X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification

Peter J. Goldmana, Tyler L. Groveb, Lauren A. Sitesb, Martin I. McLaughlina,b, Squire J. Bookerb,c, and Catherine L. Drennana,d,e,1

Departments of aChemistry and dBiology and eHoward Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139; and Departments of bChemistry and cBiochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802

Edited by Vern L. Schramm, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY, and approved April 12, 2013 (received for review February 5, 2013) Arylsulfatases require a maturating to perform a co- or in terms of structure and mechanism (6, 7), far less is known posttranslational modification to form a catalytically essential about their anaerobic cousins, the anSMEs. Here we provide formylglycine (FGly) residue. In organisms that live aerobically, structural insights into these unusual AdoMet radical . molecular oxygen is used enzymatically to oxidize to FGly. The AdoMet radical enzyme family catalyzes a diverse array of Under anaerobic conditions, S-adenosylmethionine (AdoMet) rad- radical-based reactions, including sulfur insertions, complex chem- ical chemistry is used. Here we present the structures of an anaer- ical transformations and rearrangements, DNA and RNA mod- obic sulfatase maturating enzyme (anSME), both with and without ifications, and in the case of anSMEs, dehydrogenation (13) (Fig. peptidyl-substrates, at 1.6–1.8 Å resolution. We find that anSMEs 1). Members of this family have historically been identified by differ from their aerobic counterparts in using backbone-based aCX3CXΦC motif (where Φ is an aromatic residue), which ligates hydrogen-bonding patterns to interact with their peptidyl- three of the four irons of a [4Fe-4S] cluster (14, 15), leaving the substrates, leading to decreased sequence specificity. These fourth iron free to bind AdoMet (16). Radical chemistry is initi- anSME structures from Clostridium perfringens are also the first of ated by the injection of an electron via the [4Fe-4S] cluster into an AdoMet radical enzyme that performs dehydrogenase chemistry. AdoMet, resulting in the homolytic cleavage of the molecule into BIOCHEMISTRY Together with accompanying mutagenesis data, a mechanistic pro- and a 5′-deoxyadenosyl radical (5′dA•, Fig. 1). This posal is put forth for how AdoMet radical chemistry is coopted to radical species subsequently abstracts a hydrogen atom from sub- perform a dehydrogenation reaction. In the oxidation of cysteine or strate, resulting in 5′-deoxyadenosine (5′dA) and a substrate rad- serine to FGly by anSME, we identify D277 and an auxiliary [4Fe-4S] ical. Differentiation among the family members is a result of the cluster as the likely acceptor of the final proton and electron, re- action of this substrate radical. In anSMEs, the removal of a pro- spectively. D277 and both auxiliary clusters are housed in a cysteine- ton and an electron from the radical intermediate completes ca- rich C-terminal domain, termed SPASM domain, that contains ho- talysis (10, 17) (Fig. 1). mology to ∼1,400 other unique AdoMet radical enzymes proposed The AdoMet radical dehydrogenase subfamily includes anSMEs to use [4Fe-4S] clusters to ligate peptidyl-substrates for subsequent and the carbohydrate natural product biosynthetic enzyme BtrN modification. In contrast to this proposal, we find that neither aux- (18, 19). Interestingly, both enzymes harbor additional [4Fe-4S] iliary cluster in anSME bind substrate, and both are fully ligated by clusters that are necessary for turnover (20). In the case of BtrN, cysteine residues. Instead, our structural data suggest that the place- one auxiliary cluster has been identified (21), while anSMEs have ment of these auxiliary clusters creates a conduit for electrons to two (10, 17). For anSME, the sequence surrounding these two travel from the buried substrate to the protein surface. clusters, including a previously identified 7-cysteine motif (CX9–15 GX4C—gap—CX2CX5CX3C—gap—C) (17), places it in a ∼1,400- iron–sulfur cluster fold | radical SAM dehydrogenase membered AdoMet radical subfamily that was recently described by HaftandBasuthroughbioinformatic analysis and thought to fi osttranslational modification expands the chemical repertoire function in the modi cation of ribosomally translated (22). Pof enzymes, in some cases by generating modified amino This subfamily has been designated TIGR04085 and named acids that are well suited to perform specific reactions. Arylsul- SPASM for its biochemically characterized founding members fatases, for example, require the co- or posttranslational for- AlbA, PqqE, anSMEs, and MtfC, which are involved in subtilosin mation of a catalytically essential formylglycine (FGly) moiety to A, , anaerobic sulfatase, and perform their hydrolysis function, removing sulfate groups from maturation, respectively (22, 23). While the function of these a wide array of substrates (e.g., sulfated polysaccharides, sulfo- auxiliary clusters is unknown, the 7-cysteine motif prompted lipids, and steroid sulfates) (1–3). In humans, lack of sulfatase speculation that members of the SPASM subfamily, including activity can lead to disease (4), while in bacteria, inhibition anSMEs, use an available ligation site on one of the [4Fe-4S] impairs colonizing the mucosal layer of the host’s gut (5). The clusters for substrate binding (10). Direct binding of substrate maturation of these sulfatases involves two classes of enzymes, one that requires molecular oxygen and another that can func- tion in its absence. FGly generating enzymes (FGEs), found in Author contributions: P.J.G., T.L.G., L.A.S., S.J.B., and C.L.D. designed