Auxiliary Iron–Sulfur Cofactors in Radical SAM Enzymes☆

Biochimica et Biophysica Acta 1853 (2015) 1316–1334

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Biochimica et Biophysica Acta

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Review Auxiliary iron–sulfur cofactors in radical SAM enzymes☆

Nicholas D. Lanz a, Squire J. Booker a,b,⁎ a Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, United States b Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, United States article info abstract

Article history: A vast number of enzymes are now known to belong to a superfamily known as radical SAM, which all contain a Received 19 September 2014 [4Fe–4S] cluster ligated by three cysteine residues. The remaining, unligated, iron ion of the cluster binds in Received in revised form 15 December 2014 contact with the α-amino and α-carboxylate groups of S-adenosyl-L-methionine (SAM). This binding mode Accepted 6 January 2015 facilitates inner-sphere electron transfer from the reduced form of the cluster into the sulfur atom of SAM, Available online 15 January 2015 resulting in a reductive cleavage of SAM to methionine and a 5′-deoxyadenosyl radical. The 5′-deoxyadenosyl

Keywords: radical then abstracts a target substrate hydrogen atom, initiating a wide variety of radical-based transforma- – Radical SAM tions. A subset of radical SAM enzymes contains one or more additional iron sulfur clusters that are required Iron–sulfur cluster for the reactions they catalyze. However, outside of a subset of sulfur insertion reactions, very little is known S-adenosylmethionine about the roles of these additional clusters. This review will highlight the most recent advances in the identiﬁca- Cofactor maturation tion and characterization of radical SAM enzymes that harbor auxiliary iron–sulfur clusters. This article is part of a Radical-dependent post-translational Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases. ﬁ modi cation © 2015 Elsevier B.V. All rights reserved. SPASM/twitch domain

1. Introduction RS enzymes containing auxiliary Fe/S clusters [26]. Therefore, this work will not strive to reproduce what has already been written, but The radical-SAM (RS) superfamily of enzymes is deﬁned by the rather focus on the most recent advances made in the ﬁeld. While the ability of its members to cleave S-adenosyl-L-methionine (SAM) to me- function of many of the auxiliary Fe/S clusters remains unknown, recent thionine and a 5′-deoxyadenosyl radical (5′-dA•), a potent oxidant, work does provide insight into their roles in several enzymes. Notably, using an electron provided by a reduced, [4Fe–4S]1+, cluster (Fig. 1) diverging roles for the auxiliary clusters in sulfur-donating enzymes

[1–7]. Cysteines residing in a CX3CX2C motif ligate three of the four have been presented, multiple crystal structures of enzymes containing iron ions of the cluster, leaving an available coordination site to which the SPASM/twitch domain have been reported, mechanistic insight has SAM binds in a bidentate fashion via its α-carboxylate and α-amino been provided for the enzymes responsible for the maturation of the groups [8–12]. All structurally characterized RS enzymes that reside in molybdenum cofactor and various hydrogenase cofactors, and several pfam04055 contain either a full or partial Triose-phosphate Isomerase new RS enzymes have been added to the class (vide infra). Mutase (TIM) barrel fold in which the SAM/[4Fe–4S] cluster cofactor is bound. Additional domains that interact with the core fold are also frequently found in various subclasses of RS enzymes [13–25]. While 2. Sulfur donating enzymes RS enzymes share a common fold, they diverge greatly in the reactions they catalyze and the mechanisms by which they operate. Two distinct types of sulfur-insertion reactions have been described A subset of RS enzymes contains one or more iron–sulfur (Fe/S) clus- to date (Fig. 2). The first type involves the insertion of one or two sulfur ters in addition to the cluster required for SAM cleavage [26].Thisgroup atoms into an organic substrate. Biotin synthase (BioB) generates a of enzymes highlights both the diversity of reactions catalyzed by RS en- thioether bond between carbons 6 and 9 of dethiobiotin in the final zymes as well as the range of functions of Fe/S clusters. The authors have step of the biosynthesis of the biotin cofactor [27–29]. The other mem- already penned a review describing the identification and functions of ber of the group, lipoyl synthase (LipA), inserts sulfur atoms at carbons 6 and 8 of a protein-bound n-octanoyl chain [30–33]. The second type of reaction involves the generation of a methylthio moiety and its subsequent attachment to a substrate carbon atom [34]. Three classes of ☆ This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, func- methylthiotransferases (MTTases), represented by RimO, MiaB, and tion, biogenesis and diseases. MtaB, catalyze this type of reaction, of which only RimO and MiaB ⁎ Corresponding author at: 104 Chemistry Building University Park, PA 16802, United States. Tel.: +1 814 865 8793; fax: +814 865 2927. have been studied mechanistically [34–36]. RimO catalyzes the E-mail address: [email protected] (S.J. Booker). methylthiolation of the β-carbon of a specific aspartyl residue of the

attachment to an unactivated carbon atom, evidence now points to differing roles of the auxiliary clusters in these enzymes.

2.1. Lipoyl synthase

LipA and BioB catalyze highly similar reactions. The reaction of BioB (Fig. 2A), the insertion of a single sulfur atom between C6 and C9 of dethiobiotin, requires the presence of a [2Fe–2S] cluster in addition to the RS [4Fe–4S] cluster. The [2Fe–2S] cluster has a unique ligation environment comprised of three cysteines and one arginine [14].Anumber of studies were conducted to determine the cluster content of BioB [38–42], the sacrificial role of its [2Fe–2S] cluster [43–50], as well as intermediates in its reaction [42,51]. As there have been no recent devel- opments, the focus of this section will be on the related enzyme, LipA. LipA catalyzes the stepwise insertion of two sulfur atoms into an octanoyl chain attached to a lipoyl carrier protein (LCP) (Fig. 2B) [30–33]. As is the case for BioB, LipA cleaves two molecules of SAM for each molecule of product produced [52]. However, in place of the [2Fe–2S] cluster found in BioB, LipA contains an auxiliary [4Fe–4S] cluster [52]. In the proposed mechanism, the first 5′-dA• generated abstracts ahydrogenatom(H•) from C6 of the octanoyl chain, resulting in a transient carbon-centered substrate radical (Fig. 3) [31,52]. The substrate radical attacks a bridging μ-sulfido ion of the auxiliary [4Fe–4S] cluster of LipA, generating a covalently cross-linked intermediate between the octanoyl chain of the LCP and the [4Fe–4S] cluster of LipA. The second 5′dA• abstracts a H• from C8 of the octanoyl substrate, and the resulting C8 radical attacks a second bridging μ-sulfido ion of the auxiliary cluster, complete the synthesis of the lipoyl cofactor upon the addition of two protons. Signifi cant mechanistic detail of the LipA reaction was obtained from several studies on the enzyme. Cicchillo et al. provided evidence that both sulfur atoms are derived from the same polypeptide in an Fig. 1. Cleavage of SAM by RS enzymes.

S12 subunit of the ribosome [37]. MiaB performs a similar reaction, catalyzing the methylthiolation of an adenosine nucleotide at position 37 (A37) on several tRNAs [35,36]. While the reactions all involve sulfur

