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Published in final edited form as: Curr Opin Struct Biol. 2012 Nov 8;22(6):701–710. doi: 10.1016/j.sbi.2012.10.005

Emerging themes in radical SAM chemistry

Krista A Shisler 1, Joan B Broderick 1
PMCID: PMC4083504  NIHMSID: NIHMS593306  PMID: 23141873

Abstract

Enzymes in the radical SAM (RS) superfamily catalyze a wide variety of reactions through unique radical chemistry. The characteristic markers of the superfamily include a [4Fe–4S] cluster coordinated to the protein via a cysteine triad motif, typically CX3CX2C, with the fourth iron coordinated by S-adenosylmethionine (SAM). The SAM serves as a precursor for a 5′-deoxyadenosyl radical, the central intermediate in nearly all RS enzymes studied to date. The SAM-bound [4Fe–4S] cluster is located within a partial or full triosephosphate isomerase (TIM) barrel where the radical chemistry occurs protected from the surroundings. In addition to the TIM barrel and a RS [4Fe–4S] cluster, many members of the superfamily contain additional domains and/or additional Fe–S clusters. Recently characterized superfamily members are providing new examples of the remarkable range of reactions that can be catalyzed, as well as new structural and mechanistic insights into these fascinating reactions.

Introduction

The radical S-adenosylmethionine (radical SAM, hereafter RS) superfamily of enzymes carry out a wide variety of biological functions including synthesis of cofactors, modification of RNA, DNA repair, and synthesis of antibiotics [1]. There are currently tens of thousands of predicted RS superfamily members spanning the phylogenetic kingdom; however, only a small fraction of these have been characterized. Although the chemistry they catalyze is diverse, the RS enzymes utilize a common mechanism for initiation of catalysis that involves generation of a primary carbon-centered radical intermediate, the 5′-deoxyadenosyl radical (dAdo•), which abstracts a hydrogen atom from the substrate [2–4,5••]. The substrate radical can then undergo radical-mediated and often complex transformations to generate product.

In order to generate the dAdo• radical from SAM, RS enzymes utilize an enzyme-bound [4Fe–4S] cluster. Three of the four irons in the [4Fe–4S] cluster are coordinated by cysteine thiolates (C or Cys) present in a triad motif, generally CX3CX2C although variations exist. The fourth iron of the cluster, often referred to as the unique iron due to its distinct coordination, has no cysteine ligand but rather is coordinated by SAM (Figure 1). The iron–sulfur cluster is active in its reduced [4Fe–4S]+ state, from which it can transfer an electron to the sulfonium of SAM to promote homolytic cleavage of the S–5′C bond of SAM, producing methionine and a 5′-deoxyadenosyl radical intermediate that abstracts a hydrogen atom from substrate (Figure 1) [6]. The highly reactive nature of the dAdo• radical intermediate [7] confers on these enzymes the ability to carry out diverse and difficult chemical transformations requiring abstraction of a hydrogen atom from generally unactivated positions on the substrate. The utilization of this primary carbon radical in catalytic chemistry, however, also requires the RS enzymes to employ exquisite control and protection of the radical intermediate in order to avoid damaging side reactions. Recent insights into the structures of the RS enzymes, and how these structures relate to mechanism and control of reactivity, will be addressed in this review.

Figure 1.

Figure 1

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The reductive cleavage of SAM catalyzed by RS enzymes. SAM is coordinated via its amino and carboxyl moieties to the unique iron of a [4Fe–4S] cluster (left). The reduced [4Fe–4S]+ cluster transfers an electron to SAM, thereby promoting homolytic cleavage to generate methionine and a 5′-deoxyadenosyl radical. The 5′-deoxyadenosyl radical abstracts a hydrogen atom from substrate (SH) to produce a substrate radical (S•), methionine, and 5′-deoxyadenosine (right).

