Structural insights into auxiliary cofactor usage by radical S-adenosylmethionine enzymes

Abstract

Radical S-adenosylmethionine (SAM) enzymes use a common catalytic core for diverse transformations. While all radical SAM enzymes bind a Fe₄S₄ cluster via a characteristic tri-cysteine motif, many bind additional metal cofactors. Recently reported structures of radical SAM enzymes that use methylcobalamin or additional iron-sulfur clusters as cosubstrates show that these auxiliary units are anchored by N- and C-terminal domains that vary significantly in size and topology. Despite this architectural diversity, all use a common surface for auxiliary cofactor docking. In the sulfur insertion and metallocofactor assembly systems evaluated here, interaction with iron-sulfur cluster assembly proteins or downstream scaffold proteins is an important component of catalysis. Structures of these complexes represent important new frontiers in structural analysis of radical SAM enzymes.

1. Introduction

Radical SAM enzymes are versatile catalysts found throughout all three kingdoms of life [1,2]. All deploy SAM in initiating remarkably diverse transformations that are generally radical-mediated [2]. These catalysts additionally bind a Fe₄S₄²⁺ cluster, typically via a CX₃CX₂C motif found within an α₆β₆ core fold [2,3]. SAM coordinates the fourth iron. After one-electron reduction of the iron-sulfur cluster by a protein partner or chemical reductant, SAM is reductively cleaved to generate a 5′-deoxyadenosyl 5′-radical (5′-dA•). The 5′-dA• intermediate is a potent oxidant capable of initiating hydrogen atom transfer (HAT) reactions with substrates or co-substrates, generally from unactivated carbons. Recent efforts to trap and characterize the 5′-dA• [4-6] have revealed that this key intermediate may be initially caged as an organometallic complex, Ω, containing an Fe-C5′ covalent bond between the 5′-dA• and the unique iron of the Fe₄S₄ cofactor [4]. Studies of synthetic models of Ω suggest that it might function to confer selectivity in reaction outcome for radical SAM enzymes by sequestering the 5′-dA• until substrates can be properly positioned for catalysis [7].

Many radical SAM enzymes bind additional transitional metals [8]. A number have been structurally characterized, including those with one or more additional iron-sulfur clusters [9-18], individual metal ions [19], or complex cofactors such as methylcobalamin [20]. Structural characterization of radical SAM enzymes containing auxiliary cofactors that are proposed to perform simple tasks, such as electron transfer [9,10,14,15] or substrate binding [16], reveals that dedicated domains are required to bind and utilize these entities [21]. Here we describe insights from recent structural characterization of four radical SAM enzymes that use additional metallocofactors for complex functionalization of substrates (Fig. 1). These include TokK, one of the first structurally characterized radical SAM enzyme that performs radical transformations involving a cobalamin (Cbl) cofactor [22]. Here the auxiliary cofactor serves as a functional group donor in a chemically challenging carbon methylation. We also review biochemical and structural analyses of two radical SAM sulfur insertion enzymes, lipoic acid synthase (LipA) [12] and MiaB [23]. Finally, we discuss structures of NifB, a radical SAM enzymes involved in complex cofactor biosynthesis. NifB is a methylase that fuses two auxiliary [4Fe-4S] clusters and facilitates insertion of an interstitial carbide [24,25] in construction of the nitrogenase active site cofactor [26]. Despite their structurally distinct auxiliary motifs and divergent reactivity, all of these enzymes use a common surface of the radical SAM core fold for docking additional cofactors. Several enzymes summarized here additionally employ unusual ligands to their auxiliary cofactors, and this strategy is likely key in enabling the chemically challenging reactivities of these systems.

Figure 1.

Reactions catalyzed by radical SAM enzymes with auxiliary metallocofactors discussed in this review.