Fig. 2. Sulfur insertion and methylthiolation reactions catalyzed by RS enzymes. Fig. 3. Proposed mechanism for lipoyl synthase. 1318 N.D. Lanz, S.J. Booker / Biochimica et Biophysica Acta 1853 (2015) 1316–1334 experiment where two forms of LipA, overproduced separately in media demonstrating the catalytic competence of the cross-linked intermediate. containing either natural abundance sulfide or 34S-labeled sulfide, were Furthermore, the rate of formation of the lipoyl product from the cross- mixed together and allowed to react under turnover conditions [32]. linked species was consistent with the overall rate of the reaction, indicat- The sulfur atoms in the resulting lipoyl products were predominantly ing the kinetic competence of the reaction from the cross-linked species. both 32S-labeled or 34S-labeled. If each polypeptide contributed only For each of the methods of isolating the cross-linked species, one sulfur atom, the lipoyl products would have been expected to samples were prepared with 57Fe-labeled LipA for analysis by EPR and have mixtures of the two isotopes in a ratio of 1:2:1 for the 32S/32S-, Mössbauer spectroscopies [54]. Although very small quantities of 32S/34S-, and 34S/34S-labeled species, which were not observed. paramagnetic species were detected, the Mössbauer spectra provided Importantly, a significant isotope effect was observed on the LipA interesting insight. As expected, the spectrum of the as-isolated enzyme reaction when a substrate containing a perdeuterated octanoyl moiety was composed of a quadrupole doublet with parameters indicative of was used [52]. Approximately one-half of an equivalent of singly- the presence of [4Fe–4S] clusters. Unexpectedly, the Mössbauer spectra deuterated 5’-deoxyadenosine (5′-dA) was observed after 1 h of incuba- revealed the presence of a [3Fe–4S] cluster present in the cross-linked tion time; however, the lipoyl product was not detected. It was deter- species regardless of the method of isolation. Though the fraction of mined that only one of the two deuterium atoms was abstracted, [3Fe–4S] cluster present in the anion-exchange purified sample effectively arresting the reaction at the halfway point. Work by Bryant suggested that greater than one equivalent of 3Fe species was present, et al. demonstrated that a thermostable LipA from Sulfolobus solfataricus the amount in excess of the first equivalent was presumed to be caused could act on a short tripeptide substrate containing the requisite by the purification method. In the other three cases, where gentler octanoyl-lysine [53]. This substrate provided a more manageable purification methods were used or no purification was necessary, the means of measuring LipA activity, because it could be readily synthe- amount of 3Fe species present correlated with the amount of sized and reliably quantified by chromatographic methods. Douglas cross-linked species. The remaining iron in the spectrum was split et al. first identified the 6-mercapto-octanoyl-peptide intermediate between ferrous iron that corresponded to the iron lost from the auxil- using a combination of mass spectrometry and two-dimensional iary cluster, and a mixture of [4Fe–4S] and site-differentiated [4Fe–4S] nuclear magnetic resonance (NMR) spectroscopy after quenching the clusters (SAM or methionine-bound) that correspond to the unaffected reaction in acid and purifying the intermediate by high-performance RS cluster. Upon completion of the reaction, the Mössbauer spectra liquid chromatography HPLC [31]. The authors measured the mass indicated the presence of a single [4Fe–4S] cluster remaining in LipA. 2 shift of the products of a reaction in the presence of 8,8,8- H3- Again, this observation is consistent with the RS cluster remaining octanoyl-peptide. All three deuteriums were retained in the stable throughout the reaction. The remainder of the iron is a mixture monothiolated product, indicating that a H• was not abstracted from of thiolate-ligated ferrous iron, N/O-ligated ferrous iron, and C8. Sulfur insertion at C6 of the intermediate was confirmed by charac- 2Fe-clusters that are likely the byproduct of the destruction of the terization of the intermediate isolated from a reaction containing unla- auxiliary cluster. Like the [2Fe–2S] cluster of BioB, the auxiliary cluster beled substrate by COSY NMR spectroscopy. of LipA appears to be sacrificed for the sake of a single turnover. More recently, the auxiliary cluster of LipA was characterized at the The recent crystal structure of LipA from Thermosynechococcus intermediate stage of the reaction by a combination of mass spectrom- elongates showed that the protein is composed of a partial TIM barrel etry and EPR and Mössbauer spectroscopies [54]. The intermediate was that is typical of RS enzymes, but also revealed a novel serine ligand to 2 generated using an 8,8,8- H3-octanoyl-LCP, and the protein complex, the auxiliary cluster (Fig. 4A) [21]. As expected, the RS cluster is coordi- which was observed as a cross-link between LipA and the octanoyl- nated by the CX3CX2C motif, and the auxiliary cluster is ligated by the LCP, was purified by anion-exchange chromatography. Formation of N-terminal CX4CX5C motif as well as by a conserved serine residue at the cross-link required incubation of LipA with the octanoyl-LCP sub- the C-terminus of the enzyme (Fig. 4B). Two structures of LipA were strate under turnover conditions. Moreover, the cross-link was labile reported. In one structure, the enzyme was crystallized in the presence in the presence of acid or detergent, but stable under chromatographic of SAH instead of SAM. S-adenosylhomocysteine (SAH), which lacks the conditions that separate LipA and the LCP, which bind tightly, before sulfonium methyl group of SAM, was bound to the cluster similarly to initiating the reaction. This data suggests that the cross-link is mediated how SAM binds in other RS enzymes. In the second case, LipA was by an iron–sulfur cluster of LipA rather than a direct covalent interaction crystallized in the presence of SAM, but a breakdown product of SAM, between the two polypeptides. 5′-methylthioadenosine (5′-MTA), was bound in the active site, and The cross-linked intermediate was also generated with an dithiothreitol was coordinated to the unique iron site of the RS cluster. eight-amino acid peptide substrate under three different condi- Using the information in these structures, SAM could be modeled into 2 tions [54].Inthefirst instance, an 8,8,8- H3-octanoyl-peptide addition- the structure as well as octanoyl-lysine methylamide as a substrate ally tagged with biotin was used. LipA from Thermus thermophilus was surrogate. In this model, the sulfonium ion of SAM is 4.4 Å from the incubated with this peptide under turnover conditions, and the nearest iron ion of the RS cluster, and the 5′ carbon of SAM is 4.4 Å resulting cross-linked species was separated from unreacted enzyme and 5.2 Å from C6 and C8 of the octanoyl chain, respectively, positioning using a streptavidin-mutein column. In the second instance, the same the substrates suitably for the reaction to occur. peptide was used to arrest turnover after the first half of the reaction Substitution of the serine ligand to the auxiliary cluster with either using enzyme from Escherichia coli (EcLipA). The yield of the intermedi- alanine or cysteine abolished the ability of the enzyme to generate a ate species was sufficiently high that further purification was unneces- lipoyl product [21]. However, abortive cleavage of SAM was still ob- sary. In the last instance, taking advantage of the fact that the first half served. The importance of the serine ligation to the auxiliary cluster is of the LipA reaction proceeds much more rapidly than the second half, consistent with the spectroscopic data previously observed in which EcLipA was reacted with unlabeled octanoyl-peptide in the presence an iron ion is lost from the auxiliary cluster during the course of the for- of only one equivalent of SAM, resulting in the formation of the cross- mation of the monothiolated intermediate (vide supra) [54]. Serine has linked intermediate as the predominant species. Using the unlabeled ahigherpKa value than cysteine, and its protonation, while coordinated peptide substrate, the authors were also able to demonstrate the chem- to the unique Fe ion, might trigger its dissociation from the cluster, ical and kinetic competence of the isolated intermediate. Upon forma- allowing for release of the Fe ion to which it was ligated. tion of the cross-link using one equivalent of SAM, excess peptide and Perhaps the most intriguing question left to be answered with re- other small-molecule reaction components were removed by gel- gard to the BioB and LipA reactions is that of the mechanism of cluster filtration chromatography. After reintroduction of SAM and dithionite, reassembly. One possibility is that the enzymes responsible for building a low-potential reductant used to initiate turnover, the lipoyl product and inserting Fe/S clusters are also capable of reassembling the auxiliary was observed to form in the absence of additional octanoyl-peptide, clusters after each turnover. These proteins are encoded in the isc or suf N.D. Lanz, S.J. Booker / Biochimica et Biophysica Acta 1853 (2015) 1316–1334 1319

ABRS Cluster

Substrate binding Auxiliary Cluster channel

RS Cluster Methionine Ser283

Auxiliary Cluster

Fig. 4. Structure of LipA (A) and coordination of methionine and a serine residue to the RS and auxiliary clusters of LipA, respectively (B) (PDB accession code4U0P).TheRSdomainofLipA is shaded in green while the N-terminal and C-terminal extensions that contain the ligands for the auxiliary cluster are shaded in blue and pink, respectively.

operons in E. coli and other organisms. ISA1 and ISA2, two of the en- 2.2. Methylthiotransferases zymes from the mitochondrial Fe/S cluster assembly system in Saccha- romyces cerevisiae, were found to support catalysis of BioB, but were The MTTases are often compared with LipA and BioB, because the not required for the de novo biosynthesis of the Fe/S clusters of the pro- reactions they catalyze involve sulfur attachment to unactivated carbon tein. It is possible that these proteins aid in the regeneration of the sac- atoms. Two MTTases, RimO and MiaB, have been studied mechanistically. rificial [2Fe–2S] cluster of BioB [55]. It has also been proposed that ISA1 RimO catalyzes the methylthiolation of the β-carbon of Asp89 and ISA2 may be required for the biosynthesis of [4Fe–4S] clusters while (E. coli numbering) of the S12 subunit of the ribosome (Fig. 2D) a second set of proteins, ISU1 and ISU2, are responsible for generating [37]. Like BioB and LipA, this reaction requires the abstraction of a H• [2Fe–2S] clusters as well as the precursors for [4Fe–4S] clusters [56]. from an sp3-hybridized carbon and the subsequent formation of a In this model, ISU1 and ISU2 may be necessary to regenerate the auxil- carbon\sulfur bond. Similarly, MiaB catalyzes the methylthiolation of iary cluster of BioB. C2 of an isopentenylated adenosine (i6A) immediately 3′ to the The well-studied iron–sulfur cluster assembly systems of both E. coli anticodon on several tRNAs, forming 2-methylthio-i6A(ms2i6A) and S. cerevisiae include an ATP-dependent chaperone, HscA (Ssq1 in S. (Fig. 2C) [66–68]. In this case, the methylthio group replaces an H• on cerevisiae) [57]. Unlike homologous chaperones, HscA is constitutively an sp2-hybridized carbon atom. A significant difference between the expressed and likely plays an important housekeeping role not associated MTTases and sulfur insertion enzymes lies in their usage of SAM. Both with stress [58,59]. In addition to the previously studied interaction of LipA and BioB cleave two molecules of SAM in a stepwise fashion, HscA with the iron–sulfur cluster scaffold IscU, it was found that HscA generating two equivalents of 5′-dA. MTTases also utilize two also interacts with BioB [60]. HscA can form a complex with both IscU equivalents of SAM; however, only one equivalent is used to generate and BioB simultaneously and displays a higher affinity for BioB lacking a5′-dA•, while the second equivalent is used as the methyl donor to both iron–sulfur clusters. These results suggest that HscA may recruit cap the sulfur atom. The co-product of this typical SN2-based methyl IscU to BioB and aid in the regeneration of the auxiliary cluster. transferase reaction is SAH. A gene encoding an interacting partner of ISA1 and ISA2 in S. Like LipA, RimO and MiaB harbor two [4Fe–4S] clusters. Given the cerevisiae, IBA57, was identified by Gelling et al. [61]. Deletion of the similarities in the reaction to LipA and BioB, it is tempting to assign a IBA57 gene prevented insertion of the iron–sulfur clusters of aconitase sacrificial role to the auxiliary clusters of the MTTases as well. Because and homoaconitase and also inhibited the functions of BioB and LipA early studies reported no more than one turnover per polypeptide for in vivo. Defective versions of the human homologs, ISCA1, ISCA2, and MiaB and RimO, it was postulated that MTTases may insert the sulfur IBA57, are associated with severe disease and premature death as well atom into the substrate in a mechanism that parallels that of LipA, and [62,63]. Misformed mitochondria and decreased activity of a subset of that this inserted sulfur atom would be subsequently capped with a mitochondrial Fe/S proteins, including LipA, were observed when methyl group. Early evidence supported this model [69].Strainsof these three genes were silenced using RNAi [62]. Given that these E. coli auxotrophic for methionine and cultured under methionine genes are also found in E. coli, this pathway is likely equally important starvation conditions produced a derivative of the isopentenylated in prokaryotes [61]. adenosine that could be methylated in a separate reaction. This observa- Two additional human genes, NFU1 and BOLA3, have similar pheno- tion led to the conclusion that the unknown adenosine derivative was a types [64,65]. Defects in these genes result in decreased protein sulfurated intermediate. lipoylation and low activity of lipoyl-dependent multienzyme com- An additional mechanism for methylthiolation invokes the genera- plexes, again leading to disease and premature death [64,65]. NFU1 is tion of a protein-bound methylthio moiety followed by its transfer to proposed to be an alternative Fe/S scaffold, and BOLA3 is thought to the substrate in a radical reaction. The observation of SAH production be a reductase that aids in the transfer of clusters from the scaffold to in the absence of a low-potential reductant or substrate favored this the target protein [64]. RNAi knockdown of NFU1 did not affect the ac- possibility [25,70]. Landgraf et al. reported the transfer of a methyl tivities of most mitochondrial Fe/S proteins, but impacted LipA and suc- group from SAM to RimO or MiaB in the absence of a low-potential cinate dehydrogenase, suggesting that the role of NFU1 may be specific reductant, and identified a protein-bound methylthio intermediate, for LipA. It is yet unknown whether NFU1 and BOLA3 act in the de novo lending credence to the latter mechanism [71]. The chemical and kinetic biosynthesis of the iron–sulfur clusters in LipA or if they may be in- competence of the protein-bound methylthio intermediate was tested volved in cluster regeneration during the catalytic cycle. in differential labeling experiments. MiaB and RimO were incubated 1320 N.D. Lanz, S.J. Booker / Biochimica et Biophysica Acta 1853 (2015) 1316–1334 with unlabeled SAM in the absence of substrate and reductant to allow of SAH and methanethiol produced was tightly correlated and approxi- for methyl transfer. Excess SAM and SAH were removed by gel-filtration mately half an equivalent with respect to enzyme concentration. Taken chromatography and the enzymes were reacted with substrate and together, these results demonstrate the presence of a protein-bound 2 2 S-adenosyl-L-[methyl- H3]methionine ( H3-SAM) under turnover methanethiol intermediate, which implies that the mechanism in conditions. For both MiaB and RimO, a burst of unlabeled product was which the methylthio group, formed by transfer of a methyl group 2 observed followed by formation of H3-labeled product, indicating from SAM to a protein sulfur atom, is transferred intact to the substrate that the unlabeled methyl group is transferred initially to the product in a radical-dependent reaction. Furthermore, the source of the sulfur followed by the labeled methyl group in subsequent turnovers atom must either be sulfide ligated to one of the Fe/S clusters or a sulfur (Fig. 5A). Transfer of the methyl group from SAM to the enzyme was atom from the Fe/S cluster itself, given that the intermediate becomes 14 14 also studied using S-adenosyl-L-[methyl- C]methionine (methyl- C- labile when the enzyme is denatured. SAM) or S-[8-14C]adenosyl-L-methionine ([adenosyl-14C]SAM). The Forouhar et al. also studied the mechanism of the RimO and MiaB reactions were then subjected to gel-filtration chromatography, and reactions, and reported that MiaB catalyzed formation of up to four the radioactivity in each fraction was counted. When MiaB or RimO equivalents of ms2i6A per mol of enzyme [24]. The authors noted that were reacted with methyl-14C-SAM, the radioactivity eluted in the pro- the enzyme was reconstituted with iron and sulfide and contained sul- tein fractions. In the control experiments, in which adenosyl-14C-SAM fide in excess of that required for the two [4Fe–4S] clusters. Upon fur- was used, the radioactivity was found in the small-molecule fractions, ther supplementation of the reaction with sodium sulfide, MiaB was indicating that the first result was not due to tight binding of SAM to observed to catalyze 12 or more turnovers. Similarly, addition of the enzymes. If reactions with MiaB or RimO and methyl-14C-SAM methanethiol or methaneselenol to reactions also increased the amount were treated with urea prior to separation on the gel-filtration column, of product generated, though not to the same extent as the addition of the radioactivity appeared in the small-molecule fraction. Thus, the sulfide. In similar experiments, multiple turnovers were also observed methyl acceptor is labile to denaturation and is not likely to be an for RimO. These observations suggest that, unlike BioB and LipA, the amino acid of the protein. MTTases do not use their auxiliary Fe/S clusters in a sacrificial manner. It was also determined that exogenously added methanethiol could Consistent with this premise, HYSCORE experiments performed on compete with the enzymatically-derived methylthio moiety [71]. MiaB and RimO variants containing Cys → Ser substitutions at all cyste- 2 Reactions containing MiaB or RimO, H3-SAM, reductant, and substrate ines in the CX3CX2C motif that ligate the RS cluster, revealed a coupling were supplemented with methanethiol, and the RNA or peptide between the 77Se isotope in methaneselenol and the electron spin in the 2 products containing either an unlabeled- or a H3-methyl group were reduced form of the auxiliary cluster. The observed spectrum was con- quantified by LC–MS. A mixture of the labeled and unlabeled products sistent with methaneselenol binding to the open coordination site of was observed, indicating that both enzymes are capable of taking up the auxiliary cluster and DFT calculations predict that the selenide is and utilizing free methanethiol. Finally, direct detection of methanethiol not intercalating into the cluster by replacing one of the bridging produced by RimO and MiaB was observed. In this experiment, μ-sulfido ions. reactions of RimO and MiaB with SAM were conducted in sealed vials Forouhar et al. also reported a crystal structure of holo-RimO. and quenched in acid, and then samples of the headspace were analyzed Although a crystal structure had been previously determined, it lacked by GC–MS for the presence of methanethiol. Parallel samples were both of the Fe/S clusters and portions of the polypeptide containing analyzed by LC–MS in order to quantify SAH production. The amount the cysteines that ligate the clusters [25]. Perhaps the most intriguing