Structural insights into the radical SAM superfamily

Binding diverse substrates with a common fold

Several RS enzymes have been structurally characterized; these reveal a common fold consisting of a triosephosphate isomerase (TIM) barrel, which is a full (α/β)8 TIM barrel in some cases (e.g. biotin synthase (BioB) [8], HydE [9], and ThiC [10]) and a partial (α/β)6 TIM barrel in most other cases (Figure 2) [11,12••,13–17,18••]. The size of the TIM barrel inversely correlates with substrate size: enzymes that act on larger substrates tend to have less complete TIM barrels, thus making the barrel opening larger [4,5•]. The glycyl radical generating enzyme pyruvate formate lyase activating enzyme (PFL-AE), for example, is the smallest RS protein structurally characterized to date with little secondary structure outside the partial (α/β)6 TIM barrel (Figure 2a) [11]. The substrate for PFL-AE is among the largest RS substrates known: the 170 kDa homodimeric protein pyruvate formate lyase (PFL). On the basis of the evidence for direct H-atom abstraction from PFL G734 by the dAdo• generated in the PFL-AE active site, it is clear that minimally the glycyl radical domain of PFL must bind within this TIM barrel such that G734 is in close proximity to the [4Fe–4S] cluster and bound SAM in PFL-AE; this is consistent with the presence of an incomplete and splayed TIM barrel that provides a larger binding pocket for substrate [11,19•].

Figure 2.

Figure 2

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Crystal structures of representative RS enzymes and Dph2. The Fe–S clusters are shown in rust for iron and yellow for sulfur. SAM molecules (teal carbons) and substrates (purple carbons) are also shown. (a) PFL-AE with SAM and the substrate PFL peptide (PDB ID: 3CB8). (b) SPL with SAM and substrate dinucleoside 5R-SP (PDB ID: 4FHD). (c) BioB with SAM, DTB substrate, and the additional [2Fe–2S] cluster (PDB ID: 1R30). (d) RlmN with SAM (PDB ID: 3RFA). (e) HydE with SAM and additional [2Fe–2S] cluster (PDB ID: 3IIZ). (f) Dph2 with the iron coordinating cysteines in red (PDB ID: 3LZD).

Spore photoproduct (SP) lyase (SPL) is another example of a RS enzyme that acts on a large substrate; in this case, the substrate is the SP (5-thyminyl-5,6-dihydrothymine) thymine dimer contained within double-stranded DNA (dsDNA). A recent structure of SPL reveals that, like PFL-AE, SPL contains a partial (α/β)6 TIM barrel with a wide lateral opening capable of binding large substrates (Figure 2b) [18••,20]. In contrast to PFL-AE and SPL, the RS enzyme BioB acts on a small-molecule substrate, dethiobiotin (DTB), and contains a full (α/β)8 TIM barrel; this full barrel still allows access of the small molecule substrate to the SAM-bound [4Fe–4S] cluster at the active site within the barrel, while also presumably shielding reactive radical intermediates from deleterious side reactions (Figure 2c) [8].

Positioning SAM and substrate for catalysis

In all RS structures to date, the catalytic [4Fe–4S] cluster bound to the characteristic RS cysteine motif resides at the C-terminal end of the TIM barrel, with the unique iron of the cluster pointing into the barrel. This unique iron is coordinated by the amino nitrogen and the carboxylate oxygen of SAM (Figure 1), an interaction first identified by electron nuclear double resonance (ENDOR) spectroscopic studies of PFL-AE [21,22] and subsequently observed in every RS crystal structure in which SAM is bound [5••]. This SAM–cluster interaction is unique to the RS enzymes and provides the key point of contact between the catalytic cluster and SAM for the production of dAdo•. Generally, in the crystal structures solved to date, the active site is exposed to solvent; however, upon binding of substrate, sections of the C-terminal region and the N-terminal region and/or the substrate itself provide a lid for the active site, subsequently blocking off solvent access to the active site [4,23•]. Such closure of the active site cavity during catalysis provides protection for the radical intermediates, including dAdo•, implicated in RS mechanisms. Without such shielding of reaction intermediates, the radicals involved in the catalytic mechanisms of these enzymes might not survive to react with their intended targets due to quenching by solvent. Further, exclusion of solvent would alter the dielectric of the active site, a factor that may be important in modulating the energetics of the catalytic steps [24•].