2.1. Cobalamin-dependent radical SAM methylase, TokK.

TokK methylates an unactivated carbon within the β-lactam core of asparenomycin A, a potent carbapenem antibiotic [27,28]. The products generated by TokK suggest that the enzyme uses a 5′-dA• to activate its substrate via HAT. The resulting substrate radical then attacks the methyl substituent of methyl-Cbl. TokK can perform serial methylations of its substrate [28], adding up to three methyl units to elaborate the C6 position of the β-lactam. This modification enhances the potency of carbapenems by preventing inactivation by β-lactamases [29].

TokK employs an N-terminal Rossmann-fold domain (Fig. 2) to dock the Cbl cofactor adjacent to the radical SAM domain [22], as observed previously in the structure of OxsB [20]. Cbl sits on top of the β2 secondary structure in the radical SAM core (Fig. 3), ~10 Å away from the SAM binding site. The enzyme also contains a third C-terminal domain that does not interact with Cbl but instead forms a substrate binding channel. TokK binds Cbl in the base-off conformation, an approach that allows for modulation of the coordination environment of the Co(III) ion by the protein environment. TokK lacks a lower axial ligand to the Co ion, using W⁷⁶ to block solvent access underneath the cofactor. This arrangement likely renders the Co-C bond in MeCbl more reactive by preventing coordination with a sixth ligand.

Figure 2.

A comparison of the domain architectures of radical SAM enzymes with auxiliary metallocofactors. The enzymes are colored by domain with the radical SAM core fold shown in blue. Substrates are shown as yellow sticks and cosubstrate SAM or its cleavage product (5′-dAH) are shown as green sticks. (A) Cobalamin-(Cbl)-dependent radical SAM enzyme, TokK, involved in carbapenem (sub) methylation (PDB ID 7KDY). (B) Lipoyl synthase, LipA, involved in sulfur insertion into a protein-linked fatty acid substrate (PDB ID 5EXK). (C) RNA modification enzyme, MiaB, implicated in methylthiolation of an adenine base in the anticodon stem loop (ACSL) of tRNA (PDB ID 7MJV). (D) Nitrogenase cofactor biosynthesis enzyme, NifB, involved in carbide and sulfur insertion to generate the Fe₈S₉C NifB-co product (PDB ID 7BI7). Selected amino acids are shown in stick format.

Figure 3.

Comparison of the metallocofactor and/or cosubstrate binding sites within the α₆β₆ radical SAM core domain reveals a common auxiliary cofactor binding site near β2 adjacent to the radical SAM Fe₄S₄ cluster. (C) TokK binds Cbl near the substrate pocket opposite the SAM cosubstrate binding site (PDB ID 7KDY). (D) LipA binds an auxiliary Fe₄S₄ cluster adjacent to the octanoyl-peptide binding site (PDB ID 5EXK). (E) NifB binds a Fe₈S₈ cluster near the N-terminal side of the radical SAM core fold (PDB ID 7B17). (F) MiaB binds an auxiliary Fe₄S₄ cluster near the SAM binding site above the tRNA docking interface (PDB ID 7MJV).

The arrangement of reactive groups in the active site of TokK was revealed by characterization of the enzyme bound to its carbapenam substrate and coproducts of the SAM cleavage reaction, 5′-dA and methionine [22]. Whereas several other Cbl-dependent radical SAM enzymes had been structurally characterized previously [20,30], none contained the primary substrate bound in a productive conformation. The carbapenem threads through a long channel at the interface between the three domains of TokK [22]. The β-lactam binds adjacent to the cobalamin, with C6 position ~4 Å away from the top ligand of the Co ion and the 5′-C of the 5′-dA. The arrangement demands that methyl transfer occur at the bottom face of the β-lactam, consistent with the known stereochemistry of the C6 substituent. The structure additionally implies that the TokK active site architecture primarily serves to position substrates and cofactors for radical methyl addition with very little intervention from other amino acids.

