S-Adenosylmethionine: more than just a methyl donor

Covering: from 2000 up to the very early part of 2023 S-Adenosyl-l-methionine (SAM) is a naturally occurring trialkyl sulfonium molecule that is typically associated with biological methyltransfer reactions. However, SAM is also known to donate methylene, aminocarboxypropyl, adenosyl and amino moieties during natural product biosynthetic reactions. The reaction scope is further expanded as SAM itself can be modified prior to the group transfer such that a SAM-derived carboxymethyl or aminopropyl moiety can also be transferred. Moreover, the sulfonium cation in SAM has itself been found to be critical for several other enzymatic transformations. Thus, while many SAM-dependent enzymes are characterized by a methyltransferase fold, not all of them are necessarily methyltransferases. Furthermore, other SAM-dependent enzymes do not possess such a structural feature suggesting diversification along different evolutionary lineages. Despite the biological versatility of SAM, it nevertheless parallels the chemistry of sulfonium compounds used in organic synthesis. The question thus becomes how enzymes catalyze distinct transformations via subtle differences in their active sites. This review summarizes recent advances in the discovery of novel SAM utilizing enzymes that rely on Lewis acid/base chemistry as opposed to radical mechanisms of catalysis. The examples are categorized based on the presence of a methyltransferase fold and the role played by SAM within the context of known sulfonium chemistry.

1 Introduction S-Adenosyl-L-methionine (SAM or AdoMet, 1) is a sulfoniumcontaining primary metabolite found in both prokaryotic and eukaryotic cells. 1 SAM is best known for the role it plays in one-carbon metabolism where it serves as a methyl donor in many biological reactions, [2][3][4] and a search in InterPro reveals that approximately 3 million protein sequences have been annotated as SAM-dependent methyltransferases. 5The molecular mechanism of SAM-dependent methylation generally involves a nucleophilic substitution at the sulfonium methyl carbon of SAM to generate a variety of C-, O-, N-and Smethylated products. 6ot all enzymes, however, utilize SAM to catalyze methylation reactions.In particular, a signicant number of enzymes also bind an [Fe 4 S 4 ] cluster typically via a highly conserved CX 3 CX 2 C motif in the active site that serves to reduce SAM leading to the homolytic cleavage of the C5 ′ -S linkage. 7,8The resulting 5 ′ -deoxyadenosyl radical equivalent (2) can then initiate a broad range of radical-mediated transformations.][20][21] Among these enzymes, SAM may serve as an alkyl donor as opposed to a strict methyl donor or stabilize reaction intermediate(s) via the positive charge of its cationic sulfonium moiety.Alternatively, SAM may only play a structural role helping to maintain the integrity of the enzyme and its active site and thus remain intact throughout the catalytic cycle.While many of these enzymes are structurally related to the SAM-dependent methyltransferases, [22][23][24] this is not universally the case.
This review emphasizes the varied enzymology of SAM in biological transformations that are not expected to involve radical intermediates (Fig. 1).The enzymes will therefore be discussed based on whether they are structurally related to the SAM-dependent methyltransferases (Section 2) or appear to represent different classes of enzymes altogether (Section 3).As will become evident, however, even structurally related enzymes can exhibit unexpected behavior thereby complicating bioinformatics efforts to infer their activities and placing an Yu-Hsuan Lee was born in Taipei (Taiwan) and received her bachelor's degree from National Taiwan University in 2016, where she spent the last year as an exchange student at the Technical University of Munich.She then joined the graduate program in chemistry at the University of Texas at Austin.In the research group led by Prof. Hung-wen (Ben) Liu, she has studied the mechanisms of radical SAM enzymes involved in the biosynthesis of nucleoside natural products.
Byungsun Jeon received his PhD from the University of Texas at Austin, where he studied the enzymes and biosynthetic pathway of spinosyn A using kinetic analysis and organic synthesis under the guidance of Prof. Hung-wen (Ben) Liu.Upon completing his studies in 2017, he worked with Prof. Christopher J. Chang at University of California, Berkeley as a postdoctoral researcher.In 2019, he started his independent career at Korea Institute of Science and Technology (KIST).His current research focuses on organic synthesis of natural products and biased-ligand development for G-protein coupled receptors.emphasis on experimental investigation.Research has nevertheless made steady progress, and since 2010 there has been a considerable expansion in the recognized scope of SAM enzymology observed in nature.Furthermore, continued study in the future is certain to ll in many of the gaps in the current understanding of these enzymes as well as identify new and potentially unforeseen biological reactions involving SAM.

Enzymes with a methyltransferase (MT) fold
The electrophilicity of the SAM sulfonium moiety is illustrated by its involvement in a number of different biological alkylation reactions (Fig. 2).][27][28] Consequently, the regioselectivity of enzyme catalyzed alkyl transfer from SAM appears to originate in variations in the enzyme active sites that tune the relative positioning between the alkyl donor and the nominal substrate, which serves as the alkyl acceptor.Moreover, a number of enzymes have also evolved active site arrangements that preferentially recognize derivatives of SAM thereby further increasing the scope of biological alkylation reactions.Protein crystallography has thus become a critical tool in understanding the details of SAMdependent alkylation reactions.The following section is

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Natural Product Reports focused mainly on more recent ndings regarding transfer of the 3-amino-3-carboxypropyl group and the modied methyl group from SAM, each catalyzed by an enzyme adopting a methyltransferase fold.Finally, a brief discussion is included of the adenosyl transfer enzymes, which lack a methyltransferase fold yet utilize SAM as the adenosyl donor.

SAM-dependent alkylation reactions
A number of enzymes have been discovered to selectively catalyze transfer of the 3-amino-3-carboxypropyl (ACP) group from the SAM sulfonium to an acceptor nucleophile expelling methylthioadenosine (MTA) as the leaving group.6][37] Substrate binding is of particular importance for regioselective alkyl transfer, and thus the emphasis of the following discussion will be placed on how SAM binding allows for selective C-S bond cleavage.
2.1.1 3-Amino-3-carboxypropyl transfer (small molecules).Nicotianamine ( 8) is a precursor to phytosiderophores, which facilitate metal acquisition by plants. 38,392][43][44] Although NAS had previously only been found in plants and fungi, recent studies have indicated that NAS-like enzymes exist in both archaea and bacteria as well. 45,46rchaeal NAS from Methanothermobacter thermautotrophicus (MtNAS) was found to copurify with thermonicotianamine (tNA, 9), which is structurally distinct from nicotianamine in that the rst condensation partner is a glutamate (10) rather than an ACP moiety (Fig. 4B). 45Various crystallographic snapshots of MtNAS in complex with its substrate, a proposed intermediate and its product have led to a mechanistic proposal involving two rounds of ACP transfer to glutamate leading to tNA. 45,47In particular, the active site of MtNAS extends deep into the protein interior from a solvent exposed entrance allowing for sequential binding of glutamate and SAM. 45As SAM binds, glutamate translocates from the donor site at the entrance of the reaction chamber to the buried acceptor site.Transfer of the ACP moiety from SAM to glutamate or the intermediate AP 2 -Glu 1 (11) is facilitated by Glu81 and Tyr107, which reside in the middle of the active site and deprotonate the nucleophilic amine on the acceptor substrate (i.e., Glu or AP 2 -Glu 1 ) (Fig. 4C). 45g. 3 Aminopropyl group transfer during the biosynthesis of spermidine.SAM is first decarboxylated to form decarboxy-SAM (dc-SAM, 6) before the 3-aminopropyl group is transferred to putrescine to yield spermidine (7).