Fig. 5. Proposed mechanisms of methylthiolation of Asp88 of the S12 subunit of the E. coli ribosome by RimO. In both proposed mechanisms, a 5′-dA• abstracts a hydrogen atom from Cβ of the substrate aspartyl residue. The resulting carbon-centered radical attacks either a bridging μ-sulfido ion of the auxiliary cluster capped with a methyl group (A) or a methyl-capped persulfide species coordinated to the unique iron ion of the auxiliary cluster (B). N.D. Lanz, S.J. Booker / Biochimica et Biophysica Acta 1853 (2015) 1316–1334 1321 aspect of the updated RimO structure is a pentasulfide bridge that enzymes that contain either the SPASM or twitch domains, which connects the two unique iron ions of each cluster. Although the bridge accounts for a significant percentage of the greater RS superfamily is most likely an artifact of the reconstitution procedure used to [79]. While a large number of enzymes contain this motif, only a few generate the holo-form of the enzyme, it does provide possible insight have been well characterized. Among those, two dehydrogenases, the an- into the mechanism of catalysis utilized by MTTases. First, it explains aerobic sulfatase maturating enzymes (anSMEs) and 2-deoxy-scyllo- the source of the extra sulfide found in reconstituted RimO and MiaB inosamine dehydrogenase (BtrN), as well as the heme b synthase for and accounts for the multiple turnovers performed by these enzymes. Desulfovibrio vulgaris will be highlighted here. While the pentasulfide bridge would likely preclude SAM from binding to the RS cluster, a sulfide ion or a shorter polysulfide likely binds to the 3.1. Anaerobic sulfatase maturating enzymes open coordination site of the auxiliary cluster and is then capped by a methyl group donated by SAM (Fig. 5B). The enzyme then uses radical Arylsulfatases require a distinctive formylglycyl (FGly) residue in chemistry to transfer the methylthio moiety to the substrate. A new sul- their active sites to carry out their function of hydrolyzing organosulfate fide ion can then be appended to the cluster and catalysis can resume monoesters. The FGly residue is generated by a two-electron oxidation without the need to repair or reassemble an iron–sulfur cluster. of either a cysteine or serine residue of the arylsulfatase (Fig. 6A). Two The difference between the mechanisms of LipA and BioB versus the distinct types of maturases catalyze this reaction, the first of which, MTTases may derive from the inherent difference in the abundance of known as FGly generating enzymes or FGEs, is oxygen-dependent and their products. Relatively small quantities of cofactors such as lipoic found both in eukaryotes and prokaryotes [80,81]. The oxygen- acid and biotin are required for cellular survival. Therefore, the ineffi- independent maturases, designated anaerobic sulfatase maturating cient process of rebuilding an Fe/S cluster after its destruction between enzymes (anSMEs), are SPASM domain-containing RS enzymes each turnover may be permissible. A sacrificial role for the auxiliary [82–84]. Initial characterization of the anSMEs from Clostridium cluster may not be tolerated for enzymes such as MiaB and RimO, perfringens(anSMEcp) and Bacteroides thetaiotaomicron (anSMEbt) which must operate on the cellularly-abundant tRNA and ribosome sub- suggested that these enzymes contained only one [4Fe–4S] cluster strates. Therefore, nature may have evolved a distinct mechanism for per protein [85,86]. A more rigorous characterization of the enzyme the MTTases that allows for multiple turnovers without the need to re- from Klebsiella pneumoniae (anSMEkp) revealed that it harbored three build an iron–sulfur cluster after each round. [4Fe–4S] clusters per polypeptide, which was later confirmed for the While RimO and MiaB may be catalytic, some evidence suggests one enzyme from B. thetaiotaomicron [77,78]. However, the function of the or both of the clusters of MiaB may be unstable during turnover. Recent auxiliary clusters was not immediately apparent. studies have found additional factors that are required for optimal activ- The mechanisms proposed for anSMEs involve abstraction of H• ity of MiaB in vivo. YgfZ, the E. coli homolog of IBA57 (vide supra), is a from the β-carbon of the cysteine or serine residue followed by loss of folate-dependent enzyme that can maintain activity in Fe/S enzymes, a proton and an electron to generate the aldehyde or a thioaldehyde including MiaB [72,73]. While it has been determined that it is that undergoes hydrolysis to the aldehyde and H2S(Fig. 6). Benjdia tetrahydrofolate-dependent, its mechanism of action is yet unknown et al. demonstrated direct abstraction of the Cβ H•, providing evidence [73–75]. Two additional proteins, a monothiol glutaredoxin (GrxD) for this mechanism [87]. The role of the auxiliary clusters in this mech- and an iron–sulfur carrier protein (NfuA), have been shown to interact anism is more cryptic. Two proposed possibilities invoke a role in elec- with MiaB [76]. It was discovered that untagged MiaB could be tron transfer for the auxiliary clusters, while a third suggests that they co-purified with his-tagged GrxD or NfuA or vice versa. Additionally, play only a structural role in the enzyme. In one instance, it has been surface plasmon resonance was used to measure a KD of 12 μMfor suggested that the auxiliary clusters provide an electron-transport apo-MiaB binding to apo-GrxD and a KD of 44 μM for apo-MiaB binding pathway to provide the requisite electron to reduce the RS cluster and to apo-NfuA. Holo-GrxD and NfuA were also tested for the ability to subsequently cleave SAM (Fig. 6C). Benjdia et al. found that variants of transfer an iron–sulfur cluster to apo-MiaB. GrxD was unable to do so; the cysteines that coordinate the auxiliary clusters of anSMEbt exhibited however, NfuA was able to transfer a [4Fe–4S] cluster to apo-MiaB a 50- to 100-fold decrease in SAM cleavage, hinting that reduction of the and the triple variant containing Cys → Ala substitutions in the RS cluster is affected in the absence of the auxiliary clusters [78].