research; P.J.G. and M.I.M. performed the crystallization and crystal structure determination; T.L.G. provided eukaryotes or aerobically living prokaryotes, generate FGly by His6 and native protein samples; L.A.S. generated mutant constructs and performed activ- oxidizing a cysteine residue on the target sulfatase using mo- ity assays; and P.J.G. and C.L.D. wrote the paper. lecular oxygen (6, 7), whereas anaerobic sulfatase maturating The authors declare no conflict of interest. enzymes (anSMEs) generate FGly from either cysteine or serine This article is a PNAS Direct Submission. residues on their target sulfatases using S-adenosyl-L-methionine Data deposition: The atomic coordinates and structure factors have been deposited in the (AdoMet) radical chemistry (8–11). In addition to their impor- Protein Data Bank, www.pdb.org (PDB ID codes 4K36–4K39). tance for sulfatase chemistry, FGEs have commercial applications 1To whom correspondence should be addressed. E-mail: [email protected]. fi “ ” for generating site-speci c aldehyde tags to use in protein- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. labeling technology (12). While FGEs have been characterized 1073/pnas.1302417110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1302417110 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 [4Fe-4S]+1 1 2 Results AdoMet Met + 5'dA 5'dAH H Iron anomalous signal from a dataset collected at a home Cu-Kα +2 - source was used to solve an initial structure of His6-tagged [4Fe-4S] SH S anSMEcpe (“His6, AdoMet-bound” structure; Table S1). Due to 3 BBH occupancy of the His6 tag in the substrate binding site, additional 4 structures of native, untagged anSMEcpe were solved with and H 5 H Electron without two substrate peptides (“AdoMet-bound,”“Kp18Cys, O S acceptor AdoMet,” and “Cp18Cys, AdoMet” structures; Table S1). All FGly H SHO structures of anSMEcpe contain three [4Fe-4S] clusters in- 2 2 cluding the AdoMet cluster housed in the N-terminal AdoMet Fig. 1. anSME reaction. (1) Electron donation to the AdoMet radical cluster radical domain and two auxiliary clusters located in the C-terminal initiates homolysis of AdoMet and 5′dA• formation in the presence of SPASM domain. The two domains are connected by the α6a helix bound substrate, (2) substrate radical generation, (3) deprotonation of the and the protein terminates with the α6′ helix (Fig. 2 A–C). substrate Cys sidechain, (4) substrate oxidation, and (5) hydrolysis of the thioaldehyde intermediate yields the FGly moiety of the activated sulfatase. Structural Features of the anSMEcpe AdoMet Radical Domain. The N-terminal domain of anSMEcpe is a parallel (β/α)6 partial triose phosphate isomerase (TIM) barrel, spanning residues 3–234 (Fig. 2 to an auxiliary [4Fe-4S] cluster has been observed in the A and C, magenta). This fold is common to nearly all other molybdenum biosynthetic enzyme MoaA, another structurally characterized members of the AdoMet radical super- AdoMet radical enzyme (24, 25). Other possible functions spe- family (28, 29). We will refer to this partial barrel as the AdoMet fi ci c to anSMEs include the second oxidation of substrate or domain. Here, the AdoMet radical sequence motif (CX3CXΦC) is substrate deprotonation (Fig. 1). found in a loop following the β1 strand. C15, C19, and C22 each In this work, we report structures of anSMEcpe, a biochemi- ligate an iron atom in one of the [4Fe-4S] clusters, referred to here cally characterized anSME from Clostridium perfringens, which as the AdoMet cluster. The fourth, so-called “unique iron,” is li- can oxidize either serines or into FGly sidechains (26, gated by the amine nitrogen and carboxyl oxygen from the methi- 27). We have solved the structure of a His6-tagged protein onine moiety of AdoMet, as expected (16). All structures contain construct with AdoMet bound and three structures of an un- clear density for AdoMet bound in the active site (Fig. S1A), with tagged protein construct in the presence of AdoMet, one sub- the exception of a substrate-bound (Cp18Cys) cocrystal structure strate free and two others with different substrates where the shape and position of density for AdoMet in chain B is bound. The overall structure and substrate binding character- minimal and inconsistent with other models. istics of anSMEcpe differ significantly from the aerobic FGE Four previously described AdoMet binding motifs are con- system, which clarifies the difference in promiscuity between the served in anSMEcpe, including the “GGE” motif, the ribose two enzyme families. Surprisingly, the structures show full cys- motif, the “GXIXGXXE” motif, and the β6 motif (Figs. S1C and teine ligation of both auxiliary clusters, which has important S2) (28, 29). In addition, the backbone of Y21 (the hydrophobic implications for the anSMEcpe mechanism, as well as for SPASM residue in the AdoMet radical CX3CXΦC motif) hydrogen family members as a whole. bonds with the N6 position of adenine, and R143, just following

A B C 4a A AdoMet Cluster

26.7 Å 16.9 Å 6 6 6 5 5 4 4 3 3 2 2 1 1 1 2

Auxiliary 2 12.9 Å Cluster I 1 6a I 3 Auxiliary 7 Cluster II 8 6 5 II 2 4 D From 6a Conserved To 6 AdoMet domain SPASM domain 1 8 G271 [4Fe-4S] cluster CX2CX5CX3C motif 3 2 7 Y274 Fe ligating cysteine Aux II G329 Aux I Average 6 1 G328 2 5 4 1 - C255 2 - C261 3 - C276 4 - C317 2 567 - C320 - C326 - C330 8 - C348 Variable