Similar to other RS enzymes, the [4Fe–4S] cluster in SPL is buried at the top of the barrel, in an environment rich in hydrophobic residues, with SAM bound through the amino and carboxylate groups [18•]. A structure with the dinucleoside 5R-SP bound shows that this substrate binds in proximity to SAM, accompanied by distinct conformational changes that appear to seal off the substrate binding pocket and provide a protected environment for subsequent radical chemistry (Figure 2b) [18••]. The conformational changes occur mainly in the catalytic pocket and the β-hairpin that resides just outside the catalytic pocket. Two residues (Arg304 and Tyr305) in the β-hairpin may help recognize and flip out the SP region from dsDNA through the insertion of the β-hairpin into dsDNA or through interactions with the DNA backbone [18••]. Other residues near the active site change their orientation to allow access of SP to the active site. Another interesting outcome of this structure is the revelation of the structural basis for the previously reported stereospecificity of SPL [20,25], which resides in the steric clashes that would occur when orienting the 5S-SP correctly for H-atom abstraction.

Similar to SPL, the crystal structures of PFL-AE in the presence and absence of a peptide analog of the PFL substrate reveal that binding of the peptide shields the active site from solvent and helps to stabilize and orient SAM in the active site (Figure 2a) [11]. The structures also reveal that binding of substrate induces a large conformational change in loop A of PFL-AE, wherein the loop swings up into the active site and interacts with the substrate to either stabilize and position it for H-atom abstraction or to induce conformational changes in the PFL substrate [11]. Given the evidence for direct H-atom abstraction from G734 of PFL by the dAdo• generated in the PFL-AE active site, together with the observation that G734 is buried in the interior of PFL in its crystal structures [19•,26], the involvement of significant conformational changes for PFL during activation has long been postulated. Recent biochemical and spectroscopic results support such a major conformational change in PFL upon interaction with PFL-AE. Peng et al. showed that in the presence of PFL-AE, PFL favors an open conformation in which the radical domain emerges from its buried position in the interior of PFL [19•]. PFL-AE thus appears to promote a conformational change in PFL that renders its glycyl radical domain accessible for binding in the PFL-AE active site [19•]. The elucidation of the details of this conformational change in PFL, and the detailed mechanism by which PFL-AE promotes this change to allow for glycyl radical formation, awaits further studies.

The SAM–cluster interaction and implications for mechanism

Uncoupled SAM cleavage, in which the dAdo• does not react with substrate but rather is quenched by protein or solvent, is a wasteful and potentially damaging reaction for RS enzymes. One method that RS enzymes use to prevent uncoupled cleavage of SAM is to take advantage of the large difference in redox potential between the [4Fe–4S] cluster and SAM. The reduction potential for SAM is approximately −1.8 V, while the [4Fe–4S] cluster in RS enzymes is only about −450 mV, resulting in a barrier of about 1.4 V or 32 kcal mol−1 [4,27]. Wang and Frey have shown that binding of SAM and substrate to LAM lowers the energy barrier to 9 kcal mol−1, resulting in more favorable conditions for the reductive cleavage of SAM [4,27]. Sulfur K-edge X-ray absorption spectroscopy and density functional theory calculations on PFL-AE have provided evidence for a back-bonding interaction between SAM and the cluster that is increased upon cluster reduction; this interaction is proposed to facilitate electron transfer from the [4Fe–4S]+ cluster to SAM [24•]. Computational results also indicate that reductive cleavage of SAM is sensitive to the dielectric in the active site, and this sensitivity has been proposed to play a role in triggering inner-sphere electron transfer and subsequent SAM cleavage upon substrate binding [24•].