CX3CX2C motif, indicating that the auxiliary cluster could be restored However, given that the flavodoxin/flavodoxin reductase reducing by NfuA. Finally, the ratio of ms2i6Atoi6A in tRNAs in E. coli was mea- system is capable of providing the electron to reduce the RS cluster of sured for WT and grxD and nfuA deletion strains. Both the grxD and other RS enzymes that do not have auxiliary clusters, it is unlikely that nfuA deletion strains showed a modest decrease in the ratio of the mod- anSMEs would require additional clusters for this purpose [77].A ified bases; however, the double deletion showed a five-fold difference second possibility is that the second electron during the two-electron in the ratio, suggesting that GrxD and NfuA are important for MiaB ac- oxidation is shuttled to an electron acceptor via the two auxiliary tivity in vivo. If the auxiliary Fe/S clusters of MTTases are not utilized clusters (Fig. 6B). in a sacrificial manner as Forouhar et al. suggests [24], one can speculate Grove et al. also characterized an anSME from C. perfringens and that these additional factors are not required for reassembly of an Fe/S found that it harbors three [4Fe–4S] clusters [88]. The as-isolated cluster, but instead may supply the sulfide for the reaction. enzyme contained 9.6 iron ions and 10.0 sulfide ions per polypeptide. Approximately 95% of the iron in the sample was assigned to [4Fe–4S] 3. SPASM domain-containing enzymes clusters by Mössbauer spectroscopy, yielding 2.3 clusters per protein in the as-isolated sample. After reconstitution with iron and sulfide, A subfamily of RS enzymes contains a motif of seven cysteines that anSMEcp contained 14.1 iron ions and 12.8 sulfide ions per monomer. ligate two additional Fe/S clusters. The SPASM domain, named for the Approximately 75% of the iron in the reconstituted protein was in the founding members that function in the maturation of subtilosin A, form of [4Fe–4S] clusters, resulting in 2.7 clusters per protein. These re- pyrroloquinoline quinone, anaerobic sulfatase, and mycofactocin, sults suggested that anSMEcp also has three [4Fe–4S] clusters. Triple consists of a CX9–15GXC–gap–CX2CX5CX3C–gap–C motif [20,77,78]. variants containing Cys → Ala substitutions of the cysteines in the The SPASM subfamily also includes enzymes with a truncated version CX3CX2C motif were generated and the as-isolated and reconstituted of the SPASM domain termed the twitch domain. The shorter twitch do- enzymes contained 3.2 and 8.8 iron ions and 7.5 and 15.1 sulfide ions main lacks the CX2CX5CX3C motif of the SPASM domain and coordinates per polypeptide, respectively. This stoichiometry together with the a single auxiliary Fe/S cluster; however, it contains many of the structur- Mössbauer spectrum of the as-isolated sample indicated the presence al features of the larger SPASM domain (vide supra). Bioinformatic ap- of 0.6 [4Fe–4S] clusters and 0.3 [2Fe–2S] clusters per monomer, suggest- proaches have identified more than 18,000 unique sequences of ing that the [4Fe–4S] clusters are less stable in the variant enzyme. After 1322 N.D. Lanz, S.J. Booker / Biochimica et Biophysica Acta 1853 (2015) 1316–1334

Fig. 6. Reaction (A) and potential mechanisms of anaerobic sulfatase maturating enzymes (B and C). reconstitution, the variant contained 1.5 [4Fe–4S] clusters per mono- above the limit of detection of the assay. The corresponding variant of mer, confirming the presence of multiple auxiliary clusters, though the anSMEcp, C276A, was also generated with similar results. The enzyme less than full complement of two clusters still suggests a greater insta- was soluble in this case; however, the activity was greatly diminished. bility of the clusters in the variant. The remaining seven variants were completely insoluble and could The activity of anSMEcp was measured using peptide substrates not be analyzed for activity. It was surmised that the seven cysteines containing the physiological cysteine, a serine in place of the cysteine, that yielded insoluble enzyme when substituted with alanine act as li- or a selenocysteine in place of the cysteine [88]. The enzyme was gands to the auxiliary clusters. The role of C291 in anSMEkp could not capable of oxidizing all three substrates, though the use of the be assigned given the data available. selenocysteine-containing substrate led to significant uncoupling of The crystal structures of anSMEcp in the presence and absence of its 5′-dA and product formation after 10 min. Although anSMEcp is capable peptide substrate resolved the issue concerning the cysteines that coor- of oxidizing the serine-containing substrate, the rate of the reaction is dinate the auxiliary clusters (Fig. 7) [20]. The N-terminal auxiliary clus- approximately three-fold slower than for the natural substrate. The ter (Aux I) is ligated by C255, C261, C276, and C317 (C. perfringens enzyme was also able to utilize both dithionite and the flavodoxin/ numbering), while the C-terminal auxiliary cluster (Aux II) is ligated flavodoxin reductase reducing system as the source of the electron by C320, C326, C330, and C348. One of the cysteines ligating Aux I, required to cleave SAM, though, as was the case for anSMEkp, the rate C255, lies outside of the SPASM domain, while the other three are part of the reaction with the flavodoxin reducing system was significantly of the seven-cysteine motif. The first three cysteines of the CX2CX5CX3C slower. motif ligate Aux II, with the final ligand coming from the final cysteine

Reductive cleavage of SAM requires a single electron, and the in the greater CX9-15GXC–gap–CX2CX5CX3C–gap–C motif. The crystal abstraction of a H• by the 5′-dA• is a one-electron oxidation of the structure also allows for an examination of the hypothesized role of substrate. Because the substrate ultimately undergoes a two-electron the auxiliary clusters in electron transfer. Both the RS cluster and Aux oxidation, it was hypothesized that the second electron could be I are within reasonable electron transfer distance (8.9 and 8.6 Å, respec- returned to the RS cluster to initiate the next round of catalysis, or alter- tively) of Cβ of the substrate cysteine, respectively (Fig. 7B). Assuming natively, transferred to an unknown electron acceptor. In order to ad- that the electron is passed to Aux I, the only reasonable candidate to re- dress the fate of the second electron, the authors generated the ceive this electron is Aux II, because the RS cluster is 16.9 Å away and flavodoxin semiquinone (Fld SQ) by incubating flavodoxin with a stoi- the path to solvent is likely obscured by the substrate. Aux II is 8.3 Å chiometric amount of dithionite [88]. A two-fold excess of the Fld SQ from bulk solvent, so it could transfer the electron to flavodoxin, was incubated with SAM, peptide and anSMEcp, and the amount of which in turn could pass the electron to the RS cluster. At 8.6 Å and Fld SQ consumed was measured by EPR in parallel to quantification of 20.8 Å, both Aux I and Aux II are too far from the substrate cysteine to the peptide and 5′-dA products by LC–MS. During the course of the ligate the substrate thiolate, suggesting that the auxiliary clusters are ei- 30 min reaction, approximately three equivalents of 5′-dA and product ther involved in electron transfer or play a purely structural role. were formed; however, only a slight decrease in the EPR signal for the Fld SQ was observed. Furthermore, a signal for a reduced [4Fe–4S] clus- 3.2. Butirosin biosynthesis ter was not detected, indicating that after each round of turnover, the electron was returned to flavodoxin. Thus, the electron generated in The 2-deoxy-scyllo-inosamine (DOIA) dehydrogenase from Bacillus the reaction can be used to initiate subsequent rounds of catalysis in a circulans (BtrN) catalyzes the two-electron oxidation of DOIA to form flavodoxin-dependent manner. amino-dideoxy-scyllo-inosose (amino-DOI) as a step in the biosynthesis The authors also attempted to address the coordination of cysteines of the antibacterial butirosin B [89]. BtrN contains a segment of the to the auxiliary clusters of anSMEcp and anSMEkp [88]. The enzyme SPASM domain containing four of the seven cysteines, which has been from C. perfringes contains 18 cysteine residues compared to 13 cyste- amusingly coined the twitch domain. Although it was originally thought ines present in anSMEkp. In order to limit the number of variants re- to contain only one [4Fe–4S] cluster [90], it was later demonstrated that quired, Cys to Ala variants were made of each of the ten cysteines not the enzyme actually harbors two clusters [91]. Despite the differences in found in the RS motif of the enzyme from K. pneumoniae.Whiletheau- the substrates for anSME and BtrN—a protein substrate for the former thors intended to purify and measure the activity of each variant, they and a small-molecule substrate for the latter—their mechanisms of found that only two of the variants, C127A and C245A (K. pneumoniae catalysis are believed to be quite similar [91]. In one mechanism, numbering), were soluble. Both of these variants supported activity abstraction of a hydrogen atom from the carbon center undergoing ox- equivalent to that of the WT enzyme, so it was assumed that these idation is followed by loss of a proton and an electron, while in a second two cysteines are unimportant for catalysis. A third variant, C291A, mechanism, deprotonation of the alcohol could precede hydrogen atom was sparingly soluble; however, it did not exhibit signifi cant activity abstraction [15,89]. Given that these reactions are two-electron N.D. Lanz, S.J. Booker / Biochimica et Biophysica Acta 1853 (2015) 1316–1334 1323

Substrate Peptide Aux 2

8.9 Å

SAM Aux 2

Aux 1 20.8 Å

8.3 Å RS Cluster 12.9 Å RS Cluster 16.9 Å 8.6 Å Aux 1 Solvent

Fig. 7. Structure of anSMEcpe (A) and the active site of the enzyme demonstrating coordination of SAM to the RS cluster and the distances between the iron–sulfur clusters and the peptide substrate (B) (PDB accession code 4K39). The RS domain of anSMEcpe is shaded in green while the SPASM domain is shaded in pink. The carbon backbones of SAM and the peptide substrate are depicted in cyan and blue, respectively. oxidations, an electron must be passed to an unknown acceptor. It has substrate as previously hypothesized (Fig. 8B) [91]. Because the been proposed that the auxiliary cluster is the immediate electron ac- distance between the two clusters is 15.9 Å, direct transfer of the ceptor and that it might deliver the electron back to the RS cluster for electron from the auxiliary cluster to the RS cluster is unlikely. The ratio- subsequent rounds of turnover via the intermediacy of flavodoxin [91]. nale for the lack of a third Fe/S cluster in BtrN likely lies in the nature of The crystal structure of BtrN shows the structural similarity of the its substrate. The macromolecular protein substrate of anSMEs obstructs C-terminal domains of BtrN and anSMEcpe (Fig. 8) [15]. The twitch access of Aux I to solvent. In the case of BtrN, the small molecule domain of BtrN and the SPASM domain of anSMEcpe are each composed substrate does not have this affect, rendering the extra electron transfer of a β-hairpin and an α-helix. In BtrN, the lone auxiliary cluster is step unnecessary. completely ligated by the four cysteines of the twitch domain. The first cysteine ligand is just upstream of the β-hairpin, the second ligand resides between the β-hairpin and the α-helix, and the last two ligands 3.3. Alternative heme biosynthesis fall on a loop after the α-helix (Fig. 9). This arrangement is similar to that in anSMEcpe, in which the first ligand to Aux I is just upstream of An alternative pathway for the biosynthesis of heme in sulfate- the start of the SPASM domain, the second resides upstream of the reducing Desulfovibrio bacteria and methanogenic bacteria converts β-hairpin, and the third resides between the β-hairpin and the siroheme to heme b in a four-enzyme process [92–94]. The last step, α-helix. The fourth ligand to Aux I, as well as all four ligands to Aux II, catalyzed by AhbD, is the conversion of two propionyl side groups of reside downstream of the α-helix. The structure of BtrN also reveals iron-coproporphyrin III (Fe-CpIII) to vinyl groups, yielding heme b that the role of the cluster is likely the same as that in the two auxiliary (Fig. 10) [92]. In addition to the canonical CX3CX2C RS motif, AhbD clusters in anSMEs. Both the RS and auxiliary clusters are sufficiently also contains a SPASM domain [95]. Initial characterization of the en- close to C3 of DOIA to receive an electron upon H• abstraction. However, zyme by UV/visible spectroscopy revealed features consistent with the at 9.6 Å, the auxiliary cluster is not properly positioned to ligate the presence of one or more iron–sulfur clusters [95].Afterconfirming