Fig. 2. Structure of anSMEcpe. (A) The AdoMet domain (magenta) contains the AdoMet cluster and the (β/α)6 partial TIM barrel. The SPASM domain (green) comprises most of the C-terminal segment and houses the remaining two [4Fe-4S] clusters. Two helices, α6a and α6′, are not part of either domain and are colored light blue. (B) Positions and distances between the three [4Fe-4S] clusters (stick representation with Fe in orange and S in yellow). (C) Topology of anSMEcpe. A, AdoMet cluster; I, Aux I; II, Aux II. (D) The SPASM domain, colored by the level of sequence homology between anSMEcpe and the other 280 members of TIGR04085. Conservation scores were calculated by the ConSurf server (40). Iron ligating cysteines are shown as spheres and labeled by their designation in C. Aux I and II are shown in stick representation and labeled.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1302417110 Goldman et al. Downloaded by guest on September 28, 2021 α4a, stabilizes the ribosyl and carboxyl moieties of AdoMet. An present. Two substrate-bound structures were solved using arginine following α4a makes a similar interaction in the AdoMet peptides designed to mimic a C. perfringens (Cp18Cys) and a radical proteins HemN and HydE (30, 31). On the backside of Klebsiella pneumoniae (Kp18Cys) sulfatase protein (see Fig. S4A the AdoMet domain, a patch of conservation can be found for sequences). Each structure contains density for 9–11 residues following the β2strand(Fig. S1B). This site is the proposed of the 18mers (Fig. S5A). These peptide residues stretch from binding location of the physiological reductant, commonly the exterior of the protein to the active site, both entering and flavodoxin (30, 32). exiting via the cavity at the bottom of the barrel (Fig. 3 A and B). Buried surface area of the protein–peptide interaction for the four SPASM Domain and Auxiliary Cluster Binding. The α6a helix links bound peptides to anSMEcpe (two per asymmetric unit) is 786 ± the AdoMet domain to the C-terminal SPASM domain (Fig. 2 38 Å2, corresponding to 60.7 ± 1.7% of the total surface area of A–C, green), the latter containing both auxiliary clusters. Accord- the modeled portion of the peptides (34). This binding mode ing to the designation by TIGR04085, the SPASM domain initiates differs from the aerobic sulfatase maturating enzymes, FGEs, at C261, not C255 (the first Fe ligating cysteine in anSMEcpe). which use the same CXPXR motif for substrate recognition. In However, both cysteines are conserved in anSMEs and it is com- the aerobic system, the sulfatase maturase adopts a very different mon for SPASM domain-containing proteins to have a proximal fold that lacks the internal cavity found in anSMEcpe. Instead, upstream cysteine (22). In the anSMEcpe structures, C255 and peptide binding and catalysis occurs on a surface-exposed region C261 ligate the first auxiliary cluster, Auxiliary Cluster I (Aux I), of the enzyme (Fig. 3C) (7). Further, in the aerobic system, only before the backbone folds into a beta hairpin (V266–Y274). Re- four of the 12 hydrogen bonds between FGE and its substrate sidues of this hairpin exhibit high conservation to other members use the substrate backbone [Protein Data Bank (PDB) ID code of the SPASM family (Fig. 2D). Immediately following the hair- 2AIJ]. The majority of interactions are made between peptide pin, C276 provides the third ligand to Aux I. A variable alpha sidechains and the maturase, explaining the high sequence spec- helical region follows, providing a barrier between this cluster and ificity in this system (7). solvent. Aux I lies 16.9 Å from the AdoMet cluster (measured Other than the arginine residue of the CX(A/P)XR motif (Fig. from the closest atom in each cluster). S6 A and B), this side-chain–based sequence specificity is not seen The second portion of the SPASM domain contains the in anSMEs. First, anSMEcpe is able to accommodate consider- CX2CX5CX3C part of the 7-cysteine motif. The first three cys- able substrate sequence variation on either side of the target teines (C317, C320, and C326) provide three ligands to the second cysteine (Fig. S6 C and D), allowing the two peptides (Cp18Cys

auxiliary cluster, Aux II, while the protein backbone forms small and Kp18Cys) to bind in an almost identical orientation (Fig. 3A). BIOCHEMISTRY helical interactions that surround the cluster. The fourth cysteine In both cases, backbone hydrogen bonds to anSMEcpe are the of the motif, C330, crosses back to provide the final ligation site to primary means of stabilization (Fig. S5C). Only two positions, 4 Aux I (Fig. 2D). Very high sequence conservation is found in the and 10, of the peptides differ, resulting in two additional hydrogen linear CXXXC region bridging the two clusters, where the last two bonds at the 4 position of Kp18Cys (Figs. S4 and S5). The residues before the final cysteine of the motif are glycines (G328 remaining 17 hydrogen bonding interactions are conserved be- and G329 in anSMEcpe; Fig. 2D). Aux II lies 12.9 Å away from tween the two substrates. Of these, 12 are formed between pep- Aux I and 26.7 Å away from the AdoMet cluster (Fig. 2B). A series tide backbone and anSMEcpe and five are formed between of loops follows CX2CX5CX3CbeforeC348(thefinal of the seven anSMEcpe and a single peptidyl sidechain, R11 of the conserved cysteines) occupies the final ligation site of Aux II, ending the CX(A/P)XR sulfatase motif. The extensive binding pocket cre- SPASM domain and initiating the α6′ helix. This helix lies adjacent ated for this arginine, made up of F188, E159, and L118, uses π to the α6 helix and completes the barrel. The C terminus of the stacking, electrostatics/hydrogen bonding, and van der Waals protein lies at the end of this helix (Fig. 2C). interactions, respectively (Fig. S6A). These interactions appear to be the anchor for the peptide and are conserved among the other Structural Homology to MoaA. While the AdoMet domain of anS- biochemically characterized anSMEs (Fig. S2). MEcpe is