Novel chemistry for radical SAM enzymes

In the last several years, a number of newly characterized RS enzymes have been reported. These RS enzymes carry out novel chemistry and include the C-methyltransferase YtkT involved in the production of the antitumor agent yatakemycin [28], PqqE which is involved in the biosynthesis of pyrroloquinoline quinine (PQQ) [29], HpnP which is involved in the methylation of hopanoids [30], and the methylthiotransferase Cdkal1 [31] which was found to be linked to type 2 diabetes in mice [32]. Other newly identified RS enzymes discussed in more detail below play central roles in antiviral activity [33•,34,35], antibiotic production [36,37•], methylation reactions [12,38•,39,40] and metal cofactor biosynthesis [41–44]. These newly elucidated functions add to the already remarkably diverse chemistry known for the superfamily (see Refs. [2,3,5••,23•]).

Viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible) is a mammalian protein that is upregulated in response to viral infections; however, the mechanism for its antiviral activity has yet to be determined [33•,34,35]. The proposed structure of viperin indicates a three-domain protein with a partial (α/β)6 TIM barrel RS domain, a leucine zipper domain possibly for protein folding and anchoring of the protein to the endoplasmic reticulum (ER), and a C-terminal domain which may be involved in substrate recognition or interactions with cofactors [45]. Viperin exhibits enhanced stability upon reconstitution with iron and sulfide [35,46], and UV–vis and electron paramagnetic resonance (EPR) spectroscopic analysis revealed the presence of a [4Fe–4S] cluster typical of the RS superfamily [33•]. Reductive cleavage of SAM was also observed, supporting the hypothesis that viperin is a RS enzyme [33•]. The mode by which RS chemistry aids in the antiviral response, however, is unknown. Viperin has been shown to interact with the enzyme farnesyl pyrophosphate synthase (FPPS) on the cytosolic face of the ER, decreasing its activity and disrupting the formation of lipid rafts, which are involved in budding of a number of viruses including HIV and influenza [33•,47]. While this interaction is an important observation, better understanding of the role of viperin in the antiviral response awaits identification of the reaction(s) it catalyzes.

Recent results demonstrate important roles for RS chemistry in antibiotic biosynthesis. AlbA is a RS enzyme involved in antimicrobial activity; it catalyzes the formation of three thioether bridges on the peptide SboA to produce subtilosin A, a sactibiotic (sulfur-to-α-carbon antibiotic) that has been shown to have antimicrobial activity against bacteria (Figure 3b) [48•,49]. The radical SAM protein NosL is involved in the production of the antibiotic thiopeptide nosiheptide (NOS) [37•]. Thiopeptides are sulfur rich, heterocyclic peptides with a macrocyclic core which includes a nitrogen-containing 6-membered ring central to multiple thiazoles and dehydroamino acids [37••,50]. In most polycylic thiopeptides, the functional side ring formation is independent of the precursor peptide and l-tryptophan provides the variable functional groups [37•]. NosL as well as NocL, which is 78% homologous to NosL and is involved in nocathiacin I (NOC-I) biosynthesis [36], are part of the MIA (3-methyl-2-indolic acid) synthase family that catalyze the conversion of l-tryptophan to MIA through an unusual fragmentation–recombination mechanism (Figure 3C).

Figure 3.

Figure 3

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Representative radical SAM reactions. (a) BioB catalyzes the sequential abstraction of hydrogen atoms from C9 and C6 of DTB (left), with insertion of sulfur from the [2Fe–2S] cluster. (b) AlbA incorporates three thioether bonds between three cysteines and the α-C of two phenylalanines and one threonine during the maturation of subtilosin A. Shown is a representative reaction between one cysteine and threonine. (c) NosL and NocL catalyze a fragmentation–recombination reaction of l-Trp to MIA which is incorporated into NOS in the case of NosL or NOC-I in the case of NocL. (d) RlmN and Cfr catalyze the methylation of A2503 of the 23S rRNA utilizing two equivalents of SAM. The sequential methylations catalyzed by these two enzymes can occur in either order.