2-DOIA

SAM 8.6 Å

9.6 Å

15.9 Å

Auxiliary Cluster RS Cluster RS Cluster Auxiliary Cluster

Fig. 8. Structure of BtrN (A) and the active site of the enzyme demonstrating coordination of SAM to the RS cluster and the distances between the iron–sulfur clusters and the 2-DOIA substrate (B) (PDB accession code 4M7T). The RS domain of BtrN is shaded in purple while the twitch domain is shaded in cyan. The carbon backbones of SAM and the 2-DOIA substrates are depicted in green and blue, respectively. 1324 N.D. Lanz, S.J. Booker / Biochimica et Biophysica Acta 1853 (2015) 1316–1334

B BtrN anSMEcpe

Fig. 9. SPASM and twitch domains of anSMEcpe and BtrN. The topologies of the SPASM and twitch domains of BtrN and anSMEcpe are shown in cartood form with the cysteine ligands to Aux I depicted as green dots and the cysteine ligands to Aux II depicted as pink dots (A). Structures of the twitch and SPASM domains of BtrN and anSMEcpe, respectively, are shaded in cyan (B). The cysteine ligands to Aux I are shaded in green and the cysteine ligands to Aux II are shaded in pink. that the enzyme binds Fe-CpIII, it was demonstrated that AhbD cata- of AhbD indicates the presence of the full SPASM motif and all eight cys- lyzes the conversion of CpIII to heme b and generates 5′-dA. teines that ligate the auxiliary clusters in anSMEcp. Therefore, the model EPR spectroscopy was used to probe for the presence of multiple may not accurately predict the locations of all of the cysteines, and AhbD clusters [95]. The observed EPR signal and its temperature dependence may actually contain three [4Fe–4S] clusters. A quantitative analysis of were consistent with the presence of one or more [4Fe–4S] clusters. iron and sulfide content paired with Mössbauer spectroscopy may be When the microwave power was increased, an additional resonance required to definitively assign the cluster content of AhbD. was observed. A slower relaxing signal was observed with g values of 2.057 and 1.925, while a faster relaxing signal was observed at g = 4. Complex cofactor maturases 2.083, 1.921, and 1.925 at low temperatures. When both substrates, SAM and Fe-CpIII, were incubated with the samples, one of the signals, 4.1. Molybdopterin cofactor biosynthesis with g values of 2.064 and 1.92, remained unperturbed. The other signal, with g values of 2.13 and 1.92, shifted somewhat. The changes The molybdopterin cofactor (MoCo) is a widely conserved redox co- were suggested to be caused by SAM binding to the RS cluster. factor composed of a tricyclic pyranopterin with a dithiolene moiety A model of the structure of AhbD was created based on the crystal that coordinates a mononuclear molybdenum center [96]. In humans, structures of anSMEcp and MoaA [95]. In this model, the ligands for three complexes require MoCo for their activity: sulfite oxidase, alde- the RS cluster and one of the two auxiliary clusters are oriented to hyde oxidase, and xanthine dehydrogenase [97]. The absence of MoCo bind an Fe/S cluster. The binding pocket for Aux I lacks two of the four in these complexes results in neurological symptoms and usually cysteines required to ligate the third cluster. Because the substrate is a death [98].Thefirst step in the conversion of GTP to MoCo is catalyzed small molecule, it is possible that only one auxiliary cluster is required by the RS enzyme MoaA (Fig. 11A). In conjunction with MoaC, MoaA as is the case for BtrN. It should be noted that the sequence alignment converts GTP to cyclic pyranopterin monophosphate (cPMP) [16, 97–100]. The reactions of MoaA and MoaC have proven difficult to separate due to the lability of the intermediates and products, resulting in multiple proposed structures of both the chemical inter- mediateandtheenzymeproducts[22,101–104].MoaAharborstwo [4Fe–4S] clusters, both of which are required for activity of the enzyme [105–107]. MoaA is another member of the twitch family of RS enzymes, though in contrast to BtrN, it lacks a fourth cysteine to ligate the auxiliary Fe/S cluster [15]. The open coordination site created by the absent cysteine is used to bind the GTP substrate (Fig. 12) [22,108]. The crystal structure of MoaA indicates that both N1 and the exocyclic amine are within bonding distance of the open coordination site of the auxiliary cluster; however, ENDOR studies suggest that N1 is in fact bound to the cluster [22,108]. Furthermore, spectroscopic evidence suggests that GTP is Fig. 10. The final step in the alternative pathway for heme b biosynthesis. bound in the enol form rather than the keto form, which may modulate N.D. Lanz, S.J. Booker / Biochimica et Biophysica Acta 1853 (2015) 1316–1334 1325

Fig. 11. Pathway for molybdenum cofactor biosynthesis (A) and proposed mechanism for the biosynthesis of cPMP by MoaA and MoaC. In the mechanism proposed by Mehta, et al., MoaA catalyzes all but the last step (the second shaded intermediate) in the biosynthesis of cPMP. The work of Hover et al. suggests that 3,8-cH2GTP is the product of MoaA, and MoaC is responsible for the remaining steps. the reactivity of the substrate [108]. Coordination of GTP to the Fe/S MoaC was then added to the filtrate, and cPMP formation was detected cluster may also be important for substrate specificity, because ATP, by HPLC. The authors then set out to isolate this soluble intermediate. which is present in much higher concentrations in the cell than GTP, Large-scale reactions were conducted, and the intermediate was must be discriminated against [22]. purified by anion-exchange chromatography before being analyzed by A series of recent studies probed the mechanistic details of the MoaA ESI–TOF–MS, 1H NMR, 13C NMR, and multiple 2D NMR techniques to reaction, the first of which revealed the identity of the H• abstracted by identify its molecular structure. It was ultimately determined to be the 5′-dA• [103]. A series of deuterium-labeled GTP substrates were in- (8S)-3′,8-cyclo-7,8-dihydroguanosine 5′-triphosphate (3′,8-cH2GTP, cubated with SAM and MoaA, and the incorporation of deuterium into Fig. 11B). To verify that 3′,8-cH2GTP is the product of the MoaA reaction, 5′-dA was measured by LC–MS. A single deuterium atom was trans- the purified intermediate was incubated with MoaC, which was able to ferred to 5′-dA from universally labeled GTP, demonstrating that the convert 3′,8-cH2GTP to cPMP. The authors proposed a mechanism in 5′-dA• directly abstracts an H• from the GTP substrate. No deuterium which an intermediate proposed by Mehta et al. [103] is actually the transfer was detected from [8-2H]-GTP or [2′-2H]-GTP, but a single deu- final product of MoaA, which suggests that MoaC has a much larger 2 terium was incorporated from [3′,4′,5′,5′- H4]-GTP. The position was fi- role in the conversion of GTP to cPMP than previously thought. nally narrowed down to the 3′ H• with observation of deuterium Mehta and coworkers were also able to trap a similar reaction inter- transfer from [3′-2H]-GTP. Additionally, analysis of the cPMP product mediate using a substrate analogue [101]. In the proposed mechanism, 2 of MoaA and MoaC with [3′,4′,5′,5′- H4]-GTP by LC–MS showed an in- the 3′ hydroxyl of the 3′,8-cH2GTP intermediate must be deprotonated crease of 3 Da in the pterin product, indicating that the 4′ and 5′ hydro- to allow for the rearrangement of the five-membered ribose ring to gens are retained. With the knowledge that an H• is abstracted from C3′ form a six-membered ring. The analogue, 2′,3′-dideoxy-GTP, lacks the by the 5′-dA•, the authors proposed a mechanism in which the resulting 3′ hydroxyl group and should therefore arrest the reaction at the stage

C3′ radical adds to C8 of GTP (Fig. 11B). of the 3′,8-cH2GTP intermediate. MoaA was incubated with 2′,3′- Shortly after, Hover et al. identiﬁed an intermediate product of the dideoxyGTP and SAM and analyzed by LCMS. The product of this reac- MoaA reaction that could be transferred to MoaC and then converted tion was consistent with the predicted intermediate. The potential to cPMP [104]. The reaction was performed stepwise by incubating redox role of the auxiliary Fe/S cluster was noted both by the authors MoaA with SAM and GTP, and then removing MoaA by ultraﬁltration. and the previous report by Hover et al. The radical intermediate 1326 N.D. Lanz, S.J. Booker / Biochimica et Biophysica Acta 1853 (2015) 1316–1334

5’-dA

GTP

Methionine RS Cluster Auxiliary Cluster

Fig. 12. Structure of MoaA (A) and coordination of methionine and GTP to the RS and auxiliary clusters of MoaA, respectively (B) (PDB accession code 2FB3). The RS domain of MoaA is shaded in orange while the twitch domain is shaded in blue. The carbon backbones of methionine and 5′-dA are depicted in green and the carbon backbone of GTP is depicted in salmon.