very similar to other members of the AdoMet radical family, the C-terminal SPASM domain is structurally similar (rmsd Identification of Catalytic Residues. Aided by two prolines, the 6.3 Å) to only one other AdoMet radical protein, MoaA (33). Like substrate peptide makes a tight turn in the active site, allowing anSMEcpe, MoaA ligates a C-terminal auxiliary cluster that over- the target cysteine to protrude into the deepest part of the barrel, lays well with anSMEcpe’sAuxI(Fig. S3). Following two cysteine just below AdoMet. The cysteinyl Cβ is located 4.1 Å from the 5′ ligands to its auxiliary cluster, MoaA has a beta hairpin that shares carbon of AdoMet (Fig. 4A). This distance is in agreement with high sequence homology with anSMEcpe and other SPASM do- previously reported distances between the 5′ position of AdoMet main containing proteins, including G273 (G271 in anSMEcpe) in and the substrate hydrogen abstraction site (3.8–4.1 Å) (28). The the n+3 position of the hairpin turn and Y276 (Y274 in anSMEcpe), orientation of the cysteine directs the Cβ pro-S hydrogen toward a residue that contributes to a hydrophobic pocket adjacent to the the AdoMet 5′ position, matching biochemical evidence for the hairpin. MoaA then has a third cysteine ligand and terminates after enzyme’s stereoselectivity (26). a helical region. It lacks both the CX2CX5CX3C motif and the During catalysis, a general base is needed for deprotonation of second auxiliary cluster that are common in SPASM family mem- the cysteine side chain to allow the formation of the thioaldehyde bers. With only these three protein ligands to its auxiliary cluster, (Fig. 1). Analysis of the peptide-binding pocket revealed the MoaA uses the available coordination site to bind substrate (24, 25). presence of two residues with titratable side chains within 5 Å of In anSMEcpe, the final cysteine of the CX2CX5CX3C motif is the the substrate cysteinyl sulfur position, D277 and Y24 (Fig. 4A). fourth ligand to this cluster (Fig. S3 E and F). Y24 is two residues downstream of the AdoMet radical domain’s CX3CXΦC motif, while D277 is in the SPASM domain and Binding Specificity for Substrate Peptides. A cavity underneath the adjacent to C276, an Aux I ligand. To identify the catalytic residue, barrel is the only access to the active site from the exterior of the two mutants were generated and assayed for activity, anSMEcpe protein. In all structures of anSMEcpe solved using the His6– D277N and Y24F. Compared with wild type, the Y24F mutant anSMEcpe construct, multiple histidines of the C-terminal tag retains 11.7% FGly production activity, while the D277N mutant could be modeled into residual electron density. These residues only retains 0.8% activity. Along with this decreased activity, an appear to block access to the active site. However, when native uncoupling of the production of FGly and 5′dA is observed for the protein was used for cocrystallization with AdoMet and peptide D277N mutant (Fig. 4B). The proximity to the substrate sulfur and substrates, peptide density reaching into the active site was a large decrease in activity imply a catalytic role for the D277 side

Goldman et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 ABAdoMet AdoMet Cluster Arg Cluster Fig. 3. Substrate peptide binding. (A) Cp18Cys (black) and Kp18Cys (gray) en- 5 ter and exit the active site of anSMEcpe 4 Cys via the underside of the barrel. The Cβ AdoMet carbon is 8.6 and 8.9 Å from Aux I and the 6 4 AdoMet cluster, respectively. AdoMet 8.9 Å and auxiliary clusters are shown in sticks Aux I CX(A/P)XR with carbons in gray, oxygens in red, Aux II nitrogens in blue, sulfurs in yellow, and 8.6 Å C Arg irons in orange. anSMEcpe β strands and 3 SPASM domain are shown in ribbons and colored as in Fig. 2. (B) Substrate peptides Aux Substrate Cys bound to anSMEcpe and (C)boundto I FGE(PDBIDcode2AIJ,blue)(7)were overlaid by the five residues encompass- ing the conserved sulfatase motif in each 1 2 system and are shown in the same ori- CXPXR entation.

chain. Two residues, Q64 and Q98, have a role in stabilizing the anSME from Bacteroides thetaiotaomicron is responsible for ac- catalytic conformation of D277, as well as helping with stabilizing tivating up to 28 sulfatases under anaerobic conditions (5). From AdoMet binding. These residues have distinct conformations in structural comparisons, we can now explain this substrate spec- the presence or absence of AdoMet and substrate peptide (SI ificity variation between these enzyme classes. While the aerobic Results and Fig. S5 D and E). system uses a mainly side chain–maturase hydrogen bonding network for substrate stabilization, the anSME system uses Electron Transfer Pathway. After hydrogen abstraction and a primarily backbone–maturase hydrogen bonding network, with deprotonation, formation of the thioaldehyde drives oxidation of only the Arg of the (C/S)X(A/P)XR motif involved in side- β β the C , requiring an electron acceptor to complete catalysis. C lies chain–based hydrogen bonding. This reliance by SMEs on pri- 8.6 and 8.9 Å from Aux I and the AdoMet cluster, respectively, marily peptide backbone-based hydrogen bonding interactions is indicating both clusters are within suitable distance to be electron in agreement with the much higher degree of promiscuity that transfer partners (35). If an electron transfer event results in the exists in the anSME system in relation to the FGE system. In- reduction of Aux I, removing the electron from the system would terestingly, pyruvate formate lyase–activating enzyme (PFL-AE), likely require transfer from Aux I to Aux II, as peptide binding the only other structurally characterized AdoMet radical