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9][50] Mutant strains that do not produce CntL show reduced virulence and decreased intracellular metal content. 46CntL catalyzes the transfer of ACP from SAM to D-histidine (13) forming xNA (14) (Fig. 4D). 46The structure of CntL was solved at 1.81 Å and found to be similar to that of MtNAS. 51Both MtNAS and CntL possess a Rossmann fold MT C-terminal domain with a characteristic glycine-rich motif that binds SAM as the ACP donor. 45,51An open to closed conformational change upon substrate binding was noted for CntL and involves the partially disordered interdomain linker in the CntL-SAM binary complex becoming an ordered a-helix in the CntL-MTA-xNA ternary complex. 51The mechanism of CntL catalyzed ACP transfer is otherwise analogous to that of MtNAS.][54] Microcin C (15) produced from E. coli is a potent inhibitor of aspartyl-tRNA synthetase. 55The biosynthesis of microcin C involves aminopropylation of the phosphate which enhances the inhibitory effect by approximately 10-fold. 56 MccD can be signicantly enhanced by the nucleosidase Mtn, which can hydrolyze the MTA by-product and presumably alleviate product inhibition. 57dditional ACP transferases with small molecule substrates are also involved in the biosynthesis of isonocardicin C (18), 58,59 betaine lipid (19), 60 discadenine (20) 61,62 and 2-(3-amino-3carboxypropyl)-isoxazolin-5-one (ACI) (21) (Fig. 6); 63,64 however, none of these enzymes appear to be homologous to the aforementioned ACP transferases NAS, MtNAS, CntL and MccD.With the exception of ACI, ACP transfer from SAM is respectively catalyzed in these cases by NAT, BtaA and discadenine synthase.However, while the in vitro formation of ACI is known to require a partially puried enzyme fraction from sweet pea seedlings as well as the presence of ATP and magnesium, 63,64 the responsible enzyme as well as the role of ATP remains elusive.
2.1.2ACP transfer (RNA).Another class of ACP transferases is involved in the maturation of eukaryotic and archaeal 18S rRNA, where a uridine base (U1191 in yeast, U1248 in human) in the loop capping helix 31 undergoes a 3-step modication consisting of pseudouridylation, methylation and ACP transfer to yield 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouridine (m 1 acp 3 J, 22, see Fig. 7A). 65The participation of Tsr3 in ACP transfer was revealed by differences in the nucleoside prole of 18S rRNA isolated from a collection of mutant strains with random gene deletions. 66Crystal structures of archaeal Tsr3 from Vulcanisaeta distributa (VdTsr3) and Sulfolobus solfataricus (SsTsr3) have been solved with and without SAM-bound, respectively. 66The C-terminus of Tsr3 is closely packed with the N-terminal domain and adopts a SPOUT fold, which is characterized by a deep trefoil knot and oen found in the structures of RNA methyltransferases. 67,68In contrast, its functional counterpart Tyw2 has a Rossmann fold and also catalyzes ACP transfer from SAM during the biosynthesis of wybutosine (23). 69Archaeal Tsr3 and its closest structural homolog Trm10, 70,71 which is a tRNA guanidine N-methyltransferase (G, 24, to m 1 G, 25) found in archaeans and eukaryotes (Fig. 7B), 72 employ a monomeric quaternary structure while the majority of enzymes in this superfamily are homodimers. 73omparison of the crystal structures of VdTsr3 and Trm10 with SAM bound reveals overall similarity at the interface of the N-and C-terminal domains. 66This is where SAM binds with the adenine moiety buried in a hydrophobic pocket and the ribose

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Natural Product Reports moiety hydrogen-bonded to the protein backbone.However, closer inspection of the VdTsr3 active site shows the carboxyl moiety of ACP positioned towards the entrance of the substrate binding pocket surrounded by hydrophilic residues, while the methyl group is directed into a hydrophobic pocket consisting of an unconserved aliphatic residue and a conserved tryptophan (Trp73 in VdTsr3, see Fig. 7D). 66Mutation of the Trp to Ala results in both diminished SAM affinity and ACP transferase activity in SsTsr3. 66Furthermore, Tsr3 employs a conserved residue (Asp70 in VdTsr3) that serves as a general base, whereas the equivalent Asp210 in Trm10 is not essential for methyl transfer. 74The above catalytically important residues in Tsr3 constitute the DTW motif with the conserved primary sequence (D/E)X(T/S)W (Fig. 7G), 75 in which Asp is the possible catalytic base and Trp assists in methyl group positioning. 76Proteins with this domain are classied into the TDD superfamily, which is named aer the three representative protein members Tsr3, DTWD1 and DTWD2. 75lthough ACP transfer catalyzed by Tsr3 is the nal step in m 1 acp 3 J modication during 18S rRNA maturation, studies of yeast mutants lacking enzymes for pseudouridylation and methylation have suggested that in vivo ACP transfer activity is independent of either modication such that the corresponding mutants can produce acp 3 U (27) or acp 3 J, respectively. 77,78hese results suggest that Tsr3 and its homologs can catalyze modication of unmodied uridine residues in RNA.Two recent studies involving comparative genomic and ribonucleome analysis have supported this hypothesis by showing that the bacterial Tsr3 homolog TapT from E. coli catalyzes acp 3 U (27) modication at U47 (26) in the V-loop of several bacterial tRNAs (Fig. 7F). 76,79While a structure of TapT is not yet available, sequence analyses and mutational studies imply that TapT harbors the catalytically important DTW motif similar to that of archaeal Tsr3 despite overall low sequence identity (Fig. 7G). 76.1.3Carboxylmethyl transfer.In Section 2.1.2,several SAM-dependent enzymes were described that catalyze transfer of the 3-amino-3-carboxypropyl (ACP) group to a nucleobase on tRNA thereby improving the thermostability and maintaining its normal function. 76,77,79,802][83][84] Gene manipulation studies have suggested that formation of this unusual nucleobase involves the activity of two putative SAMdependent methyltransferases CmoA and CmoB (Fig. 8A). 85moA catalyzes an unprecedented carboxylation of SAM yielding carboxy-SAM (Cx-SAM, 29), 86,87 the details of which will be discussed in Section 2.5.1.Subsequently, CmoB transfers the carboxymethyl group from 29 to 5 ′ -hydroxyl uridine (30) and generates 5 ′ -carboxymethyloxyuridine (28). 86hile CmoB can catalyze alkyl transfer from both SAM and Cx-SAM, it exhibits a strong preference for Cx-SAM. 88In order to identify the structural determinants of this selectivity, the crystal structure of CmoB was solved by Kim and co-workers (Fig. 8B). 88,89It was found that the C-terminal domain of CmoB adopts a Rossmann fold shared among typical methyltransferases, whereas the structure of the N-terminal domain, which spans around 100 amino acid residues, is unique to CmoB. 88Lys91 was found adjacent to the anionic carboxymethyl moiety and mutation of Lys to Ala completely abolished the production of cmo5U (28) in vivo, the effect of which is attributed to a 19-fold increase in affinity towards SAM and a 275-fold reduction in affinity towards Cx-SAM. 88Consequently, Lys91 likely serves to stabilize the carboxymethyl moiety in the active Natural Product Reports Review site via electrostatic interactions thereby enhancing the speci-city of CmoB towards Cx-SAM (29) versus SAM.Furthermore, unlike typical SAM methyltransferases, CmoB does not appear to employ general base catalysis to increase the nucleophilicity of the acceptor oxygen, 88 because the substrate hydroxyl has an apparent pK a of 7.68 which is sufficient for specic acid/base catalysis. 90ollowing discovery of the CmoA/CmoB carboxymethylation cascade, a homologous enzyme pair was found in the biosynthetic pathway of 3-thiaglutamate (31), a nonproteinogenic amino acid with unknown function. 91TglE/F show high sequence identity with CmoA/B, and TglF has been shown to catalyze the trans-carboxymethylation of a norcysteine thiol in a ribosomally synthesized and post-translationally modied peptide (32 / 33) prior to amide hydrolysis by the protease TglG to release 3-thiaglutamate (31) (Fig. 9A). 91Primary sequence alignment between CmoB and TglF showed that the essential residues including Lys91 for selective binding of Cx-SAM in CmoB are all conserved in TglF.Furthermore, the biosynthetic gene cluster (Mcc Bam ) of the Trojan horse peptidecytidylate antibiotic microcin C (34) encodes MccB Bam which has been shown to catalyze transfer of the carboxymethyl moiety from Cx-SAM synthesized by MccS Bam to its tRNA substrate 35 (Fig. 9B). 92The C-terminal domain of MccB Bam (MccB Bam CTD ) adopts a methyltransferase fold that does not show signicant similarity with CmoB and is strictly specic for Cx-SAM (29). 92Although the site of carboxymethylation in 34 has yet to be determined, this nding implies that the two-enzyme cascade for this modication is operative in both Gramnegative and Gram-positive bacteria. 92he discovery of Cx-SAM demonstrates that SAM can be naturally modied leading to additional diversity among the available types of biological SAM-dependent alkyl transfer reactions.Given the high intracellular concentration of SAM versus its modied counterparts such as Cx-SAM, 88,93 enzymes must employ recognition mechanisms in order to differentiate between the SAM congeners and thereby avoid inhibition or nonproductive shunt reactions.The structures of CmoB demonstrate that interactions between specic residues and the bound SAM analog at least play a part in this specicity.Consequently, it may be possible to engineer new alkyl transferases via optimized point mutations to facilitate the selective binding of other non-natural SAM analogs and thereby further expand the range of biocatalytic alkylation reactions for biotechnological purposes. 94,95.1.4Adenosyl transfer.Of the three possible alkyl transfer reactions involving SAM (Fig. 2), adenosyl transfer reactions are only known in the hydrolysis of SAM to methionine (5) and adenosine as well as the biosynthesis of 5 ′ -deoxy-5 ′ -haloadenosine. 96,97][100][101] The utilization of SAM in these biosynthetic pathways is not immediately obvious, because the resulting 5 ′ -uoro-5 ′ -deoxyadenosine (5 ′ -FDA, 36) and 5 ′ -chloro-5 ′ -deoxyadenosine (5 ′ -ClDA, 37) are each metabolized by downstream enzymes before yielding the nal halogenated natural products (39-42) with only part of the ribose skeleton from SAM remaining (Fig. 10A). 101,102rystallographic studies of both FlA and SalL revealed an overall structure distinct from SAM-dependent methyltransferases.The structures of FlA and SalL instead resemble those of proteins belonging to the DUF62 superfamily despite none of the members having been functionally characterized at