generated by addition of the C3′ radical to C8 would need to be reduced ligands, and a dithiomethylamine (DTN) moiety that bridges the two by one electron to form the stable intermediate observed by both iron ions (Fig. 13A) [109–112].The[4Fe–4S] component is built on groups. The auxiliary [4Fe–4S] cluster to which the substrate is ligated HydA first, presumably by the standard Fe/S cluster assembly machin- was suggested to serve this purpose. ery (i.e. the gene products of the isc operon) [113].Assemblyofthe A more recent study by Mehta et al. identified a different product of diiron cluster is more complicated and requires an additional three the MoaA reaction and suggested a lesser role for MoaC [102]. In this maturases. The first of the maturases, HydF, is a GTPase that is likely study, MoaA was overproduced in a moaC deletion strain to prevent the scaffold and insertase for the diiron center [114–119]. The other contamination from MoaC in MoaA preparations. When MoaA prepared two maturases, HydE and HydG, are RS enzymes that each potentially in this manner was incubated with SAM, GTP and dithionite, the prod- contain an auxiliary Fe/S cluster [120]. uct detected after derivatization was inconsistent with 3′,8-cH2GTP, While the reaction catalyzed by HydE is still uncertain, it has been but instead appeared to be the tricyclic pterin triphosphate intermedi- proposed that it may function in the synthesis of the bridging DTN moi- ate that directly precedes the cPMP product. The authors also used ety. This is in large part inferred from the fact that HydG catalyzes the two substrate analogs, 2′-chloroGTP and 2′-deoxyGTP, to trap interme- formation of the CO and CN ligands bound to the diiron center (vide diates in the MoaA reaction. The 2′-chloroGTP substrate was used to infra), which leaves only the synthesis of the DTN moiety unaccounted provide evidence for the 3′ radical that is proposed to initiate the reac- for. In addition to the canonical RS motif, HydE contains a CX7CX2Cmotif tion. Upon formation of the 3′ radical, the chlorine at the 2′ position that ligates an auxiliary cluster [121]. In the crystal structure, a [2Fe–2S] leaves as a radical species, generating a labile compound. The expected cluster is coordinated by this motif; however, it was suggested that this ribosyl derivative was detected as the product of this reaction. The 2′- may be a breakdown product of a [4Fe–4S] cluster [121]. The role the deoxyGTP substrate was predicted to prevent the dehydration reaction auxiliary cluster might play in the mechanism of HydE is entirely in which the 2′ hydroxyl is the leaving group. In the absence of the 2′ hy- unknown, and it may not be required at all. The CX7CX2C motif is not droxyl, an alternative product is formed, which lacks the third ring of conserved among HydEs, and variants with single substitutions of the cPMP. The authors proposed a mechanism for MoaA that accounts for cysteines in the motif still retained near WT activity [121].Itwas the product observed with the substrate analogue (Fig. 11B). proposed that the second iron–sulfur cluster might have a regulatory The discrepancy between the current data lies in the nature of the role or may simply be vestigial. product of the MoaA reaction and the role of MoaC in generating HydG also contains a second cysteine rich motif that harbors an Fe/S cPMP. Hover et al. suggests that MoaA generates 3′,8-cH2GTP, and cluster, and the cysteines residing in the CX2CX22C motif are essential MoaC catalyzes the remaining steps of the conversion of 3′,8-cH2GTP for HydG activity [122]. The reaction catalyzed by HydG has been to cPMP [104]. The work of Mehta et al. indicates that MoaA can catalyze much better characterized. It was first noted that HydG could cleave ty- all but the last step of the reaction, which is the hydrolysis of the tri- rosine to form p-cresol [123]. Further studies, using isotopically-labeled phosphate to form pyrophosphate and the fourth ring of cPMP [102]. tyrosine substrates, indicated that HydG generated both CO and CN Further study will be required to reconcile the existing data and eluci- from tyrosine [124,125]. Additionally, a variant in which the first two date the true product of the MoaA reaction. cysteines of the CX2CX22C motif were substituted with serine was able to generate CN but not CO [126]. It was therefore proposed that 4.2. Fe–Fe hydrogenase cofactor maturation dehydroglycine derived from tyrosine was cleaved in a radical- − dependent fashion to generate formaldehyde imine and •CO2 . − The interconversion of protons and molecular hydrogen is catalyzed The •CO2 would need to coordinate to the auxiliary cluster to be by hydrogenases using a metal center-containing active site. One of the reduced by one electron to CO. three types of hydrogenases, the Fe–Fe hydrogenase (HydA), contains a To further investigate the mechanism by which HydG operates, complex cofactor known as the H-cluster. The H-cluster is composed of Driesner et al. constructed a triple Cys → Ala variant of cysteines in a cubane [4Fe–4S] cluster bridged by a cysteine thiol to a diiron cluster the CX3CX2C motif and a truncated variant of the C-terminal domain that contains three carbon monoxide (CO) ligands, two cyanide (CN) of HydG (ΔCTD) from Clostridium acetobutylicum that lacks the N.D. Lanz, S.J. Booker / Biochimica et Biophysica Acta 1853 (2015) 1316–1334 1327

Fig. 13. Reaction pathway of H-cluster biosynthesis (A) and proposed mechanism of HydG (B).

CX2CX22C motif [127]. The truncated HydG and triple variant each used to trap and detect the radical intermediate. Formation and decay contained 3.1 and 3.4 iron ions, respectively, and an EPR spectrum of of the transient radical was observed with its maximum intensity at each sample confirmed the presence of a [4Fe–4S] cluster. The spectrum approximately 2 s after the reaction was initiated. The g = 2 radical of the ΔCTD variant was similar to that of the WT enzyme. In the ab- signal was further investigated using HYSCORE and ENDOR techniques. sence of SAM, the WT spectrum displays g values 2.03, 1.92, and 1.90, Coupling observed with 13C- and 15N-labeled tyrosine confirmed that while the addition of SAM results in a more complex spectrum with the radical signal was derived from tyrosine. The exact identity of the two distinct sets of g values (g1 = 2.02, 1.93, and 1.91, g2 = 2.00, 1.87, radical species was identified using specifically-labeled tyrosine 13 and 1.83). The spectrum of the ΔCTD variant has g values identical to substrates. Labeling of Cα of tyrosine with C does not affect the shape 2 15 the WT enzyme in the absence of SAM, and nearly identical for the of the spectrum; however, the signal collapses when [ H7, N]-labeled SAM-containing sample (g1 = 2.04, 1.92, and 1.90, g2 = 2.00, 1.88, tyrosine is used as a substrate. This observation is consistent with and 1.84). The spectrum of the triple variant enzyme was also similar previously identified tyrosyl radicals; however, further isotopic labeling to that of the WT enzyme (g = 2.03, 1.92, and 1.88), except that addition experiments indicate that the radical in HydG is different than the of SAM no longer perturbed it. protein tyrosyl radicals that have been characterized. The spectrum of 2 The binding and kinetic constants of the WT and variants of HydG HydG in the presence of 3,5- H2-tyrosine is nearly identical to that of with respect both to SAM and tyrosine were measured in order to the enzyme with unlabeled tyrosine. Conversely, a 1:2:1 pattern is 2 determine if the auxiliary cluster has a role in tyrosine cleavage, observed in the spectrum of the 2,3,5,6- H4-tyrosine-containing SAM cleavage, or both [127].BoththeΔCTD variant as well as a C386S sample, indicating the presence of two equivalently coupled protons. A 2 variant, in which one of the ligands to the auxiliary cluster was sample containing β,β- H2-tyrosine further collapses the signal, indicat- substituted with a serine, were compared with the reaction performed ing that the greatest amount of spin is centered on Cβ.Takentogether, by the WT enzyme. The effects of the variants on binding and rates of the spectra are inconsistent with a tyrosyl radical, but instead indicate cleavage of SAM were modest; however, the apparent KM for tyrosine the presence of a 4-oxidobenzyl radical. Using this knowledge, the au- was significantly higher for the C386S variant, and especially for the thors propose a mechanism that accounts for this radical intermediate ΔCTD truncation. The rate of tyrosine cleavage as measured by forma- and the previously accumulated data (Fig. 13B). tion of p-cresol was not greatly affected by disruption of the auxiliary A subsequent study adds to the mechanistic understanding of the cluster; however, the rate of CN production by the ΔCTD variant was steps that follow the formation of the 4-oxidobenzyl radical [129]. negligible, and neither variant produced any CO, furthering the notion Stopped-flow Fourier transform infrared (SF–FTIR) and ENDOR spec- that cleavage of dehydroglycine to CN and CO requires the assistance troscopies were used to track the formation of a new intermediate in of the auxiliary cluster. the formation of the diiron center of HydA. SF–FTIR is ideal for detecting Another study of HydG from Shewanella oneidensis revealed the CO and CN formation from HydG. In the WT enzyme, two discrete presence of a radical intermediate in the pathway of tyrosine cleavage intermediates were detected. The first was assigned to an Fe–CO and [128]. Rapid-mix freeze-quench coupled with EPR spectroscopy was an Fe–CN complex. This complex formed on the same timescale as the 1328 N.D. Lanz, S.J. Booker / Biochimica et Biophysica Acta 1853 (2015) 1316–1334 decay of the 4-oxidobenzyl radical previously characterized. A second cleavage products of SAM were determined. Unlike GDH-AE, HPLC complex was detected that forms concomitant with the decay of the and TLC analysis identified 5′-dA and methionine products of SAM gen- first. This complex exhibited two Fe–CO vibrational bands and an erated by HPD-AE. The amount of 5′-dA was quantified by HPLC and Fe-CN band in the FTIR spectrum. Thus, it was predicted to be a cuboidal correlated to the amount of the glycyl radical produced on HPD as de-

[3Fe–4S] cluster with an Fe(CO)2(CN) at the unique iron site of the aux- termined by integration of the EPR signal for the radical. The amount iliary cluster. ENDOR spectroscopy revealed that the iron ions of the of 5′-dA produced was similar to the amount of radical present until diiron center of HydA are derived from the [4Fe–4S] cluster of HydG. the maximum activation of HPD was achieved. No other cleavage prod- In this experiment, in vitro assembly of the diiron center was performed ucts of SAM were detected. Thus, the unusual mode of cleavage of SAM using HydG that had been overproduced in the presence of 57Fe and by GDH-AE is not conserved among GRE-AEs of this class. HydE and HydF that had been overproduced in the presence of natural abundance iron. The presence of 57Fe in HydA indicates that the iron 5.2. Choline trimethylamine-lyase activating enzyme was derived from HydG, and the signal was consistent with the diiron portion of the H cluster. The authors describe the product of HydG as a Choline trimethylamine-lyase (CutC) is an enzyme found in bacteria