enzyme provides a barrier between Aux I and solvent (Fig. S7). Aux I lies involved in protein modification, also uses primarily peptide near the bottom opening of the anSMEcpe barrel. When no pep- backbone–activase interactions (32). As more structures become tide is present, the cluster is 9.7 Å from bulk solvent, with the inside available, it will be interesting to see if this binding mode will be of the protein barrel as the closest protein–bulk solvent interface common to AdoMet radical enzymes that act on protein substrates. (Fig. S7A). In this conformation, the cluster has a similar residue These structures of an anSME also provide insight into the depth to both the AdoMet cluster (9.5 Å) and Aux II (8.3 Å). catalytic mechanism of this enzyme class. For catalysis, at least However, when substrate is present this avenue to solvent is cut off, five steps are required (Fig. 1). For the first step of 5′dA• gen- and the shortest path to bulk solvent is below the barrel, 11.0 Å away (Fig. S7B). Peptide binding does not affect the residue depth eration, the high degree of similarity between the structure of the of either the AdoMet cluster or Aux II. anSME AdoMet radical domain and structures of other AdoMet radical enzymes suggests that all components necessary for Discussion radical generation are found in this N-terminal domain, implying Here we present the structures of an anSME, anSMEcpe, which allow us to compare how nature evolved anaerobic as well as aerobic solutions for the same enzyme function. While both en- A B zyme classes are designed to bind their target sulfatase, in one case, Y24 binding must involve sequestering a Cys/Ser to afford radical-based 8 chemistry, while in the other, the target Cys must be accessible to wt FGly* interact with molecular oxygen (6–8, 17). We find that the aerobic D277 wt 5 dA* FGEs and anSMEs use different protein folds, with the N-terminal 6 Y24F FGly domain of anSMEs sharing a classic AdoMet radical partial barrel M) Y24F 5 dA fold (28, 29), consistent with the chemistry being performed. While D277N FGly 4 D277N 5 dA the active site of FGE is on the surface of that enzyme, where it is AdoMet readily accessible to molecular oxygen, the active site of anSME is Q64

buried in a cleft created between the C-terminal SPASM domain ( Product 2 and the N-terminal AdoMet radical domain. To fitintothiscavity, Substrate the target peptide adopts a relatively tight turn, perhaps explaining Aux I 0 the preference for Ala or Pro in position of the conserved (S/C)X 0 20 40 60 80 100 120 (A/P)XR motif. In contrast, peptides bound to FGE have no ap- Time (min) parent conformational restraints (Fig. 3) (7). Fig. 4. anSME active site. (A) The active site of anSMEcpe. Sticks are dis- FGEs and anSMEs also vary in their substrate selectivity. played for AdoMet, target cysteine, and residues within 5 Å of the substrate Compared with FGEs, anSMEs are able to act on a larger variety cysteine Sγ. Distances as follows: AdoMet 5′C–cysteine Cβ,4.1Å;Y24–Sγ,4.7Å; of peptide substrates. For example, anSMEcpe itself can bind D277–Sγ, 4.6 Å; Q64 – Sγ,3.3Å.ColoredasinFig.3.(B)FGlyand5′dA pro- and catalyze FGly formation on a C. perfringens substrate analog duction for the Y24F and D277N mutants. Displayed product formation is per as well as a K. pneumoniae substrate analog (26). Another μmol enzyme. *Wild-type data from ref. 26.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1302417110 Goldman et al. Downloaded by guest on September 28, 2021 that anSMEs share a common initiation mechanism with the rest In addition to the mechanistic insight provided by these of the AdoMet radical superfamily. structures, visualization of the C-terminal domain of anSMEcpe In terms of substrate radical generation, these structures show clarifies the function of the recently described SPASM domain. the Cβ of the target Cys 4.1 Å from the AdoMet 5′C, in agree- Accession TIGR04085 designates 281 sequences as SPASM sub- ment with all other structures of AdoMet radical enzymes that family members. However, searching these 281 sequences within have substrates bound (Fig. 4). Very little movement within the Structure Function Linkage Database (SFLD) of the Radical anSMEcpe is required for substrate to bind in this catalytic po- SAM superfamily shows that these sequences are found in 153 sition. Only two glutamines, which are well conserved in anSMEs different nodes, which contain 1,392 unique sequences (Fig. S8) (Fig. S2, blue), have a distinct orientation in the presence or (38). While this is the first structure of a SPASM domain-con- absence of either AdoMet or peptide substrate. These residues taining enzyme, we find that the first part of the domain, con- aid both the stabilization of AdoMet and the positioning of D277 taining two of the seven cysteines of the 7-Cys motif, has been (SI Results and Fig. S5 D and E). visualized before in the structure of the AdoMet radical protein In contrast to radical generation, substrate deprotonation is MoaA. In particular, anSMEcpe shares with MoaA a conserved only required by a small number of AdoMet radical enzymes. beta hairpin that extends the AdoMet radical beta sheet and Before this work, it was not clear in anSME whether auxiliary contains cysteines on either end of the turn (Fig. 2). In both cases, cluster(s), enzyme residue(s), or both are involved in this reaction these two cysteines ligate an auxiliary [4Fe-4S] cluster along with step. Here, the structures of anSME with peptides bound show a third upstream cysteine (C255 in anSMEcpe) (Fig. S3). This beta that the substrate does not directly ligate either auxiliary cluster as hairpin also has high sequence homology to non-SPASM