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Natural Product Reports the time (Fig. 11).Subsequently, one protein from the DUF62 superfamily, SaDUF62, was shown to catalyze hydrolysis of SAM, which is effectively adenosyl transfer from SAM to a hydroxide ion thereby producing adenosine (38) and methionine (5). 103,104Moreover, a strictly conserved His-Arg-Asp triad is only present in DUF62 proteins (Fig. 11C), which activates one of the two H-bonded waters as the hydrolytic nucleophile necessary for the displacement reaction. 105In contrast, both halogenases lack the catalytic triad and waters are instead expelled from the vicinity of the sulfonium of SAM and the adjacently trapped halide nucleophile.
Comparison of the binary FlA/SAM substrate complex with the ternary FlA/5 ′ -FDA/Met product complex has suggested that the C5 ′ of SAM is positioned between the uoride and the sulfur of SAM during the substitution reaction indicative of an S N 2type mechanism. 100A catalytically inactive SalL mutant forms a ternary complex with chloride and SAM revealing a colinear arrangement between the chloride ion, C5 ′ and the sulfur center of SAM (Fig. 10B). 101Although the biological function of hydroxide adenosyltransferases is not as obvious as their halogenase counterparts, recent research identied a DUF62containing protein, StDUF62, catalyzing the stereoselective hydrolysis of the non-native (R,S)-SAM (43), which is a stereoisomer that equilibrates with biological (S,S)-SAM (1) upon racemization of the sulfonium. 106This hints at a role played by DUF62 proteins in preventing in vivo accumulation of the nonbiological SAM diastereomer.

Intramolecular cyclization of SAM
Transformations that utilize SAM as the alkylating agent take advantage of the inherent electrophilicity of the SAM sulfonium group 107 This property renders SAM susceptible to nucleophilic attack not only intermolecularly but also intramolecularly.In fact, the major decomposition pathway of SAM under neutral or slightly acidic conditions is the intramolecular cyclization that yields homoserine lactone (44) and MTA (4) (Fig. 12). 108,109ccording to Baldwin's rules, 110 this reaction is a favored 5-exotet ring closure and likely proceeds through direct nucleophilic attack of the carboxylate oxygen at the C-g carbon.2][113] However, not all of these biosynthetic enzymes adopt a SAM-dependent   Natural Product Reports Review methyltransferase (MT) fold despite each utilizing SAM as the substrate.Therefore, only MT-like cyclases are included in the following section, and the rest will be discussed in Section 3.
2.2.1 Azetidine 2-carboxylic acid.Azetidine 2-carboxylic acid (AZE, 45) is a non-proteinogenic amino acid rst isolated from Convallaria majalis. 1142][123] The responsible enzymes (i.e., VioH and AzeJ) share only 28% sequence identity and catalyze the conversion of SAM to AZE (45).Similar chemistry has also been proposed for the homologous enzyme BnvI during the biosynthesis of bonnevillamides (50) (Fig. 13). 124Both AzeJ and VioH have been reported to display relatively low turnover numbers with the k cat of AzeJ being 17 min −1 and that of VioH being too low to readily measure. 122,123Furthermore, VioH is most active under alkaline conditions indicative of general base catalysis to facilitate nucleophilic attack of the amino group at C-g. 122 However, none of these enzymes have been structurally characterized, and homology models have been inconclusive due to the lack of a suitable template. 122Consequently, it remains uncertain how the ACP side chain of SAM is arranged in the active site to facilitate the 4-exo-tet cyclization and avoid lactonization.
2.2.2 Cyclopropane.SAM-dependent two-enzyme cascades have recently been reported to install a spirocyclopropyl moiety in CC-1065 (51) 125 and possibly gilvusmycin ( 52) 126 and yatakemycin (53) 127 as well (Fig. 14).CC-1065 biosynthesis involves the HemN-like radical SAM enzyme C10P and a SAM-dependent methyltransferase C10Q. 125In a coupled assay, the two enzymes together catalyze the cyclopropylation of 55 yielding CC-1065 (51), the mechanism of which was proposed to involve intermediate 56 generated from the addition of a SAM methylene radical (54) to the pyrrole ring of the nominal substrate (55)  followed by reduction under the action of C10P.The subsequent cyclopropane ring formation is hypothesized to be catalyzed by C10Q and proposed to involve base catalyzed intramolecular elimination of SAH. 125The reaction proposed to be catalyzed by C10Q is analogous to other intramolecular elimination reactions involving SAM; however, in this case the methyl group has

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Natural Product Reports been modied with the moiety that serves as the nucleophile during the intramolecular cyclization. 125While this biosynthetic pathway is fully consistent with all available experimental data including detection of 56 by high resolution mass spectrometry and related isotope labelling results, 125 direct characterization of C10Q with 56 in the absence of C10P has been hampered by poor stability of the latter. 125Therefore, many aspects of C10P/ C10Q chemistry remain to be fully established.