Fe(CO)2(CN) synthon, two of which must be transferred to HydA with in the human gastrointestinal tract that cleaves choline to trimethlamine loss of one CO molecule and the addition of the DTN moiety, presumably [142]. It is a GRE that has an associated activating enzyme (CutD) that from HydE, to form the diiron center of HydA (Fig. 13A). This reaction belongs to the RS superfamily. Like HPD-AE discussed above (as well as can be thought of as parallel to those of LipA and BioB. Whereas LipA BssD and GDH AE), CutD from Desulfovibrio alaskensis contains two and BioB donate one or two sulfur atoms from their auxiliary Fe/S CX2–5CX2–4CX3C sequences that have been termed “Clostridial” type clusters, HydG donates an iron ion. Some of the same questions that motifs [139,141]. As hypothesized for the other GRE-AEs of this class, have been asked of the sulfur-donating enzymes also apply to HydG. the two additional cysteine-rich motifs of CutD are also thought to coor- For instance, are both iron ions of the diiron center donated by the dinate auxiliary Fe/S clusters. When incubated with SAM and dithionite, same HydG polypeptide, how is the auxiliary cluster reassembled for purified CutD cleaved SAM, forming 5′-dA and methionine, confirming subsequent rounds of turnover, and what factors are required to repair that CutD is in fact a member of the RS superfamily. Although the CutC the cluster? substrate was not present in this experiment, abortive cleavage of SAM in the absence of substrate is relatively common among RS enzymes. 5. Glycyl radical enzyme activating enzymes When CutC was added to the reaction, formation of the glycyl radical on CutC could be measured using EPR spectroscopy. Finally, CutD was Glycyl radical enzymes (GREs) contain a specific glycyl residue that found to contain 8.4 mol of iron and 7.6 mol of sulfide per mol of protein is converted to a glycyl radical via H• abstraction from Cα by an activat- after reconstitution with iron and sulfide, suggesting that multiple ing enzyme [130–133]. The radical is stabilized by the captodative ef- clusters may be present [139]. fect, and can have a half life of several hours under anaerobic The presence of three distinct cysteine motifs has led many to be- conditions [134]. The enzymes that generate the glycyl radical cofactor lieve that the GRE-AEs with these extra motifs contain three [4Fe–4S] belong to the RS superfamily. Two of the best studied GRE-activating en- clusters. Indeed, the iron content of the enzymes that have been charac- zymes (GRE-AEs), pyruvate formate-lyase activating enzyme (Pfl-AE) terized hints that one or more auxiliary Fe/S clusters may be coordinat- and anaerobic ribonucleotide reductase activating enzyme (Nrd-AE), ed by the cysteines in these motifs. However, definitive evidence that require only the RS [4Fe-4S] cluster to carry out this transformation the GRE-AEs harbor multiple clusters, or that the extra cysteine motifs [131,135]. The activases for benzylsuccinate synthase (BssD), are required for activity is still lacking. Furthermore, the function of

4-hydroxyphenylacetate decarboxylase (HPD-AE), B12-independent the putative Fe/S clusters is also a mystery, especially given that the bet- glycerol dehydratase (GDH-AE), and the more recently discovered cho- ter studied GRE-AEs, Pfl-AE and Nrd-AE do not need an auxiliary cluster line trimethylamine-lyase (CutD) each contain two additional cysteine- to generate the glycyl radical cofactor on their respective substrates. rich motifs that may harbor one or more additional Fe/S clusters [136–139]. Here, we will focus on the reactions catalyzed by HPD-AE 6. Complex heterocycle biosynthesis and CutD for which new information has become available. 6.1. Wybutosine biosynthesis 5.1. 4-Hydroxyphenylacetate decarboxylase activating enzyme Wybutosine (yW) is a tricyclic base found 3′ to the anticodon of cer- A study of GDH-AE suggested that instead of cleaving SAM to gen- tain tRNAs in archaea and eukaryotes [143]. The heavily modified yW erate 5′-dA• and methionine, the enzyme produced a 3-amino-3- base promotes translational reading frame fidelity due to its high hydro- carboxypropyl radical and 5′-deoxy-5′-methylthioadenosine [140]. phobicity, which reinforces the interaction of the codon and anticodon. Given that none of the GRE-AEs that contain extra cysteine motifs had The modifications also prevent the base from forming Watson–Crick been characterized, it was postulated that the others may share this base pairing interactions with the first base of the codon [144–148]. unusual mechanism. In order to investigate this hypothesis, HPD-AE Wybutosine is synthesized from guanosine via methylation of N1 by was characterized further [141]. The as-isolated enzyme contained 4.0 the SAM-dependent enzyme TRM5 (Fig. 14A) [149–152]. Formation of iron ions per polypeptide, but could be reconstituted with up to the tricyclic ring structure (4-demethylwyosine or imG-14) is catalyzed 12.0 mol of iron per mol of protein when treated with cell lysate in by TYW1, which is an RS enzyme [23,153]. TYW2, TYW3, and TYW4 cat- which the Fe/S cluster assembly machinery had been over-expressed. alyze formation of the remaining substituents on C7 of yW [154].TYW1

The EPR spectra revealed the presence of small quantities of [3Fe–4S] contains the canonical CX3CX2C RS motif and an additional CX12CX12C clusters, but suggested that the majority of the iron was in the form of motif that may harbor an auxiliary iron–sulfur cluster. Eukaryotic, but [4Fe–4S] clusters. When reduced with dithionite, the reconstituted not archaeal TYW1 enzymes have an additional ﬂavodoxin-like domain sample contained 1.6 [4Fe–4S]+ clusters per protein, strongly indicating that may be required for reduction of the RS cluster [23]. the presence of at least two [4Fe–4S] clusters in the enzyme. The Prior to any biochemical characterization of TYW1, multiple crystal Mössbauer spectrum was also consistent with the majority of the iron structures provided a hint of how this enzyme might work [13,23]. residing in the form of [4Fe–4S] clusters, with the caveat that the The ﬁrst structure of the enzyme, from Methanocaldococcus jannaschii, sample on which Mössbauer spectroscopy was performed only showed that TYW1 is composed of the partial TIM barrel typical of contained 4 to 6 mol of iron per mol of protein. The enzyme was tested other RS enzymes [13]. The Fe/S clusters were not strongly present in for the ability to activate its cognate protein substrate, HPD, and the the structure, given that the crystals were grown under aerobic N.D. Lanz, S.J. Booker / Biochimica et Biophysica Acta 1853 (2015) 1316–1334 1329

Fig. 14. Biosynthetic pathway of wybutosine (A) and proposed mechanisms of TYW1 (B and C).

conditions. Even after soaking with iron and sulfide under anaerobic reactions containing TYW1, tRNAPhe, dithionite, as well as TRM5 and conditions cluster incorporation was poor. Very weak density for the SAM to catalyze the initial methylation of N1. Only the addition of pyru- RS cluster was observed, and no electron density was observed for the vate allowed for formation of imG-14. Furthermore, pyruvate labeled predicted auxiliary cluster. However, the cysteines of this motif were with 13C at C2 or C3 resulted in imG-14 with a mass increase of 1 Da, aligned such that they could coordinate a [4Fe–4S] cluster, and there- while C1-13C labeled pyruvate did not result in a mass shift. Addition 13 fore, the authors modeled two [4Fe–4S] clusters in the structure. In of C1,2,3- C3 labeled pyruvate to the reaction resulted in a mass shift this model, the unligated iron site faces towards the active site and the of 2 Da, indicating that two carbons from pyruvate are incorporated RS cluster, potentially as a site for substrate binding as observed for into imG-14. In the mechanism proposed, the conserved lysine forms MoaA. Moreover, activity of TYW1 was absolutely dependent on the aSchiffbasewithpyruvate(Fig. 14B). Abstraction of a H• from the N1 presence of all six conserved cysteines as well as a conserved lysine in methyl of N-methylguanosine (m1G) is followed by radical recombina- the active site. A second structure provided evidence that both motifs tion of the N1 methyl with C2 of pyruvate and homolytic cleavage of the ligate an Fe/S cluster [23]. In this study, anomalous data was used to C1\C2 bond. Finally, transamination and deprotonation releases imG- map the Fe/S clusters. Two anomalous peaks corresponding to the 14. cavities created by the cysteine motifs were observed. The anomalous Spectroscopic characterization revealed more about the potential peak corresponding to the RS motif was much larger than the other role of the auxiliary cluster of TYW1 [156]. Aerobic purification of peak, and was assigned to a [4Fe–4S] cluster. Because the peak for the TYW1 from Pyrococcus abyssi resulted in less than 0.7 iron and sulfide auxiliary cluster was much weaker, it was modeled as a [2Fe–2S] clus- ions per monomer; however, anaerobic reconstitution of the protein ter; however, iron and sulfide content was not measured, so the lack yielded 8.2 iron and sulfide ions per protein. Analysis by Mössbauer of density for the auxiliary cluster may be due to poor incorporation. spectroscopy indicated that approximately 95% of the iron in the sample Young and Bandarian elucidated the source of the final two carbons was in the form of a [4Fe–4S] cluster; thus, TYW1 harbors two [4Fe–4S] and proposed a mechanism for the formation of the third ring of imG-14 clusters per protein as the sequence analysis would suggest. A complex [155]. Activity of TYW1 from M. jannaschii was measured with a number EPR spectrum of dithionite-reduced TYW1 results from two distinct of potential two-carbon donors in order to determine the source of the species that can be simulated well. Addition of SAM alters the spectrum two carbons that complete the third ring of imG-14. Acetyl-CoA, acetyl of the RS cluster while addition of both SAM and pyruvate results in only phosphate, phosphoenolpyruvate, and pyruvate were each added to one signal corresponding to the SAM-bound RS cluster. These results 1330 N.D. Lanz, S.J. Booker / Biochimica et Biophysica Acta 1853 (2015) 1316–1334

imply that SAM and pyruvate interact with the RS and auxiliary clusters, Characterization of both types of F0 synthases revealed that two Fe/S respectively. The HYSCORE spectrum of SAM bound to TYW1 also clusters are present in each system, and provided insight into the mech- showed strong interactions of a nitrogen atom interacting with the anism of F0 biosynthesis [163].FbiCfromThermobifida fusca contained reduced [4Fe–4S] cluster, indicating that SAM likely binds in a only 1.2 iron ions per polypeptide, but could be reconstituted with up similar manner as shown for other RS enzymes with the α-amino and to 9.5 iron ions per monomer. Quantification of the products of a reac- α-carboxylate groups ligated to the unique iron site (Figs. 7B and 8B, tion containing FbiC, SAM, tyrosine, diaminouracil, and the flavodoxin/ structures of SAM bound to anSMEcpe and BtrN). Further characteriza- flavodoxin reductase reducing system yielded approximately 2.5 equiv- tion of the SAM and SAM plus pyruvate bound species was carried out alents of 5′-dA produced for every equivalent of F0. This result is by Mössbauer spectroscopy. The subtraction of the two spectra revealed consistent with the proposal that one molecule of SAM is cleaved at that the interaction of pyruvate to the auxiliary cluster induces reoxida- each of the two RS cluster sites. When dithionite is used as the reducing tion of the cluster to the S = 0 [4Fe-4S]2+ state in addition to altering reagent in place of the flavodoxin/flavodoxin reductase reducing the environment of the cluster. The results of this study strongly imply system, 3.6 equivalents of 5′-dA was produced for each equivalent of that pyruvate binds to the auxiliary [4Fe–4S] cluster of TYW1 and led F0 produced; however, this stoichiometry is likely due to abortive cleav- to an alternative mechanism to that proposed by Young and Bandarian, age of SAM. Abortive cleavage of SAM was also observed for each en- in which the Fe/S cluster is used in place of the lysine to facilitate zyme of the CofG and CofH pair. In this study, CofG from M. jannaschii catalysis (Fig. 14C). and CofH from Nostoc punctiforme were purified and characterized and shown to abortively cleave SAM in vitro. CofG and CofH contained