Methylation of ribosomal RNA (rRNA) usually occurs via an SN2 reaction with the donation of the methyl group from SAM. In the case of the RS enzymes RlmN and Cfr that methylate carbons 2 and 8, respectively, of adenosine 2503 of 23S rRNA, methylation occurs via radical subsequent to transfer of a methyl group to a non-cluster Cys (Figure 3d) [38••,39]. Two equivalents of SAM are needed: one for methylation of the Cys to form mCys, and one for dAdo• production necessary for H-atom abstraction from mCys [38••,39]. In the crystal structure of RlmN, the β7 loop moves to dip into the active site upon the addition of SAM, positioning the catalytic Cys355 closer to the active site for methyl transfer (Figure 2d) [12••]. This Cys355 residue was methylated in the structure, apparently by the first molecule of SAM, suggesting that the second molecule of SAM was bound, awaiting reductive cleavage to initiate H-atom abstraction [12••]. Unlike methylthiotransferases (MTTases) that utilize two [4Fe–4S] clusters for the insertion of a methylthio group into substrate, RlmN and Cfr contain only one [4Fe–4S] cluster bound to the typical RS CX3CX2C motif, and both SAM molecules presumably bind to this same [4Fe–4S] cluster at different steps in the catalytic cycle [12••,38••,39].

Another emerging function for RS enzymes is the biosynthesis of complex metal cofactors including the iron–molybdenum cofactor (FeMo-co) of nitrogenase and the H-cluster of [Fe–Fe]-hydrogenase. NifB is a RS enzyme that inserts the central carbide in an essential step in FeMo-co maturation, with the carbide originating from the methyl group of SAM via novel chemistry [51]. The role of RS chemistry in H-cluster biosynthesis has been partially delineated in recent years [44]. The H-cluster consists of a [4Fe–4S] cluster bridged by a cysteine residue to a 2Fe cluster coordinated by three CO molecules, two CN− ions, and a bridging dithiolate; this cluster is unique to [FeFe]-hydrogenase and is the site where protons are reduced to H2 [42,52,53]. While the [4Fe–4S] cluster portion of the H-cluster is synthesized by the housekeeping Fe–S cluster assembly machinery, the 2Fe subcluster is assembled by three proteins, two of which (HydE and HydG) are RS enzymes [42,43]. HydG has been shown to synthesize the CO and CN− ligands from tyrosine using RS chemistry (Figure 4) [54–56], and HydE is presumed to catalyze formation of the dithiolate ligand, although the substrate and reaction catalyzed remain a mystery. HydG and HydE deliver these synthesized ligands to the GTPase HydF where the 2Fe subcluster is assembled [41,57]. Once constructed, the 2Fe subcluster is transferred to hydrogenase already containing a [4Fe–4S] cluster in order to generate the H-cluster and the active enzyme [52,58,59].

Figure 4.

Figure 4

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Proposed biosynthesis pathway for HydA H-cluster. HydE uses an unknown substrate to synthesize a dithiolate ligand on a [2Fe–2S] cluster of HydF. HydG catalyzes the decomposition of tyrosine to produce p-cresol, CO, and CN−; the latter two diatomics bind to the H-cluster precursor 2Fe cluster on HydF. HydF then transfers the 2Fe H-cluster precursor to HydA to produce the complete H-cluster and the active hydrogenase. Colors of atoms are as follows: green, carbon; red, oxygen; blue, nitrogen; rust, iron; yellow, sulfur; purple, unknown bridging atom; black, unknown residue coordinating to protein.

