization of two enzymes: the established radical SAM enzyme, HemN or oxygen-independent coproporphyrinogen III oxidase from Escherichia coli, and littorine mutase, a presumed radical SAM enzyme, responsible for the conversion of littorine to hyoscyamine in plants. The enzymes are compared to other radical SAM enzymes and in particular the three reported crystal structures from this family, HemN, biotin synthase and MoaA, are discussed.     

Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of Radical SAM enzymes

'Radical SAM' enzymes generate catalytic radicals by combining a 4Fe-4S cluster and S-adenosylmethionine (SAM) in close proximity. We present the first crystal structure of a Radical SAM enzyme, that of HemN, the Escherichia coli oxygen-independent coproporphyrinogen III oxidase, at 2.07 A resolution. HemN catalyzes the essential conversion of coproporphyrinogen III to protoporphyrinogen IX during heme biosynthesis. HemN binds a 4Fe-4S cluster through three cysteine residues conserved in all Radical SAM enzymes. A juxtaposed SAM coordinates the fourth Fe ion through its amide nitrogen and carboxylate oxygen. The SAM sulfonium sulfur is near both the Fe (3.5 A) and a neighboring sulfur of the cluster (3.6 A), allowing single electron transfer from the 4Fe-4S cluster to the SAM sulfonium. SAM is cleaved yielding a highly oxidizing 5'-deoxyadenosyl radical. HemN, strikingly, binds a second SAM immediately adjacent to the first. It may thus successively catalyze two propionate decarboxylations. The structure of HemN reveals the cofactor geometry required for Radical SAM catalysis and sets the stage for the development of inhibitors with antibacterial function due to the uniquely bacterial occurrence of the enzyme.     

Spore Photoproduct Lyase from Bacillus subtilis Spores Is a Novel Iron-Sulfur DNA Repair Enzyme Which Shares Features with Proteins such as Class III Anaerobic Ribonucleotide Reductases and Pyruvate-Formate Lyases

The major photoproduct in UV-irradiated spore DNA is the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP). An important determinant of the high UV resistance of Bacillus subtilis spores is the accurate in situ reversal of SP during spore germination by the DNA repair enzyme SP lyase. To study the molecular aspects of SP lyase-mediated SP repair, the cloned B. subtilis splB gene was engineered to encode SP lyase with a molecular tag of six histidine residues at its amino terminus. The engineered six-His-tagged SP lyase expressed from the amyE locus restored UV resistance to spores of a UV-sensitive mutant B. subtilis strain carrying a deletion-insertion mutation which removed the entire splAB operon at its natural locus and was shown to repair SP in vivo during spore germination. The engineered SP lyase was purified both from dormant B. subtilis spores and from an Escherichia coli overexpression system by nickel-nitrilotriacetic acid (NTA) agarose affinity chromatography and was shown by Western blotting, UV-visible spectroscopy, and iron and acid-labile sulfide analysis to be a 41-kDa iron-sulfur (Fe-S) protein, consistent with its amino acid sequence homology to the 4Fe-4S clusters in anaerobic ribonucleotide reductases and pyruvate-formate lyases. SP lyase was capable of reversing SP from purified SP-containing DNA in an in vitro reaction either when present in a cell-free extract prepared from dormant spores or after purification on nickel-NTA agarose. SP lyase activity was dependent upon reducing conditions and addition of S-adenosylmethionine as a cofactor.     

UV-radiation-induced formation of DNA bipyrimidine photoproducts in Bacillus subtilis endospores and their repair during germination.

The spore photoproduct (SP) is the main DNA lesion after UV-C irradiation, and its repair is crucial for the resistance of spores to UV. The aims of the present study were to assess the formation and repair of bipyrimidine photoproducts in spore DNA of various Bacillus subtilis strains using a sensitive HPLC tandem mass spectrometry assay. Strains deficient in nucleotide excision repair, spore photoproduct lyase, homologous recombination (recA), and with wild-type repair capability were investigated. Additionally, one strain deficient in the formation of major small, acid-soluble spore proteins (SASPs) was tested. In all SASP wild-type strains, UV-C irradiation generated almost exclusively SP (>95 %) but also a few by-photoproducts. In the major SASP-deficient strain, SP and by-photoproducts were generated in equal quantities. The status time of 60 min, >75% of the SP was repaired in wild-type strains and in the SASP-deficient strain, while half of the photoinduced SP was removed in the recA-deficient strain. SP-lyase-deficient spores repaired 20% of the SP produced. Thus, SP lyase, with respect to nucleotide excision repair, has a remarkable impact on the removal of SP upon spore germination.     

Spore Photoproduct Lyase: The Known,the Controversial,and the Unknown

Spore photoproduct lyase (SPL) repairs 5-thyminyl-5,6-dihydrothymine, a thymine dimer that is also called the spore photoproduct (SP), in germinating endospores. SPL is a radical S-adenosylmethionine (SAM) enzyme, utilizing the 5′-deoxyadenosyl radical generated by SAM reductive cleavage reaction to revert SP to two thymine residues. Here we review the current progress in SPL mechanistic studies. Protein radicals are known to be involved in SPL catalysis; however, how these radicals are quenched to close the catalytic cycle is under debate.     