6.2. F0 synthase 1.7 and 1.8 iron ions per polypeptide, respectively, indicating incom- plete incorporation of the [4Fe–4S] clusters in these enzymes but still

The F420 cofactor is a widely found deazaﬂavin cofactor found in demonstrating that both enzymes are likely to contain an Fe/S cluster. numerous organisms that participates in redox reactions [157–161]. Reactions containing both enzymes produced F0, though the ratio of It is composed of a tricyclic core that is structurally similar to that of 5′-dA to F0 was 4:1 or greater. It was also demonstrated that CofH ﬂavins, and a variable number of glutamic acid residues linked together generates a diffusible intermediate that can be converted to F0 by by γ-glutamyl bonds appended to the core via the phospholactate group CofG. Each enzyme was independently reacted with SAM, dithionite,

(Fig. 15A). The core of the cofactor is generated by F0 synthase, tyrosine and diaminouracil and then subjected to ultrafiltration. The which fuses tyrosine with 5-amino-6-ribitylamino-2,4(1H,3H)- filtrate was then reacted with the opposite enzyme, SAM, dithionite, pyrimidinedione (diaminouracil), a precursor to riboflavin [162–164]. tyrosine, and diaminouracil. F0 was produced only when the product F0 synthase is a single polypeptide in actinobacteria (FbiC), but is of CofH was provided to CofG. also found as two separate subunits in archaea and cyanobacteria Based on the available information, the authors proposed a

(CofG and CofH) [165]. mechanism for F0 synthase (Fig. 15B) [163]. Abstraction of the F0 synthase contains two distinct RS motifs, either within the same phenoylic H• of tyrosine by 5′-dA• leads to radical fragmentation of protein for FbiC, or split between the CofG and CofH subunits. tyrosine to a glycyl radical and a quinine methide, which can then

Fig. 15. Structure of the F420 cofactor (A) and the proposed mechanism for F0 biosynthesis (B). Shaded in blue and yellow are the reactions catalyzed by CofH and CofG, respectively, in the multi-subunit system. N.D. Lanz, S.J. Booker / Biochimica et Biophysica Acta 1853 (2015) 1316–1334 1331 add to diaminouracil. The second 5′-dA• abstracts a H• from the intermediates in the reductive cleavage reaction suggests that both bridging methylene carbon, which allows for release of ammonia, methylations are catalyzed by the same enzyme. deprotonation of the phenol, and subsequent cyclization. Loss of an Like F0 synthase, MJ0619 contains two canonical RS motifs [172]. additional H• (or a proton and an electron) results in F0. The authors Single Cys → Ala variants in each of the cysteine motifs were created propose that the electron is transferred back to the [4Fe–4S] cluster, in order to determine if both motifs are essential. The variant in which but do not account for the fate of the glycyl radical that is generated. one of the cysteines in the N-terminal motif was substituted with an alanine was able to generate 7-methylpterin; however, the dimethylated product was not observed. No methylated products were observed in 6.3. Methanopterin biosynthesis extracts of E. coli expressing the variant of the C-terminal motif. It was hypothesized that the N-terminal cluster was required for methylation Methanopterin (MPT) and its analogs are one-carbon carrier of C9 and that methylation of C7 precedes methylation at C9. coenzymes found in methanotrophic bacteria and methanogenic The nature of the methyl donor was also probed [172].A archaea (Fig. 16A) [166–169]. It is structurally similar to folate; however, methylcobalamin intermediate was ruled out because MJ0619 does not the enzymes that synthesize and use MPT are not homologous to those contain a cobalamin-binding motif. Moreover, when the mj0619 gene is of the folate cofactor [170,171]. MPT is distinguished by methyl groups expressed in E. coli cultured in minimal media, 7-methylpterin was gener- on C7 and C9 of its heterocyclic ring structure. The enzyme responsible ated even though E. coli cannot synthesize cobalamin. SAM was predicted for methylation of the pterin precursor was only recently identified in to be the methyl donor as is the case in the well-characterized RlmN and M. jannaschii as MJ0619 [172]. Cfr systems [173,174]. However, when E. coli expressing the mj0619 gene 2 MJ0619 was recombinantly expressed and purified from E. coli,and was supplemented with [methyl- H3]-methionine, no deuterium incorpo- the modifications to the natural folate cofactors in E. coli were identified ration was observed in 7-methylpterin. However, when the cells were [172]. Folate derivatives were extracted from E. coli expressing MJ0619 supplemented with deuterated acetate, a pattern of deuterium incorpora- and analyzed by HPLC after being oxidatively or reductively cleaved. tion was observed that was vastly different than that of the methyl group Oxidation of folates results in cleavage of the C6\C9 bond while of methionine extracted from cells grown in media containing deuterated reductive cleavage results in breakage of the C9\N10 bond (Fig. 16). acetate as the sole carbon source. Given that the methyl group of methio- 7-methylpterin (Fig. 16B) and 6-ethyl-7-methylpterin (Fig. 16C) nine is derived from N5-methyl-tetrahydrofolate, N5-methyl-tetrahydro- were found in the respective reactions, indicating that MJ0619 is capa- folate is not likely to be the source of the methyl group for MJ0619. ble of methylating folates in E. coli. The lack of singly-methylated Given these results, the authors proposed a mechanism for MJ0619 that

Fig. 16. Structures of methanopterin (A), 7-methylpterin (B) and 6-ethyl-7-methylpterin (C). The proposed mechanism for MJ0619 in E. coli (D). In the native organism, M. jannaschii,the N5,N10-methylene-tetrahydrofolate is likely replaced with N5,N10-methylene-methanopterin. 1332 N.D. Lanz, S.J. Booker / Biochimica et Biophysica Acta 1853 (2015) 1316–1334 accounts for the isotopic labeling data, in which methylenetetrahydrofo- [16] P. Hänzelmann, H. Schindelin, Crystal structure of the S-adenosylmethionine- dependent enzyme MoaA and its implications for molybdenum cofactor deficiency late donates the appended methyl carbon (Fig. 16D). in humans, Proc. Natl. Acad. Sci. U. S. A. 101 (35) (2004) 12870–12875. [17] A.K. Boal, et al., Structural basis for methyl transfer by a radical SAM enzyme, 7. Conclusions Science 332 (2011) 1089–1092. [18] J.L. Vey, C.L. Drennan, Structural insights into radical generation by the radical SAM superfamily, Chem. Rev. 111 (2011) 2487–2506. The number of RS enzymes for which extra cysteine-containing [19] J.L. Vey, et al., Structural basis for glycyl radical formation by pruvate formate–lyase motifs have been identified in their primary structures continues to activating enzyme, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 16137–16141. grow. Many of these enzymes have been purified and shown to harbor [20] P.J. Goldman, et al., X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification, Proc. Natl. Acad. Sci. auxiliary clusters. In almost every case, the auxiliary cluster(s) has U. S. A. 110 (21) (2013) 8519–8524. proven to be essential for the activity of enzymes that contain them. [21] J.E. Harmer, et al., Structures of lipoyl synthase reveal a compact active site for con- – The greater challenge lies in assigning a function to the auxiliary clusters trolling sequential sulfur insertion reactions, Biochem. J. 464 (2014) 123 133. [22] P. Hänzelmann, H. Schindelin, Binding of 5′-GTP to the C-terminal FeS cluster of in each of the enzymes that require one. For most of the known systems the radical S-adenosylmethionine enzyme MoaA provides insights into its mecha- the function of the auxiliary clusters is still undefined. For LipA and BioB, nism, Proc. Natl. Acad. Sci. U. S. A. 103 (18) (2006) 6829–6834. it has become clear that the auxiliary cluster is required as a source of [23] S. Goto-Ito, et al., Structure of an archaeal TYW1, the enzyme catalyzing the second step of wye-base biosynthesis, Acta Crystallogr. D Biol. Crystallogr. 63 (Pt 10) sulfur for the reaction. In the cases of MoaA and TYW1, the cluster likely (2007) 1059–1068. binds and/or activates the substrate for catalysis. In a number of systems [24] F.Forouhar,etal.,TwoFe–S clusters catalyze sulfur insertion by radical-SAM such as the SPASM domain-containing enzymes, it is hypothesized that methylthiotransferases, Nat. Chem. Biol. 9 (5) (2013) 333–338. [25] S. Arragain, et al., Post-translational modification of ribosomal proteins: structural the auxiliary clusters are required for electron transfer, but experimen- and functional characterization of RimO from Thermotoga maritima, a radical S- tal evidence is limited. Beyond the functions already listed, Fe/S clusters adenosylmethionine methylthiotransferase, J. Biol. Chem. 285 (2010) 5792–5801. may function in substrate positioning or Lewis acid catalysis. We also [26] N.D. Lanz, S.J. Booker, Identification and function of auxiliary iron-sulfur clusters in radical SAM enzymes, Biochim. Biophys. Acta 1824 (11, Sp. Iss. SI) (2012) 1196–1212. have to consider that some Fe/S clusters may be structurally important, [27] A. Marquet, B.T. Bui, D. Florentin, Biosynthesis of biotin and lipoic acid, Vitam. or in rare cases, simply vestigial. The example of HydG in which the Horm. 61 (2001) 51–101. auxiliary Fe/S cluster both coordinates the substrate and sacrifices an [28] A. Marquet, et al., In vivo formation of C\S bonds in biotin. An example of radical – – iron ion to the H-cluster of HydA demonstrates the variety of ways in chemistry under reducing conditions, J. Phys. Org. Chem. 11 (8 9) (1998) 529 535. [29] C.J. Fugate, J.T. Jarrett, Biotin synthase: insights into radical-mediated carbonâ€- which nature uses Fe/S clusters. The functional diversity of Fe/S clusters sulfur bond formation, Biochim. Biophys. Acta (BBA), Proteins Proteomics 1824 may not yet be fully understood, which makes study of RS enzymes (11) (2012) 1213–1222. containing auxiliary clusters even more appealing. [30] J.R. Miller, et al., Escherichia coli LipA is a lipoyl synthase: in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein, Biochemistry 39 (49) (2000) 15166–15178. Acknowledgments [31] P. Douglas, et al., Lipoyl synthase inserts sulfur atoms into an octanoyl substrate in a stepwise manner, Angew. Chem. Int. Ed. 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