Methylation induced cascade reaction
SAM is also involved in enzyme catalyzed cyclization reactions that proceed in tandem with alkylation. 128,129The majority of examples of this chemistry characterized to date come from the study of terpene biosynthesis.Terpenoid lipids are characterized by extremely diverse cyclic scaffolds constructed from the two unsaturated precursors isopentenyl-pyrophosphate and dimethylallyl-pyrophosphate. 130,131The structural diversity is attributed to the formation of carbocation intermediates such as 57 that can rearrange in a number of ways within the active sites of terpene cyclases. 132,133The activated carbocation intermediates are typically generated via departure of pyrophosphate (58), which may still be associated with the carbocation species as an ionic complex, from the respective precursors (59) (Fig. 15A); however, synthetic methodologies have been developed that employ a Lewis acid to induce carbocation formation including epoxide ring opening and alkene protonation. 134This type of chemistry has also been found in natural product biosynthesis where SAM serves as the Lewis acid catalyst by essentially donating a methyl cation to the electron rich psystem of the terpenoid precursor.Cyclization of the resulting carbocation thus proceeds in tandem with alkylation before deprotonation, which in the case of cyclopropyl fatty acid synthases is performed by an active site bicarbonate ion (60) (Fig. 15B). 135,136.3.1 Terpene cyclization.Sodorifen ( 61) is a 16-carbon terpene natural product isolated from Serratia plymuthica, 137 which possesses the four-gene sod cluster responsible for sodorifen biosynthesis. 138,139Biosynthesis involves SodC catalyzed cyclization of farnesyl pyrophosphate (FPP, 59) to yield the biosynthetic intermediate pre-sodorifen pyrophosphate (PSPP, 62), which is subsequently converted to sodorifen under the action of SodD (Fig. 16). 139,140The tertiary structure of SodC was predicted by in silico homology modeling, which allowed for further computation of the reaction mechanism. 140It has thus been proposed that PSPP formation proceeds through a cyclopropane intermediate ( 65) that is generated upon the carbocationic rearrangement of FPP following SodC catalyzed pmethylation by SAM (59 / 63). 140The initially generated cyclohexane carbocation rst undergoes ring contraction in a stepwise process involving a neutral bicyclic intermediate (64 / 65 / 66). 140The resulting carbocation 66 can then undergo additional hydride and methyl shis directed by an active site Tyr-His dyad leading to PSPP formation with an overall release of energy. 140Bioinformatics analysis of clustered sodC and sodD homologs has identied 28 sod-like biosynthetic gene clusters that may also encode additional methyltransferases, Rieske proteins and avodoxin proteins. 141This suggests a number of additional exotic terpenoid ring systems remain to be discovered.
2.3.2Pictet-Spengler rearrangement.Another example of an alkylation induced carbocationic rearrangement and cyclization comes from the biosynthesis of teleocidins (69-71). 128,142,143TleD is a SAM-dependent methyltransferase that catalyzes C25 methylation of the isobutenyl moiety of teleocidin A1 (72) to generate a putative tertiary carbocation intermediate (73, Fig. 17A).As in the case of terpene biosynthesis, the resulting carbocation may then undergo a [1,2]-hydride shi (73 Natural Product Reports Review / 74), which is consistent with deuterium incorporation experiments, followed by intramolecular electrophilic aromatic substitution to yield an intermediate containing an unstable spiro-ring system (75). 144Subsequent Pictet-Spengler rearrangement 145,146 and deprotonation would then yield 69-71.
Crystal structures of TleD have been solved with SAH bound (2.5 Å) as well as both SAH and substrate bound (2.8 Å) in the active site. 147These structures place the sulfur of SAH approximately 4.4 Å away from C25 of the teleocidin A1 (72), which is consistent with the proposed methyltransfer mechanism. 147nlike typical SAM methyltransferases, TleD appears to be active as a hexamer with each active site formed at the interface of two TleD monomers in a domain-swapped fashion (Fig. 17B  and C). 147The active site of TleD is tightly lined with hydrophobic residues and thus water is excluded and prevented from quenching the putative intermediate carbocations. 147The geranyl moiety of the substrate appears to be exible and assumes two different conformations in the crystal structure; however, molecular dynamics analysis suggests that only one of them is likely to account for the observed product distribution. 147.3.3Stevens rearrangement.Alkylation induced carbocationic rearrangements can also be initiated via the methylation of moieties other than alkenes and do not necessarily need to involve cyclization.9][150] The structure of echinomycin features a methyl thioacetal linkage, which is introduced via a redox-neutral rearrangement of the disulde bridged precursor triostatin (78) 151 and is believed to lend additional stability to the dimer compared to its disulde counterpart (Fig. 18A). 152,153The enzyme responsible for this transformation is the SAM-dependent methyltransferase Ecm18 (Qui8) [154][155][156] or Swb8 in the case of the related compound SW-163D (77) and its derivatives. 157,158he catalytic cycle of Ecm18 is hypothesized to proceed through an intermediary sulfonium ion generated via SAMdependent methylation of one of the two sulfur atoms of the disulde bridge in triostatin (78 / 79). 154The sulfonium ion can then undergo a-deprotonation in the presence of a general base to yield an ylide (80), which is capable of a Stevens rearrangement to yield the methylthioacetal linkage (80 / 76).To probe the mechanistic details of Ecm18 catalysis, Hotta and coworkers solved the 1.5 Å crystal structure of Ecm18 with the reaction products SAH and echinomycin (76) bound in the active site. 159Ecm18 was thus found to adopt a characteristic SAM binding Rossmann fold featuring a compact hydrophobic substrate/product binding site (Fig. 18B). 159The proposed methylation reaction is consistent with the linear arrangement of the sulfur center of SAH, the transferred methyl group and