In this structure (and nearly all others of Cbl-dependent radical SAM enzymes) [20,22,30], the top ligand of cobalamin is modeled as a solvent-derived water or hydroxide, rather than the native methyl component of cobalamin. This substitution could alter the internal water network near the top ligand, thereby modifying the electrostatic environment of the substrate binding pocket. Incorporation of the bona fide Cbl methyl ligand could enable solution of additional structures with substrates bound. Interestingly, concomitant with the recent report of the TokK enzyme substrate complex, Mmp10, a peptide-modifying Cbl-dependent radical SAM enzyme was characterized in complex with its substrate [31]. This structure revealed analogous distances between reactive groups to those found in TokK. Mmp10 has a distinctive domain architecture compared to other Cbl-dependent radical SAM enzymes, including a different fold for the Cbl-binding domain and a different docking site within the radical SAM core for the Cbl cofactor. Mmp10 was reportedly cocrystallized with MeCbl, which may have promoted substrate incorporation in this system. In the absence of substrate and SAM, Mmp10 appears to bind an unusual Tyr ligand to the unique iron of the radical SAM cluster. The authors suggest that this interaction could be important for re-methylation of Cbl, a phenomenon that is not well understood in any Cbl-dependent radical SAM enzyme. Additional structural and spectroscopic characterization of these unusual interactions will be critical for experimental validation of this proposal.

2.2. Sulfur insertion radical SAM enzymes with auxiliary iron-sulfur clusters.

All radical SAM enzymes that insert sulfur into unactivated carbon centers contain additional iron-sulfur clusters that are proposed to serve as the sulfur source in these enzymes [8]. Several recent landmark studies by Booker and colleagues [12,32,33] have provided the comprehensive experimental evidence for such a role in the vitamin biosynthetic enzyme, LipA. LipA converts a carrier-protein-linked octanoyl side chain to lipoyl cofactor [34], which contains two thiol substituents at C6 and C8 (Fig. 1). The transformation proceeds as two distinct SAM-dependent transformations, with a 5′-dA• intermediate targeting C6 initially. Arrest of the reaction after the first step yields a cross-linked enzyme-substrate complex [32]. Analysis of this form of the enzyme by Mössbauer spectroscopy [32] and x-ray crystallography [12] revealed conversion of the auxiliary Fe₄S₄ cofactor to a Fe₃S₄ complex (Fig. 4). Comparison to structures solved in the absence of substrate shows that the lost iron in the auxiliary cluster was originally coordinated by a serine side chain located in a conserved C-terminal sequence motif. These interactions yield weaker bonds between the cofactor and the protein, likely enabling cofactor disassembly for sulfur donation.

Figure 4.

Zoomed-in views of the auxiliary iron-sulfur clusters in the sulfur-/carbide-insertion enzymes show the unusual metal binding motifs employed in the apo forms of each enzyme. (A, B) LipA uses a Ser ligand to the auxiliary Fe₄S₄ cluster (PDB ID 5EXJ) to facilitate formation of a monothiolated intermediate in which C6 of the octanoyl substrate side chain has added to a sulfide of the auxiliary cluster (PDB ID 5EXK). (C, D) MiaB contains a pentasulfide bridge between the unique irons of the auxiliary and radical SAM Fe₄S₄ clusters (PDB ID 7MJZ). Binding of SAM and the tRNA substrate results in a complex poised for methylation of sulfide in the auxiliary cluster and subsequent transfer to C2 of i⁶-A37 (PDB ID 7MJV). (E, F) In K1-bound NifB, an insertion loop in the radical SAM core contributes Glu and Cys ligands to the K1 and radical SAM Fe₄S₄ clusters, respectively (PDB ID 6Y1X). These unusual coordination interactions are disrupted upon occupancy of the enzyme with a Fe₈S₈ cluster (PDB ID 7B17).