AdoMet previously suggested (10), and that both auxiliary clusters are far radical dehydrogenase BtrN. Like MoaA, BtrN contains an aux- from the target Cys (8.6 Å and 20.8 Å), making it highly unlikely iliary [4Fe-4S] cluster and, while BtrN has not been structurally these clusters are involved in this deprotonation and/or inner- characterized, the MoaA auxiliary cluster superimposes very well sphere electron transfer. Instead, the structure reveals two resi- with Aux I in anSMEcpe (Fig. S3). Thus, this beta hairpin motif fl dues with titratable side chains that are close to the target Cys that is anked by cysteines appears to be associated with binding (Y24 from the AdoMet radical domain and D277 from the an auxiliary [4Fe-4S] cluster in more than just SPASM-domain SPASM domain), and mutagenesis studies are consistent with containing proteins. Since this hairpin is a subdomain of SPASM, D277 as the catalytic base. It is interesting that the SPASM do- we will refer to it as a twitch subdomain. main, and not the AdoMet radical domain, contributes this key Following the twitch subdomain, MoaA completes two helices catalytic residue that differentiates anSME’s chemistry from that and terminates, while anSMEcpe continues the SPASM domain BIOCHEMISTRY of other AdoMet radical enzymes, as this finding is consistent with with one helix leading to the next set of four cysteines arranged previous structural studies that also showed the importance of in a CX2CX5CX3C motif. Interestingly, while all four cysteines of residues outside of the partial barrel radical fold to the di- this motif bind to an auxiliary cluster, they do not ligate the same cluster. The first three cysteines ligate Aux II, while the final versification of AdoMet radical chemistry (28, 29). cysteine ligates Aux I. Therefore, Aux I is coordinated by one The next step, substrate oxidation, is again only required by upstream cysteine, two twitch subdomain cysteines, and one certain subfamilies of AdoMet radical enzymes such as the cysteine from the SPASM domain’sCXCX CX C motif. MoaA dehydrogenases studied here, and others, like the bio- 2 5 3 shares this cluster coordination except for the last Cys; this Fe synthetic enzyme HemN (30). While the Aux I and AdoMet site is available for substrate binding (Fig. S3 C and F). In clusters are nearly equidistant and within acceptable electron anSME, the bridging sequence between the final two cysteines of transfer distances (8.6 and 8.9 Å) from the substrate Cβ (Fig. 3), CX2CX5CX3C is Lys–Gly–Gly. This “XGG” sequence is highly we propose that Aux I is the immediate electron acceptor for this conserved in TIGR04085 and could function to ensure a viable oxidation (Fig. 1, step 4). With few exceptions (36, 37), reduction electron transfer pathway environment between the two auxiliary of the AdoMet cluster during catalysis has only been proposed in clusters. The final Cys of the 7-Cys motif coordinates Aux II. systems that, unlike anSMEcpe, use AdoMet catalytically. As- Cysteine ligation of the auxiliary clusters was accurately predicted suming that Aux I is the electron acceptor, for a subsequent by TIGR04085, which establishes seven cysteine ligands for the turnover, it would need to be reoxidized. Here we further pro- two auxiliary clusters. However, an upstream cysteine, C255 in pose that Aux II, 12.9 Å away, performs this function. Other anSMEcpe, is also involved in cluster binding, resulting in the options for the reoxidation of Aux I are more problematic: the unexpected full protein ligation of both clusters. This result AdoMet cluster is too far from Aux I for direct electron transfer refutes the idea that all SPASM domains have an available ligation (16.9 Å), and the closest protein surface to which an external site for substrate binding and indicates that at least anSMEs do electron acceptor could bind appears blocked by bound substrate not use an auxiliary cluster for this purpose. An analysis of (Fig. S7). Thus, Aux II is the most viable candidate. TIGR04085 reveals that 37% of SPASM domain proteins have a We can further consider if electrons are recycled in this re- cysteine that is both upstream of the SPASM domain and down- — action that is, an electron used to homolytically cleave AdoMet stream of the AdoMet radical domain, indicating that full ligation in one cycle is derived from a previous cycle’s substrate oxidation. of the auxiliary cluster may be common among members of the By monitoring the level of flavodoxin semiquinone depletion SPASM subfamily. Without the role of substrate ligation, these during anSMEcpe catalysis, Grove et al. have recently established SPASM family members may use these clusters to facilitate electron that an electron can indeed be recycled in this fashion (26). While flow in or out of the active site during turnover, insinuating that their Aux II is also too far from the AdoMet cluster for direct electron mechanisms involve some kind of chemistry. transfer, an external electron acceptor, like flavodoxin, could ac- In the case of SPASM family members lacking an upstream cept an electron from Aux II and redeposit it into the AdoMet cysteine, the anSMEcpe structure indicates how substrates might cluster. Electrochemical characterization of all three clusters coordinate Aux I. In the absence of a cysteine equivalent to would provide validation that (i) Aux I is the substrate radical C255, an available iron coordination site would be exposed to electron acceptor during catalysis and (ii) electron transfer be- this substrate binding region (Fig. S3C). Importantly, due to the tween Aux I and Aux II is possible. In the meantime, the struc- distance between the ligation site and the AdoMet binding site, tures described here reveal distances that support a role for these the auxiliary cluster binding and hydrogen abstraction locations clusters in substrate oxidation by electron transfer, and refute must be distal (in anSMEcpe, C255 is 12 Å from the substrate other possible functions, including a role in substrate binding and hydrogen abstraction site). In the case of MoaA, where substrate deprotonation, as discussed above. does directly ligate cluster, the ligation site (the N1 position of