Emerging themes in radical SAM chemistry

Multiple Fe–S clusters

All RS enzymes require a [4Fe–4S] cluster in the active site for the binding and reductive cleavage of SAM; however, an emerging theme in the RS field is the presence of a second Fe–S cluster in certain subclasses of RS enzymes. In some cases, the second cluster appears to be a source of sulfur that is inserted into substrate during catalysis, a role first proposed for biotin synthase (BioB) which catalyzes the conversion of DTB to biotin by a sulfur insertion reaction [8,60,61••,62]. BioB contains an additional [2Fe–2S] cluster positioned such that DTB is sandwiched in between SAM and the [2Fe–2S] cluster (Figure 2c) [8]. The [2Fe–2S] cluster was found to undergo reduction concomitant with the formation of a 9-mercaptodethiobiotin (9-MDTB) intermediate; such cluster reduction is consistent with sulfur donation from the cluster thus providing the most recent experimental evidence that the cluster serves as a sulfur source during biotin synthesis (Figure 3a) [60,62]. Lipoate synthase catalyzes a reaction that is quite similar to that of biotin synthase: the insertion of sulfur into an unactivated C–H bond (in this case the C6 and C8 of the octanoyl group) to form the lipoyl cofactor. Lipoate synthase contains two [4Fe–4S] clusters, one of which is the RS active cluster and the other appears to serve as the source of the two sulfides inserted during catalysis [63]. The methylthiotransferases such as RimO [64], MiaB [65], and MtaB [66] also require a sulfur source and contain a second Fe–S cluster; the implication is that the second cluster is the source of the sulfur required in catalysis, but direct biochemical evidence has yet to be reported.

Other potential purposes for auxiliary Fe–S clusters in RS enzymes have also been indicated, including such roles as electron acceptors or anchors for substrates. In the case of BtrN, Grove et al. found that the second [4Fe–4S] cluster cannot be reduced by chemical means, suggesting that this cluster is inaccessible to exogenous reductants [67]. During turnover, however, an EPR signal was observed that is proposed to arise as a result of the second cluster accepting an electron from the RS cluster during catalysis [67]. The second [4Fe–4S] cluster in MoaA binds its substrate 5′-GTP through the N1 of the purine ring to the unique Fe [68]. AlbA also contains a second [4Fe–4S] cluster that is proposed to be an electron acceptor during turnover [48••]. Anaerobic sulfatase-maturating enzymes (anSMEs), on the other hand, contain two additional [4Fe–4S] clusters that are thought to be involved with the reduction of the RS [4Fe–4S] cluster [69]. The anSMEs catalyze the oxidation of cysteine and serine residues of the sulfatase enzymes to produce Cα-formylglycine (FGly) [69]. Mutation studies to knock out the additional clusters resulted in loss of activity and it was proposed that either: first, one of the clusters binds substrate and acts as an electron acceptor while the second cluster transfers an electron from an external electron donor to the RS cluster [70]; or second, in a more recent hypothesis, both clusters act to transfer the electron to the RS cluster and neither cluster coordinates substrate [69].

The [FeFe]-hydrogenase maturation proteins HydE and HydG also contain additional Fe–S clusters. Although spectroscopic studies had indicated the presence of only [4Fe–4S] clusters in HydE, in the crystal structure of HydE there was a [2Fe–2S] cluster in addition to the RS [4Fe–4S] cluster (Figure 2e) [6,9,71]. The [2Fe–2S] cluster of HydE is bound in a site about 20Å from the active site and on the exterior of the barrel separated from the active site by a water-filled cavity, quite different from the [2Fe–2S] cluster in biotin synthase, which was within the barrel and in close proximity to the active site [9]. Furthermore, the cysteine residues that coordinate the [2Fe–2S] cluster in HydE are not conserved across all HydE proteins, suggesting that this second cluster does not play an essential role in catalysis [9]. HydG contains two distinct [4Fe–4S] clusters upon reconstitution with iron and sulfide, and both of these clusters are coordinated by conserved cysteine motifs and have been shown to be essential for HydG activity [55••]. EPR spectroscopic characterization indicates that SAM binds to one of these clusters [55••]. Variants of HydG lacking the ligands for the second cluster were able to produce CN− and p-cresol upon incubation under assay conditions with SAM and tyrosine; however, no CO production was observed, suggesting a role for the second cluster in CO production [53].