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Natural Product Reports the sulfur center on the product.A polar histidine residue resides sufficiently close to the thioacetal and could serve as a general base for a-deprotonation of the substrate sulfonium to yield the putative sulfur ylide 80 (Fig. 18C). 159Rearrangement of the ylide may then proceed via a 3-endo-tet cyclization and ringopening to yield the methyl thioacetal in 76 (Fig. 18A, mechanism a). 159However, two other mechanistic hypotheses for the rearrangement have also been proposed that do not require intermediary cyclization via 81 to effect the Stevens rearrangement as shown in Fig. 18A. 107,160,161One involves heterolytic cleavage of the S-S bond followed by thiolate addition to the resulting thionium 82 (mechanism b).The other starts with homolytic cleavage of the S-S linkage and a subsequent radicalradical recombination to construct the C-S bond in 76 (mechanism c).
2.3.4Alkaloid cyclization.Biosynthesis of a subclass of pyrroloindoline alkaloids has also been shown to involve an alkylation-annulation cascade. 162Natural products in this family such as physostigmine (84) are characterized by complex, fused ring systems derived from tryptophan. 163Liu and coworkers discovered that the SAM-dependent methyltransferase PsmD is an early participant in the biosynthesis of physostigmine where it catalyzes cyclization of 85. 164 PsmD catalysis begins with methylation at C3 of the indole moiety resulting in an iminium ion 86 that undergoes intramolecular nucleophilic addition by a nearby amide nitrogen to afford the heterocycle in 84 (Fig. 19A). 164A similar methylation induced cyclization has been reported in the biosynthesis of nocardioazine B (87) and proposed in indimicin (88). 165,166Evidence for such chemistry comes from model systems, which have demonstrated that C3 alkylation of the indole moiety always results in intramolecular addition of the adjacent amide. 167,168Thus, only the alkylation step appears to require enzyme catalysis with subsequent cyclization following nonenzymatically.Such alkylation-annulation tandem reactions have also been utilized in the total synthesis of an expanded scope of alkylated pyrroloindoline scaffolds in which alkyl bromides are used as the alkyl donor (Fig. 19B). 168

SAM as a prosthetic group
Apart from direct participation in the group transfer reactions, SAM can also serve as a prosthetic group for many enzymes that are annotated as SAM-dependent methyltransferases.Among these examples, SAM does not undergo any covalent changes in its structure during the reaction.
2.4.1 LepI in leporin biosynthesis.Leporins (89) are pyridone alkaloids exhibiting mild antibacterial activity. 169Within the lep biosynthetic gene cluster, LepI is annotated as a SAMdependent O-methyltransferase and contains a C-terminal Rossmann fold for SAM binding. 170However, in vitro analysis suggests that LepI accelerates a stereoselective dehydration reaction of its nominal substrate (91, the stereochemistry of the carbon with the departing hydroxyl group is not yet known) to (E)-92, and facilitates a subsequent regioselective hetero-Diels-Alder (HDA) reaction (92 / 89) (Fig. 20A).Meanwhile, LepI can also convert a shunt intramolecular-Diels-Alder (IMDA) product 93 to 89 via a retro-Claisen rearrangement. 170Consistent with its annotation, however, catalytically active LepI copuries with SAM at almost 90% occupancy.Furthermore, sinefungin (SFG, 94), which is an isosteric analog of SAM, also supports LepI catalysis, while SAH (3) is a strong inhibitor of both the dehydration and retro-Claisen rearrangement.Therefore, the positive charge on SAM or sinefungin appears to play a key role in LepI catalysis even if SAM itself remains intact. 170he positive charge on SAM and sinefungin was originally proposed to play an electrostatic role in catalysis, since coordination of the substrate to a sulfonium moiety is suggested by computational simulations to accelerate the cyclization and drive the periselectivity towards the favored product. 170owever, the crystal structure of LepI complexed with SAM and an unreactive substrate analog (90) revealed that the distance between the two ligands is too remote for coordination (Fig. 20A and 21), 171 whereas a product complex between LepI/SAM/89 indicated that the guanidinium group of Arg295 forms a hydrogen bond with the amide carbonyl of the LepI product (89). 171In addition, the His133 residue essential for LepI catalysis is near the 4-O of 89, implying its role in deprotonation of 91 (Fig. 20B).The resulting imidazolium of His133, along with the guanidinium of Arg295, is thus hypothesized to stabilize the transition-state during cycloaddition via 92. 1713][174] Sequence and structural comparison between LepI and its closest homolog OxaC, which is an Omethyltransferase catalyzing the methyltransfer of meleagrin (95) to yield oxaline (96) (Fig. 20D), 175 revealed that Arg295 in LepI aligns with an essential histidine (His313) in OxaC (Fig. 20G). 171His313, along with Glu369, is conserved among functional O-methyltransferases for substrate deprotonation, and the substitution of the former in LepI may provide an evolutionary perspective on the mechanistic shi from performing methylation to dehydration and cyclization.
Such repurposed methyltransferases catalyzing cyclization reactions have also been found to participate in other biosynthetic pathways.For example, scherin (97) features a cis-decalin moiety, the biosynthesis of which is not immediately obvious.The dedicated cyclase FinI shows 25% sequence identity and structural homology with LepI and also co-puries with SAM as does LepI. 170,176Moreover, FinI catalyzed cyclization of 98 to 99 is believed to involve an exo-selective DA reaction Natural Product Reports Review (Fig. 20F), which is rare among known pericyclases. 176Unlike the case of LepI, however, SAM and the substrate 98 bind the FinI active site so as to place the sulfonium methyl of SAM close to the amide nitrogen of 98 at a distance of 3.4 Å suggesting an electrostatic interaction and a possible role for SAM in substrate recognition.Whereas in the case of LepI Arg295 can act as a Lewis acid to activate the diene for cyclization, 171 the homologous residue Arg313 in FinI is unable to do so given the structure of 98 (Fig. 20F).Instead, Arg310 may play a similar role serving to activate the dienophile in the FinI catalyzed reaction by forming a hydrogen bond with the carbonyl group at C7 of 98.Although Arg310 in FinI is conserved in homologous methyltransferases such as OxaC, where it interacts with SAM, this residue appears to have been evolutionarily repurposed in the FinI active site to interact and likely activate the substrate for cyclization (Fig. 20G).2.4.2SpnF and SpnL in spinosyn biosynthesis.SpnF and SpnL participate in the biosynthesis of the tetracyclic polyketide spinosyn A (100) and are both annotated as SAM-dependent methyltransferases with a SAM binding Rossmann fold at the C-terminal; 177 however, neither enzyme catalyzes a methyl transfer reaction.SpnF instead catalyzes an intramolecular [4 + 2]-cycloaddition (101 / 102), whereas SpnL catalyzes the cyclization of 103, yielding the rst biosynthetic intermediate 108 with the same ring skeleton as spinosyn A. 177 The catalytic properties of SpnF were identied when an approximately 500fold increase in reaction rate was observed for the cyclization reaction in the presence of the enzyme versus its absence (Fig. 22). 177Furthermore, measurement of secondary kinetic Fig. 20 (A) LepI catalyzed dehydration-cyclization cascade during leporin C biosynthesis.Although the stereochemistry at C7 in 91 is currently unclear, it has been shown that the diastereomer of 91 is not accepted by LepI.Comparison of crystal structures of (B) LepI/SAM/89 complex (PDB ID: 6IX9) and (C) OxaC/SFG/95 complex (PDB ID: 5W7S).In LepI, SAM is too far from the product (89, in tan) for methyl transfer; however, in the methyltransferase OxaC, the substrate meleagrin (95) (tan) is located in between SFG and the His313-Glu369 dyad.In contrast, the catalytic histidine is substituted with Arg295 in LepI such that deprotonation cannot occur.Green ribbons in LepI are residues from the second monomeric peptide.(D) OxaC catalyzed methylation during formation of oxaline (96).