Goldman et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 the guanine base) is on the opposite end of the GTP substrate anaerobically using the vapor-diffusion technique (SI Materials and Methods). than the hydrogen abstraction site (the 3′ hydrogen atom of the The structure of anSMEcpe was solved using Fe anomalous data collected from ribose; Fig. S3C, gray) (24, 25, 39). A similar mode of binding a rotating copper anode source. High-resolution data were collected at would be required for any SPASM members that use cluster li- beamlines 24-ID-E and 24-ID-C at the Advanced Photon Source and on X-29 at gation for substrate binding. As substrates in the SPASM family the National Synchrotron Light Source and solved by isomorphous replacement. are predicted to be peptides, this would entail cluster ligation ∼3 Further data collection and model building details, as well as protocols used for creating Y24F anSMEcpe and D277N anSMEcpe, can be found in the SI Materials residues up- or downstream of the hydrogen abstraction site. and Methods. Activity assays were carried out as previously described (26), with In summary, the anSMEcpe system provides a great example of modifications detailed in the SI Materials and Methods. the modularity of the AdoMet radical superfamily. The anSMEcpe AdoMet domain has very similar structural folds and cofactor ACKNOWLEDGMENTS. For helpful discussions, we thank Daniel Dowling binding motifs to the rest of the superfamily. However, the end and Marco Jost. We also thank Allison Provost (Harvard University) for reaction catalyzed is unique from all other structurally charac- assistance with sequence analysis and Dennis Dean (Virginia Polytechnic terized AdoMet radical proteins. While the AdoMet domain must Institute) for the gift of pDB1282. Data for this study were measured at Beamline X29 at the National Synchrotron Light Source. This work was provide all residues necessary for radical generation, it is the supported by National Institutes of Health (NIH) Grant GM-63847 (to addition of the SPASM domain, which includes both auxiliary S.J.B.), NIH Grant GM-103268 (to S.J.B.), and National Science Foundation clusters and the catalytic residue that steers catalysis following Grant MCB-0543833 (to C.L.D.). This work is based upon research conducted 5′dA• generation. Interestingly, aside from the anSMEs, the only attheAdvancedPhotonSourceontheNortheastern Collaborative Access Team beamlines, which are supported by Award RR-15301 from the Na- other biochemically characterized AdoMet radical dehydrogenase, tional Center for Research Resources at NIH. Use of the Advanced Photon BtrN, only contains one additional [4Fe-4S] cluster. Upon struc- Source, an Office of Science User Facility operated for the US Department of tural characterization, it will be interesting to compare this enzyme Energy (DOE) Office of Science by Argonne National Laboratory, was sup- to anSMEcpe and the full SPASM domain architecture. ported by the US DOE under Contract DE-AC02-06CH11357. Financial sup- port comes principally from the Offices of Biological and Environmental Materials and Methods Research and of Basic Energy Sciences of the US DOE, the National Center for Research Resources (P41RR012408), and the National Institute of Gen- fi His6 anSMEcpe, puri ed as described (26), and native anSMEcpe, constructed eral Medical Sciences (P41GM103473) of NIH. C.L.D. is a Howard Hughes and purified as described in the SI Materials and Methods, were crystallized Medical Institute Investigator.

1. Schmidt B, Selmer T, Ingendoh A, von Figura K (1995) A novel amino acid modifica- 21. Grove TL, Ahlum JH, Sharma P, Krebs C, Booker SJ (2010) A consensus mechanism for tion in sulfatases that is defective in multiple sulfatase deficiency. Cell 82(2):271–278. radical SAM-dependent dehydrogenation? BtrN contains two [4Fe-4S] clusters. Bio- 2. Ghosh D (2007) Human sulfatases: A structural perspective to catalysis. Cell Mol Life chemistry 49(18):3783–3785. Sci 64(15):2013–2022. 22. Haft DH, Basu MK (2011) Biological systems discovery in silico: Radical S-adenosylmethionine 3. Bojarová P, Williams SJ (2008) Sulfotransferases, sulfatases and formylglycine-gener- protein families and their target peptides for posttranslational modification. J Bac- Curr Opin Chem Biol – ating enzymes: A sulfation fascination. 12(5):573 581. teriol 193(11):2745–2755. fi 4. Dierks T, et al. (2003) Multiple sulfatase de ciency is caused by mutations in the gene 23. Haft DH (2011) Bioinformatic evidence for a widely distributed, ribosomally produced Cell – encoding the human C(alpha)-formylglycine generating enzyme. 113(4):435 444. electron carrier precursor, its maturation proteins, and its nicotinoprotein redox 5. Benjdia A, Martens EC, Gordon JI, Berteau O (2011) Sulfatases and a radical AdoMet partners. BMC Genomics 12:21. enzyme are key for mucosal glycan foraging and fitness of a prominent human gut 24. Lees NS, et al. (2009) ENDOR spectroscopy shows that guanine N1 binds to [4Fe-4S] Bacteroides. J Biol Chem 286(29):25973–25982. cluster II of the S-adenosylmethionine-dependent enzyme MoaA: Mechanistic im- 6. Dierks T, et al. (2005) Molecular basis for multiple sulfatase deficiency and mechanism plications. J Am Chem Soc 131(26):9184–9185. for formylglycine generation of the human formylglycine-generating enzyme. Cell 25. Hänzelmann P, Schindelin H (2006) Binding of 5′-GTP to the C-terminal FeS cluster of 121(4):541–552. the radical S-adenosylmethionine enzyme MoaA provides insights into its mechanism. 