Complete stereospecific repair of a synthetic dinucleotide spore photoproduct by spore photoproduct lyase

Spore photoproduct lyase (SP lyase), a member of the radical S-adenosylmethionine superfamily of enzymes, catalyzes the repair of 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)], a type of UV-induced DNA damage unique to bacterial spores. The anaerobic purification and characterization of Clostridium acetobutylicum SP lyase heterologously expressed in Escherichia coli, and its catalytic activity in repairing stereochemically defined synthetic dinucleotide SPs was investigated. The purified enzyme contains between 2.3 and 3.1 iron atoms per protein. Electron paramagnetic resonance (EPR) spectroscopy reveals an isotropic signal centered at g = 1.99, characteristic of a [3Fe–4S]⁺ cluster accounting for 3–4% of the iron in the sample. Upon reduction, a nearly axial signal (g = 2.03, 1.93 and 1.92) characteristic of a [4Fe–4S]⁺ cluster is observed that accounts for 34–45% of total iron. Addition of S-adenosylmethionine to the reduced enzyme produces a rhombic signal (g = 2.02, 1.93, 1.82) unique to the S-adenosyl-l-methionine complex while decreasing the overall EPR intensity. This reduced enzyme is shown to rapidly and completely repair the 5R diastereomer of a synthetic dinucleotide SP with a specific activity of 7.1 ± 0.6 nmol min⁻¹ mg⁻¹, whereas no repair was observed for the 5S diastereomer.     

Antioxidants from defatted Indian Mustard (Brassica Juncea) protect biomolecules against in vitro oxidation

Indian mustard seeds were defatted by distillation with hexane and the residue extracted with methanol was analyzed for potential antioxidants; ascorbate, riboflavin, and polyphenols. Gallic acid (129.796 μg), caffeic acid (753.455 μg), quercetin (478.352 μg) and kaempferol (48.060 μg)/g dry seeds were identified by HPLC analysis of the extract. DPPH free radical scavenging activity and protection of lipids, proteins and DNA against metal induced oxidation was examined. Defatted mustard seed remnant had excellent free radical scavenging activity and protects biomolecules with IC₅₀ value 2.0–2.25 mg dry seed weight. Significant content of polyphenols in methanol extract of defatted seeds accounts for high antioxidant potential. We are the first to report the detailed analysis of antioxidant composition and protection of biomolecules against oxidative damage by methanol extract of mustard seed remnant after oil extraction.     

Reconstitution of the base excision repair pathway for 7,8-dihydro-8-oxoguanine with purified human proteins

In mammalian cells, repair of the most abundant endogenous premutagenic lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is initiated by the bifunctional DNA glycosylase OGG1. By using purified human proteins, we have reconstituted repair of 8-oxoG lesions in DNA in vitro on a plasmid DNA substrate containing a single 8-oxoG residue. It is shown that efficient and complete repair requires only hOGG1, the AP endonuclease HAP1, DNA polymerase (Pol) β and DNA ligase I. After glycosylase base removal, repair occurred through the AP lyase step of hOGG1 followed by removal of the 3′-terminal sugar phosphate by the 3′-diesterase activity of HAP1. Addition of PCNA had a slight stimulatory effect on repair. Fen1 or high concentrations of Pol β were required to induce strand displacement DNA synthesis at incised 8-oxoG in the absence of DNA ligase. Fen1 induced Pol β strand displacement DNA synthesis at HAP1-cleaved AP sites differently from that at gaps introduced by hOGG1/HAP1 at 8-oxoG sites. In the presence of DNA ligase I, the repair reaction at 8-oxoG was confined to 1 nt replacement, even in the presence of high levels of Pol β and Fen1. Thus, the assembly of all the core proteins for 8-oxoG repair catalyses one major pathway that involves single nucleotide repair patches.     

Mechanistic understanding of Pyrococcus horikoshii Dph2, a [4Fe-4S] enzyme required for diphthamide biosynthesis

Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on eukaryotic and archaeal translation elongation factor 2 (EF2). The proposed biosynthesis of diphthamide involves three steps and we have recently found that in Pyrococcus horikoshii (P. horikoshii), the first step uses an S-adenosyl-L-methionine (SAM)-dependent [4Fe-4S] enzyme, PhDph2, to catalyze the formation of a C-C bond. Crystal structure shows that PhDph2 is a homodimer and each monomer contains three conserved cysteine residues that can bind a [4Fe-4S] cluster. In the reduced state, the [4Fe-4S] cluster can provide one electron to reductively cleave the bound SAM molecule. However, different from classical radical SAM family of enzymes, biochemical evidence suggest that a 3-amino-3-carboxypropyl radical is generated in PhDph2. Here we present evidence supporting that the 3-amino-3-carboxypropyl radical does not undergo hydrogen abstraction reaction, which is observed for the deoxyadenosyl radical in classical radical SAM enzymes. Instead, the 3-amino-3-carboxypropyl radical is added to the imidazole ring in the pathway towards the formation of the product. Furthermore, our data suggest that the chemistry requires only one [4Fe-4S] cluster to be present in the PhDph2 dimer.