Review
Natural Product Reports isotope effects (KIE) suggested that both non-enzymatic and SpnF-catalyzed cyclization likely proceed via a stepwise rather than a concerted mechanism, 178 in agreement with computational studies. 179,180In contrast to LepI, however, active SpnF copuries with SAH rather than SAM. 181The crystal structure of the SpnF-SAH binary complex was solved at 1.50 Å (Fig. 21B), and computational docking of substrate or product into the hydrophobic cavity of SpnF implied that neither substrate nor product exhibits direct contact with the SAH moiety. 181Therefore, SAH may serve as a component necessary for the integrity of the SpnF structure, and the positive charge of the sulfonium ion of SAM is less likely to be essential for catalysis. 181ollowing SpnF catalyzed [4 + 2] cycloaddition and SpnG catalyzed glycosylation, 182 SpnL catalyzes the transannular carbon-carbon bond formation between C3 and C14 of 103 to generate 108 (Fig. 22). 177Two possible mechanisms have been postulated, the rst involving deprotonation at C12 to form an enolate (104) poised for Michael addition at C3 giving 105.Alternatively, a nucleophilic residue in the active site may rst add to C13 to yield intermediate 106, which may then cyclize into 107 prior to elimination of the assisting nucleophile.Involvement of a nucleophilic residue during modication of the b-carbon of an enone moiety is indicative of a Rauhut-Currier (RC) reaction 146,183 and is reminiscent of thymidylate synthase chemistry. 184When the SpnL reaction was conducted in buffered D 2 O, only a single solvent deuterium was incorporated into the product; however, no signicant primary KIE was observed with the C12-deuterated substrate. 185Moreover, a C13-uorinated substrate analog 109 led to inactivation of SpnL via the formation of a covalent enzyme-substrate adduct (112)  demonstrated by mass spectrometry (Fig. 23A). 185Thus, all the biochemical evidence is consistent with an RC mechanism.SpnL shares 35% sequence identity with SpnF and also cop-uries with bound SAH. 185The binary complex of SpnL with SAH bound was solved at 3.05 Å (Fig. 21C) and demonstrated that Cys60 resides nearby the bound SAH with its thiol moiety buried and apparently inaccessible from the active site cavity (Fig. 23B). 185Consequently, a conformational change upon  Natural Product Reports Review substrate binding was proposed to allow for Cys60 exposure to the substrate thereby facilitating catalysis.
During biosynthesis of the fungal secondary metabolite ilicicolin H (113), the cyclohexene moiety is believed to be constructed via an inverse-electron demand Diels-Alder (DA) reaction catalyzed by IccD, which harbors an intact SAMbinding motif yet does not copurify with SAM (Fig. 24A). 186oreover, two additional groups of methyltransferase-like proteins were also found to catalyze pericyclic reactions.EpiI, UpiI and HpiI are proposed to catalyze a hetero-DA reaction yielding 119 on the quinone methide substrate (117) generated upon dehydration of 116, while PdxI, AdxI and ModxI catalyze conversion of the same substrate to 121 via a proposed Alderene reaction (Fig. 24B). 187The catalytic cycles of these pericyclases, however, do not depend on either SAM or SAH indicating they are mechanistically distinct from those of LepI or SpnF and SpnL. 186,187][190] 2.4.3SlnM in salinomycin biosynthesis.Salinomycin ( 122) is a broadly utilized coccidiostat featuring a bis-spiro tricyclic structure with a polyether linkage. 191Its derivatives have recently drawn attention in the eld of oncology due to their antitumor potential. 192,193Within the biosynthetic gene cluster, SlnM is annotated as a SAM-dependent O-methyltransferase containing a glycine-rich motif canonical for SAM binding. 194,195ene inactivation of SlnM results in the accumulation of a ringopened intermediate (123) suggesting that SlnM catalyzes the dehydration and subsequent spirocyclization of the putative precursor species 124 (Fig. 25). 196The presence of SAM is essential for SlnM activity, though it is not consumed over the course of the reaction, and SAM can be replaced by its isosteric analog sinefungin (94) but not SAH. 196Consequently, SAM likely serves to both help maintain the structural integrity of SlnM and also contribute directly to catalysis in some manner that requires the positively charged sulfonium moiety.On the basis of sequence alignment, mutational studies and computational modelling, Asp58 and Asp186 were identied as catalytically important residues, with one presumably serving as the general acid that facilitates the dehydration reaction. 196It has therefore been hypothesized that the sulfonium of SAM facilitates elimination of water by stabilizing the negative charge buildup on the aspartate residues as they protonate the leaving group (Fig. 25). 196However, the exact positioning between SAM, substrate and these Asp residues has yet to be fully established due to the lack of a crystal structure.
2.4.4SAM-dependent hydroxylase.The rst example of a SAM-dependent hydroxylase is RdmB, which catalyzes decarboxylative hydroxylation of 15-demethoxy-3-rhodomycin (125) to form the anthracycline precursor b-rhodomycin (126). 1978][199][200][201] These substrates all feature a carboxylate moiety at C10, which upon decarboxylation forms the anion 128 that is stabilized by extended conjugation.Moreover, sinefungin (94) but not SAH ( 3) is able to support hydroxylase activity, which led to the hypothesis that electrostatic stabilization of the anionic intermediate 128 by SAM is essential for catalysis. 200The intermediate 129 can then activate molecular oxygen to yield the peroxide 130.In the presence of reduced glutathione (GSH), 130 is converted to the hydroxylation product 131.
There is reason to believe that RdmB may have diverged from an evolutionary lineage of SAM-dependent O-methyltransferases upon losing methyltransferase activity.DnrK is a homolog of RdmB (52% sequence identity) that catalyzes methylation at O-4 of carminomycin (133) yielding

Review
Natural Product Reports daunorubicin ( 134), 202 which is a powerful chemotherapeutic agent and close structural analog of b-rhodomycin (126) (Fig. 26A). 203Comparison of the crystal structures of RdmB and DnrK demonstrated subtle changes consistent with the divergence in catalytic activities.Thus, the crystal structure of DnrK bound with the methylation product 4-methoxy-3-rhodomycin T (136, Fig. 26C) and SAH shows alignment between the acceptor hydroxyl group, the transferred methyl and the sulfur of SAH as expected for methyltransferase activity (Fig. 27A). 204In contrast, the ternary complex of RdmB bound with SAM and the hydroxylation product 11-deoxy-b-rhodomycin A (DbrA, 131 ′ ) revealed an O-C-S angle of 122.9°between the SAM sulfonium and the corresponding hydroxyl oxygen of 131 ′ suggesting misalignment for efficient nucleophilic displacement (Fig. 27A). 200ecent structural comparisons of RdmB and DnrK coupled with extensive mutational studies by Grocholski and co-workers have provided even more insight into this question. 205The Michaelis complex of RdmB is solvent inaccessible, 200 which prevents protonation of the anionic intermediate 129 by water.Closure of the active site is facilitated by a loop that includes Phe300 and blocks the channel connecting the substrate binding site to the external media (Fig. 27C). 205In contrast, the corresponding Phe296 in DnrK faces away from the channel leaving the active site of DnrK solvent accessible (Fig. 27D).Notably, DnrK catalyzes the conversion of 127 to 132 in vitro (Fig. 26B). 205Although the timing of methylation at C4-OH is unclear, this observation is consistent with the hypothesis that upon decarboxylation at C15, the resulting anion (i.e., 129) is quenched by water in the DnrK active site. 205Moreover, the different orientation of Phe300 in RdmB and Phe296 in DnrK is due to Ser301 in RdmB, which is missing in the primary sequence of DnrK 205 (Fig. 27B); however, Phe296 in DnrK can be rotated towards the channel thus blocking solvent access by insertion of a serine residue between Phe296 and Thr297, which mimics the Ser301 in RdmB (Fig. 27B). 205As expected, this DnrK insertion mutant (DnrK-Ser) did exhibit hydroxylase activity, implying the importance of solvent extrusion to support hydroxylation activity.