7. Roeser D, et al. (2006) A general binding mechanism for all human sulfatases by the Proc Natl Acad Sci USA – formylglycine-generating enzyme. Proc Natl Acad Sci USA 103(1):81–86. 103(18):6829 6834. 8. Fang Q, Peng J, Dierks T (2004) Post-translational formylglycine modification of 26. Grove TL, et al. (2013) Further characterization of Cys-type and Ser-type anaerobic bacterial sulfatases by the radical S-adenosylmethionine protein AtsB. J Biol Chem sulfatase maturating enzymes suggests a commonality in mechanism of catalysis. 279(15):14570–14578. Biochemistry, 10.1021/bi400136u. 9. Benjdia A, et al. (2007) Anaerobic sulfatase-maturating enzymes: Radical SAM en- 27. Benjdia A, et al. (2008) Anaerobic sulfatase-maturating enzymes, first dual substrate zymes able to catalyze in vitro sulfatase post-translational modification. J Am Chem radical S-adenosylmethionine enzymes. J Biol Chem 283(26):17815–17826. Soc 129(12):3462–3463. 28. Vey JL, Drennan CL (2011) Structural insights into radical generation by the radical 10. Grove TL, Lee KH, St Clair J, Krebs C, Booker SJ (2008) In vitro characterization of AtsB, SAM superfamily. Chem Rev 111(4):2487–2506. a radical SAM formylglycine-generating enzyme that contains three [4Fe-4S] clusters. 29. Dowling DP, Vey JL, Croft AK, Drennan CL (2012) Structural diversity in the AdoMet Biochemistry 47(28):7523–7538. radical enzyme superfamily. Biochim Biophys Acta 1824(11):1178–1195. 11. Berteau O, Guillot A, Benjdia A, Rabot S (2006) A new type of bacterial sulfatase reveals 30. Layer G, Moser J, Heinz DW, Jahn D, Schubert WD (2003) Crystal structure of cop- a novel maturation pathway in prokaryotes. JBiolChem281(32):22464–22470. roporphyrinogen III oxidase reveals cofactor geometry of radical SAM enzymes. 12. Rabuka D, Rush JS, deHart GW, Wu P, Bertozzi CR (2012) Site-specific chemical protein EMBO J 22(23):6214–6224. conjugation using genetically encoded aldehyde tags. Nat Protoc 7(6):1052–1067. 31. Nicolet Y, Amara P, Mouesca JM, Fontecilla-Camps JC (2009) Unexpected electron 13. Frey PA, Hegeman AD, Ruzicka FJ (2008) The radical SAM superfamily. Crit Rev Bio- transfer mechanism upon AdoMet cleavage in radical SAM proteins. Proc Natl Acad chem Mol Biol – 43(1):63 88. Sci USA 106(35):14867–14871. fi 14. So a HJ, Chen G, Hetzler BG, Reyes-Spindola JF, Miller NE (2001) Radical SAM, a novel 32. Vey JL, et al. (2008) Structural basis for glycyl radical formation by pyruvate formate- protein superfamily linking unresolved steps in familiar biosynthetic pathways with lyase activating enzyme. Proc Natl Acad Sci USA 105(42):16137–16141. radical mechanisms: Functional characterization using new analysis and information 33. Hänzelmann P, Schindelin H (2004) Crystal structure of the S-adenosylmethionine- visualization methods. Nucleic Acids Res 29(5):1097–1106. dependent enzyme MoaA and its implications for molybdenum cofactor deficiency in 15. Hiscox MJ, Driesener RC, Roach PL (2012) Enzyme catalyzed formation of radicals humans. Proc Natl Acad Sci USA 101(35):12870–12875. from S-adenosylmethionine and inhibition of enzyme activity by the cleavage prod- 34. Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline ucts. Biochim Biophys Acta 1824(11):1165–1177. state. J Mol Biol 372(3):774–797. 16. Walsby CJ, Ortillo D, Broderick WE, Broderick JB, Hoffman BM (2002) An anchoring 35. Moser CC, Anderson JL, Dutton PL (2010) Guidelines for tunneling in enzymes. Bio- role for FeS clusters: Chelation of the amino acid moiety of S-adenosylmethionine to chim Biophys Acta 1797(9):1573–1586. the unique iron site of the [4Fe-4S] cluster of pyruvate formate-lyase activating en- 36. Grove TL, et al. (2011) A radically different mechanism for S-adenosylmethionine- zyme. J Am Chem Soc 124(38):11270–11271. Science – 17. Benjdia A, et al. (2010) Anaerobic sulfatase-maturating enzyme—A mechanistic link dependent . 332(6029):604 607. with glycyl radical-activating enzymes? FEBS J 277(8):1906–1920. 37. Szu PH, Ruszczycky MW, Choi SH, Yan F, Liu HW (2009) Characterization and mech- 18. Yokoyama K, Ohmori D, Kudo F, Eguchi T (2008) Mechanistic study on the reaction of anistic studies of DesII: A radical S-adenosyl-L-methionine enzyme involved in the a radical SAM dehydrogenase BtrN by electron paramagnetic resonance spectroscopy. of TDP-D-desosamine. J Am Chem Soc 131(39):14030–14042. Biochemistry 47(34):8950–8960. 38. Gerlt JA, et al. (2011) The Enzyme Function Initiative. Biochemistry 50(46):9950–9962. 19. Yokoyama K, Numakura M, Kudo F, Ohmori D, Eguchi T (2007) Characterization and 39. Mehta AP, et al. (2013) Catalysis of a new ribose carbon-insertion reaction by the mechanistic study of a radical SAM dehydrogenase in the biosynthesis of butirosin. J molybdenum cofactor biosynthetic enzyme MoaA. Biochemistry 52(7):1134–1136. Am Chem Soc 129(49):15147–15155. 40. Ashkenazy H, Erez E, Martz E, Pupko T, Ben-Tal N (2010) ConSurf 2010: Calculating 20. Lanz ND, Booker SJ (2012) Identification and function of auxiliary iron-sulfur clusters evolutionary conservation in sequence and structure of proteins and nucleic acids. in radical SAM enzymes. Biochim Biophys Acta 1824(11):1196–1212. Nucleic Acids Res 38(Web Server issue):W529–W533.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1302417110 Goldman et al. Downloaded by guest on September 28, 2021