A second comprehensive structural study of a radical SAM sulfur insertion enzyme was published earlier this year [23]. Methylthiotransferase, MiaB, responsible for synthesis of a methylthio substituent at C2 of a hypermodified adenine base in transfer RNA [35], was characterized bound to an RNA substrate in several different stages of its reaction cycle [23]. MiaB contains an auxiliary Fe₄S₄ cluster bound within a large N-terminal domain (Fig. 2). As in TokK and LipA, this cofactor docks against the N-terminal side of the radical SAM core (Fig. 3). Surprisingly, the structure and size of this domain differs significantly between LipA and MiaB. In LipA, the auxiliary cluster is coordinated by much smaller domains at the N- and C-termini (Fig. 2). However, MiaB shares with LipA an unusual cluster binding motif in its apo form. The auxiliary Fe₄S₄ cluster has a unique iron site that is linked to the radical SAM cluster via a polysulfide bridge in the apo state but becomes open upon SAM and RNA binding (Fig. 4).

Structures of MiaB with a small RNA fragment bound reveal a substrate binding site located directly underneath the two Fe₄S₄ cluster cofactors, approximately equidistant from the 5′-C of the 5′-dAH and one of the sulfide ions of the auxiliary cluster. The structures are consistent with a two-step mechanism for methylthiolation. In a first step, SAM methylates the auxiliary cluster at the outermost sulfide ion, yielding a Fe₃S₄ intermediate [36]. Attack of the methylated thiol upon the RNA substrate yields a transient enzyme-substrate intermediate that is proposed to be resolved by reductive cleavage of a second equivalent of SAM.

Observation of auxiliary cluster degradation during catalysis would, in theory, limit LipA and MiaB to single turnover reactions. Recently, two iron-sulfur cluster carrier proteins, NfuA and IscU, were shown to reconstitute the degraded auxiliary cluster in the LipA reaction [33,37], enabling multiple turnovers. Structural characterization of these carrier proteins in complex with LipA (and other sulfur insertion enzymes) will be an important next step in understanding the mechanism of iron-sulfur cluster reconstitution. Many other open questions remain about the structure and metal binding mode of the carrier proteins themselves. NfuA has been characterized by NMR spectroscopy, providing information about its overall fold [38], but little insight into how it binds its iron-sulfur cluster substrate and transfers it to target enzymes. Several crystal structures of IscU bound to a Fe₂S₂ cluster have been reported [39,40]. While the cluster binding motif remains ambiguous, the Fe₂S₂ unit is most likely coordinated by three cysteines and either an aspartic acid or a histidine at the fourth site. Observation of site differentiation in both the auxiliary cofactor of the target enzyme (LipA) and one of its partner iron-sulfur cluster carrier proteins suggests that this feature could play a role in cluster assembly and degradation.

2.3. Radical SAM enzymes in nitrogenase cofactor biosynthesis.

Radical SAM enzymes with auxiliary cofactors are additionally involved in complex cofactor assembly [2]. For example, a precursor to FeMo-co of nitrogenase is synthesized upon the radical SAM enzyme, NifB [41]. The core of mature FeMo-co contains two Fe₄S₃ subunits connected via three μ₂ sulfide ions and one μ₆ carbide ion [26]. The early stages of FeMo-co assembly require two proteins, NifB and NifEN [42,43]. NifB fuses two simple Fe₄S₄ clusters (K1 and K2) and inserts an additional sulfide and carbide to form a Fe₈S₉C product (Fig. 1), also known as NifB-co. NifB-co is subsequently transferred to scaffold protein NifEN for further maturation to give the active FeMo-co.

Two recently reported x-ray crystal structures of NifB provide initial insight into how two simple Fe₄S₄ clusters (K1 and K2) might be converted into NifB-co [24,25]. Fajardo et al. solved a crystal structure of NifB (PDB ID: 6Y1X) that shows the relative locations of the radical SAM iron-sulfur cluster and one of the two auxiliary [4Fe-4S] clusters, K1 [24]. The K2 cluster is missing in this dataset, along with >20 amino acids at the C-terminus of NifB. The K1 cluster is coordinated by two Cys side chains, a His, and a Glu. These ligands are found on the N-terminal side of the radical SAM core or in NifB-specific structural features (Fig. 2) that include a small N-terminal domain and insertion loop between β1 and β2 of the radical SAM fold. K1 binds near β2 in the core, similar to the auxiliary cluster binding sites in TsrM, MiaB, and LipA (Fig. 3).