Natural Product Reports Review
Human complex I is a respiratory chain protein that drives proton pumping for ATP synthesis by mediating electron transfer between NADH and ubiquinone. 206,207NDUFAF5 is an essential protein in the biogenesis of human complex I and is characterized by a conserved SAM-binding motif at the Cterminus.Suppression of NDUFAF5 expression results in reduced hydroxylation of an arginine residue in the protein NDUFS7, which is a subunit in human complex I. 208 Thus, NDUFAF5 has been hypothesized to be an arginine hydroxylase targeting NDUFS7 (137 / 138) (Fig. 28).The secondary structure of NDUFAF5 is predicted to be similar to that of RdmB in both the substrate and SAM binding regions indicating that NDUFAF5 might adopt an active site structure similar to that of RdmB. 208While the in vivo activity of NDUFAF5 has been correlated with arginine hydroxylation, the in vitro activity and mechanistic details regarding whether the hydroxylase activity is SAM or SAH dependent and how the arginine is activated for oxidation remains unclear.
2.4.5 SAM-dependent decarboxylase.Brevione E ( 139) is a hexacyclic meroditerpenoid demonstrating allelopathic activity. 209The biosynthesis of brevione E involves BrvO, which is annotated as a SAM-dependent methyltransferase and proposed to catalyze the decarboxylation of 140/141 to 145 (Fig. 29). 210Decarboxylation of 140/141 can be accelerated in the presence of BrvO and SAM.The mechanism is hypothesized to involve initial rearrangement of the 2 ′ ,3 ′ -alkene in 140/141 to the 3 ′ ,4 ′ -alkene in 143 prior to decarboxylation and generation of the oxyanionic intermediate 144.It is possible that SAM participates by helping to stabilize the negatively charged intermediate (144) via electrostatic interactions similar to the role it has been proposed to play during the catalytic cycles of SlnM and RdmB. 196,200However, the characterization of BrvO is currently incomplete including whether SAH and SFG can also support BrvO activity.Consequently, further studies will be required to understand the mechanism of this enzyme.
2.5 SAM as a nucleophile 2.5.1 Carboxy-SAM.In Section 2.1.3,the formation of 5 ′carboxymethyloxyuridine (28) residues at the wobble position of tRNA catalyzed by CmoB was discussed as an example of an alkylation reaction involving carboxy-SAM (Cx-SAM, 29) as the alkyl donor. 86However, the biosynthesis of Cx-SAM itself requires carboxylation of the SAM methyl group catalyzed by CmoA with prephenate (146) being the carboxylate donor. 86moA homologs have also been found in a broad range of organisms and natural product biosynthetic pathways including 3-thiaglutamate (31) in Pseudomonas (TglE) 91 as well as microcin C (34) in Bacillus (MccS) 92 (Fig. 8, Section 2.1.3).The CmoA catalyzed reaction was proposed to involve the nucleophilic addition of a sulfonium methylide (148), generated from a-deprotonation of SAM, to the carbon dioxide liberated upon decarboxylative dehydration of prephenate (Fig. 30A). 86Therefore, the CmoA catalyzed reaction differs from other SAMdependent alkylation reactions in that the methyl substituent on the sulfonium moiety is acting as a nucleophile rather than an electrophile.
The rst half of the CmoA catalyzed reaction is hypothesized to proceed analogous to that of prephenate dehydratase, where decarboxylation of prephenate facilitates elimination of a hydroxide ion thereby generating CO 2 and phenylpyruvate (147). 211In the catalytic cycle of prephenate dehydratase, the eliminated hydroxide is believed to abstract a proton from an active site threonine residue thereby completing the reaction. 211s the pK a of the threonine hydroxyl is close to that measured for the trimethylsulfonium ion, 212 the same hydroxide is expected to be competent for deprotonating the methyl of SAM.The resulting sulfonium methylide (148) can then add to the eliminated molecule of CO 2 to yield Cx-SAM (29).Moreover, the X-ray crystal structure of Cx-SAM-bound CmoA from E. coli revealed an overall Rossmann fold 86,87 and a cavity adjacent to the Cx-SAM binding site that was predicted to be the prephenate (146) binding site by molecular docking. 86Notably, the region of the active site adjacent to the eliminated prephenate hydroxide is lined primarily with hydrophobic residues leaving the methyl group of SAM the only available proton source nearby (Fig. 30B). 86omputational substrate docking has also suggested an arrangement of the prephenate hydroxyl and SAM methyl that disallows a typical methylation reaction. 86This is consistent with the observation that CmoA does not catalyze O-methylation O/H-C hydrogen bond between the prephenate hydroxyl group and the SAM methyl, which is more consistent with the proposed deprotonation reaction. 86Formation of the putative sulfonium methylide ( 148) was also supported by the observed exchange of only a single deuterium from CD 3 -labelled SAM with solvent under turnover conditions.4][215][216] This would address the large difference in pK a between water and sulfonium a-carbons measured in polar solvents. 212,217Likewise, a hydrophobic substrate binding site has also been proposed in the case of Ecm18 to lower the pK a value of a sulfonium intermediate in assisting the formation of a sulfur ylide (80) (Section 2.3.3). 159hile current evidence points to an intermediary sulfonium ylide (148), the reaction could also occur in a concerted manner avoiding the formation of a discrete ylide intermediate (Fig. 30A, route b).2.5.2Queuosine.Apart from CmoA, formation of a sulfonium ylide derived from SAM has also been proposed in the catalytic mechanism of QueA from queuosine (149) biosynthesis. 218,219QueA catalyzes transfer and isomerization of the ribose moiety of SAM to a modied guanidine base on tRNA (150).The mechanism was proposed to proceed via an initial depurination triggered by deprotonation (1 / 151).Subsequent addition of the substrate amine to the vinyl sulfonium (151) yields sulfur ylide intermediate (152), which can then undergo a Corey-Chaykovsky reaction to give the epoxide product 154 and eliminate methionine (Fig. 31). 218The proposed mechanism of QueA is also consistent with kinetic studies showing ordered binding of tRNA followed by SAM to QueA, whereas the order of product release is adenine, methionine and then 154. 220owever, the only other experimental investigations of QueA catalysis have involved crystallographic studies of the Thermotoga maritima and Bacillus subtilis enzymes, both structures of which lack bound substrates. 221,222Thus, it remains unclear precisely how either tRNA or SAM binds to the enzyme.While the detailed mechanism remains to be explored, QueA represents another potential example of sulfur ylide formation during an enzymatic reaction, which would further expand the chemistry of SAM from an exclusive electrophile to a nucleophile as well.