Unexpected cysteine motifs

One of the characteristic features of RS enzymes is the cluster-binding CX3CX2C triad motif; however, a number of variations of this cluster-binding motif have now been reported. A CX2CX4C motif was found in 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase (ThiC), a RS enzyme that converts 5-aminoimidazole ribonucleotide (AIR) into HMP-P during thiamine biosynthesis [10]. HmdB is a RS enzyme involved in the biosynthesis of the [Fe]-hydrogenase through an undetermined reaction, and exhibits a CX5CX2C motif [72]. Dph2, which is involved in diphthamide biosynthesis, has not been classified as a RS enzyme, although biochemical evidence strongly supports a catalytic mechanism in which a [4Fe–4S] cluster interacts with SAM to generate a 3-amino-3-carboxypropyl (ACP) radical intermediate; such chemistry is clearly analogous to RS reactions [73••,74]. Dph2 exhibits neither the cysteine triad motif nor the typical RS TIM barrel structure (Figure 2f); rather, each of the three cysteines coordinating the Fe–S cluster reside on a separate domain, with over one hundred amino acids separating each cysteine residue [73••,74]. The observation of non-canonical cluster binding motifs in RS enzymes, together with the characterization of RS-like chemistry in a protein that has neither the sequence nor the structural signatures of the superfamily, suggests the likelihood that many additional, as-yet unidentified proteins will ultimately be discovered which catalyze radical reactions using an Fe–S cluster and SAM.

Reductive cleavage of alternate C–S bonds of SAM

RS enzymes have been described as cleaving the S–5′C bond to form methionine and a dAdo• radical intermediate, where dAdo• abstracts a hydrogen from substrate, producing 5′-deoxyadenosine (dAdo) and a substrate radical (Figure 1). Recently, Dph2 and glycerol dehydratase activating enzyme (GDH-AE) were reported to produce alternatively 5′-deoxy-5′-methylthioadenosine (MTA) and a 3-amino-3-carboxypropyl (ACP) radical intermediate, thus implicating the reductive cleavage of an alternate S–C bond of SAM [73••,74,75]. Dph2, as stated previously, is not classified as a RS enzyme but yet it still catalyzes a radical reaction using SAM and an Fe–S cluster [73••]. GDH-AE, on the other hand, is a member of the RS superfamily and is predicted to contain a partial TIM barrel structure similar to PFL-AE [75]. It is unclear why these proteins cleave an alternate S–C bond of SAM although the regioselectivity of the reductive cleavage of SAM has been proposed to be a result of the orientation of SAM with respect to the [4Fe–4S] cluster and substrate [74,76•].

Concluding remarks

The RS superfamily contains an amazing variety of enzymes that carry out diverse and difficult radical reactions that are essential to the metabolic processes in all kingdoms of life. Despite the presence of little sequence homology among superfamily members, these enzymes exhibit a common TIM barrel fold, a common location of a catalytically essential [4Fe–4S] cluster within that barrel, and a mode of binding the [4Fe–4S] cluster through three cysteines to generate a site-differentiated Fe that can be coordinated by SAM. Functional and structural diversity is conferred in some cases by additional domains and by additional Fe–S clusters that can serve a variety of roles in catalysis. New insights into mechanism, as well as newly characterized functions, continue to emerge for this fascinating group of enzymes.

Acknowledgements

Research on RS enzymes in the Broderick Laboratory is supported by the National Institutes of Health (GM54608) and the Department of Energy (DE-FG02-10ER16194). The Astrobiology Biogeocatalysis Research Center is supported by NASA (NNA08C-N85A).

References and recommended reading

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