The radical SAM cluster of NifB also exhibits unusual four-Cys coordination. Three ligands are provided by the predicted CX₃CX₂C cluster signature motif, but a fourth Cys (C⁶²) is distinct to NifB, and binds to the iron that is typically “open” for SAM to bind (Fig. 4). C⁶² is in the insertion loop that also contains the Glu ligand to K1. The radical SAM binding motif is reminiscent of that of TsrM, which uses a Glu to bind the fourth iron. However, unlike TsrM, NifB is a dual-purpose enzyme. In NifB, SAM is used both as a methyl donor and for hydrogen atom abstraction via reductive cleavage [43]. Methyl donation by SAM provides the interstitial carbide in NifB-co. But NifB produces both S-adenosylhomocysteine (SAH) and 5′-dAH as byproducts, indicating that a second SAM molecule is subsequently used to produce the 5′-dA•. The exact mechanism of carbide insertion by NifB remains unknown. The structure of NifB shows that the distance between the radical SAM cluster and K1 [24] is 12 Å, likely too far for direct methyl transfer to K1. The authors propose that the K2 cluster must therefore be the initial target for methylation and it probably resides in between the two iron-sulfur clusters observed in the structure.

A second structure of NifB was reported by Kang et al. [25], purported to contain the radical SAM cluster and both K1 and K2 clusters (PDB ID 7JMB). This structure would seem to show the K2 binding site and coordinating side chains. However, due to modest resolution (3.0 Å) and poor fit of the auxiliary cluster models to both omit and anomalous difference electron density maps, information about these features of the active site is limited. Refinement of this NifB structure by another research group [44] indicated that the electron density assigned to the auxiliary cofactors instead likely corresponds to a P-cluster-like fused Fe₈S₈ cluster with a μ₂-bridging sulfide ion (PDB ID: 7BI7). The significance of this cluster form as a possible intermediate in the NifB reaction remains unclear, although the bridging sulfide resides proximal to the radical SAM iron-sulfur cluster (Fig. 4), consistent with its initial target for methylation. Occupancy of the K2 binding site is accompanied by ordering of the C-terminus, which could provide additional ligands for binding a second auxiliary cluster. The insertion loop also changes conformation to move away from the cavity between the radical SAM cluster and K1. Important open questions remain about the oxidation states of the K1 and K2 clusters, both in the x-ray crystal structures and during catalysis. Finally, the SAM binding site remains to be elucidated in any structure of NifB. Given the dual role for SAM in NifB, structures in complex with this cosubstrate will be particularly important for understanding the mechanism of carbide insertion and cluster fusion.

3. Conclusions and outlook

Our analysis of recently reported x-ray crystal structures of radical SAM methylases and sulfur insertion enzymes highlights a common binding site for their auxiliary iron-sulfur cluster and cobalamin cofactors (or cosubstrates). All use the N-terminal side of the α₆β₆ radical SAM core fold to anchor the auxiliary moiety opposite the SAM binding site. Interestingly, this general binding location is shared by several other radical SAM enzymes that use auxiliary iron-sulfur clusters for electron transfer or substrate binding such as MoaA [16] and SuiB [14] (Fig. 2). Many of the enzymes discussed here also require interface with iron-sulfur-cluster assembly proteins. Use of a common auxiliary cluster binding site could provide a common docking surface for partner proteins. Structural characterization of these protein-protein complexes remains an important goal for future work. Additionally, we show that auxiliary domains at the N- and C-termini contribute important ligands to the auxiliary cofactors in these systems. The auxiliary metal binding motifs often include labile ligands that are likely responsible for the unique reactivities of the sulfur and carbon modification reactions catalyzed by this subset of radical SAM enzymes.