SAM as an amino donor
SAM is also recognized as an amino donor; however, only two instances of such enzymology have been reported.BioA depends on PLP and catalyzes the oxidative deamination of Fig. 30 (A) Proposed mechanism for CmoA catalysis.(B) Clip view of Cx-SAM/CmoA complex structure (PDB ID: 4GEK) highlighting the inner cavity for Cx-SAM binding.Computational substrate docking models have suggested that SAM adopts a similar binding configuration as that of the product Cx-SAM.Prephenate ( 146) is predicted to bind in the hydrophobic pocket adjacent to SAM (green arrow).In the zoomed in region, the surface coloring indicates hydrophobicity (hydrophobic in gold and hydrophilic in cyan).The red arrow indicates the position of the methyl group of SAM, which is surrounded by a hydrophobic environment.Natural Product Reports Review SAM to its a-keto acid congener while reductively aminating 7keto-8-aminopelargonic acid (KAPA, 156) to 7,8-diaminopelargonic acid (DAPA, 157) during the biosynthesis of biotin (155) (Fig. 32A). 223,224The mechanism of BioA is thus no different from other PLP transaminases, 225 despite SAM serving as the amine donor rather than a free amino acid.In contrast, the second example appears to be unrelated to PLP chemistry and contributes to the biosynthesis of rhodoquinone in Rhodospirillum rubrum. 226hodoquinone (159) differs structurally from ubiquinone (160) by replacing an O-methoxy group with an amine in a reaction that requires the enzyme RquA. 227,228RquA was recently characterized as a manganese-dependent enzyme that catalyzes transfer of the a-amino group from SAM to ubiquinone (160) to yield rhodoquinone (159). 229While RquA resembles SAM-dependent methyltransferases, only a subset of the conserved residues in the predicted Rossmann fold generally associated with SAM binding are required for activity (Fig. 32B). 227,229In addition to a divalent metal ion, RquA catalysis also depends on aerobic conditions as well and is expected to involve a redox component.Indeed, isotope labelling experiments have suggested the oxidative decomposition of SAM to CO 2 , MTA and an aldehyde fragment during the transformation. 229Mechanistic hypotheses for this transformation are shown in Fig. 32C and D; however, much work is still le to be done before the chemistry of this unique enzyme is fully understood.

Enzymes without a typical MT-fold
As discussed in the previous sections, evolutionary diversication of SAM-dependent methyltransferases has led to the emergence of several different types of chemistry.However, there also exist groups of SAM utilizing enzymes that lack the MT-fold making their evolutionary relationship with the SAMdependent methyltransferases less clear.These enzymes also show a broad range of different activities again emphasizing the mechanistic versatility and intrinsic reactivity of SAM as a biological substrate.
3.1 Pyridoxal 5 ′ -phosphate-dependent enzymes 3.1.1Cyclopropylation.The sulfonium moiety of SAM represents a good leaving group for substitution reactions thereby explaining its prevalence in various biological alkylation reactions.However, this feature of SAM also suggests that it may be susceptible to intramolecular cyclization reactions.Indeed, under alkaline conditions, SAM undergoes nonenzymatic decomposition to yield methylthioadenosine (MTA, 4) and homoserine lactone (44) as one of its main decomposition products. 108,1092][233] This difficulty with deprotonation has been overcome in the case of plant ACC synthase by acidifying C1 via formation of an external aldimine with PLP (167). 234eprotonation at C1 thus creates a highly conjugated quinonoid (168) and is favorable (Fig. 33).The reaction mechanism of plant ACC synthase has been extensively studied and reviewed; 235 however, the existence of a bacterial counterpart had been previously unknown despite a number of

Review
7][238] Therefore, it is of interest to know whether similar chemistry is found in bacteria.
Recently, guangnanmycin (GNM, 172), which is a structural homolog of the antitumor antibiotic leinamycin (LNM), was discovered through genome mining. 239Apart from a hybrid peptide-polyketide backbone, GNM also features an unusual ACC moiety (Fig. 34). 239Comparative analysis of lnm-type biosynthetic gene clusters between guangnanmycin producers and non-producers revealed that the gene product of gnmY may catalyze the formation of ACC from SAM.This functional assignment was then validated by in vivo inactivation of gnmY, which rendered the mutant strain unable to produce guangnanmycin unless exogenous ACC is supplemented. 240Sequence analysis revealed that GnmY belongs to the aspartate aminotransferase superfamily, and subsequent in vitro characterizations conrmed that GnmY catalyzes the conversion of SAM to ACC in a PLP-dependent manner. 240GnmY also exhibits steady-state kinetic parameters comparable to those of plant ACC synthases and utilizes a catalytic lysine residue (Lys251) to form an internal aldimine (166) with PLP during catalysis. 240Nevertheless, bioinformatics analyses have suggested that GnmY is phylogenetically distinct from the family of plant ACC synthases despite sharing a similar mechanism of catalysis. 240omologs of GnmY have also been found in the putative biosynthetic gene clusters for two other peptide antibiotics, SW-163C (173) and Q6402A (174), 157,241 which are each decorated with an unusual 2-methyl-ACC (MeACC, 170) moiety (Fig. 34). 242,243In the biosynthetic gene cluster of Q6402A, the gene product of orf30 shows homology with GnmY suggesting that its activity may involve the formation of MeACC (170). 241nitial characterization of puried Orf30 revealed its ability to catalyze the conversion of SAM to ACC. 241 However, Orf30 exhibits a 1900-fold lower value of k cat compared to GnmY such that SAM may not be the native substrate of Orf30. 241The B 12dependent radical SAM methylase Orf29 encoded by a gene fragment upstream of Orf30 was later demonstrated to catalyze methylation of SAM yielding (4 ′′ R)-4 ′′ -methyl-SAM (171). 244This SAM derivative ( 171) can be efficiently cyclized to give MeACC (170) under the action of Orf30 (Fig. 33). 244It remains to be established how Orf30 distinguishes between SAM and methylated-SAM.
3.1.2Elimination.The muraymycins (175), sphaerimicin A (177) and caprazamycins/liposidomycins (178) are peptidyl nucleoside natural products that inhibit bacterial cell wall assembly. 245,246These antibiotics are structurally characterized by a unique disaccharide core consisting of 5 ′′ -amino-5 ′ ′ -deoxyribose (ADR) and 5 ′ -C-glycyluridine (GlyU) sugar residues (Fig. 35). 247These nucleoside antibiotic families are distinguished, however, by highly diverse appendages at the amino acid end of GlyU.Feeding experiments with a variety of amino acids enriched with stable isotopes revealed that the aminopropyl linker in muraymycin D1 ( 176) is derived from L-Met (Fig. 35). 2480][251][252] These clusters instead encode a PLP-dependent enzyme (e.g., Mur24, Cpz13, LipJ, SphL) with a sequence similar to enzymes annotated as ACC synthases also suggesting SAM as the precursor. 248urication and in vitro characterization of Mur24 and its homologs LipJ 250 and SphL 252 indicated that all three enzymes do indeed catalyze ACP transfer from SAM to the substrate (182) amine coupled with the elimination of MTA (Fig. 36). 248To investigate the role of PLP in this reaction, the conserved lysine residue necessary for internal aldimine formation with PLP was mutated leading to a complete loss of ACP transfer activity. 248he catalytic mechanism of Mur24 was further studied using SAM isotopologs, the results of which provided evidence that two protons are washed out in the product (183) including the C-a proton and either the C-b or C-g proton. 248Furthermore, reactions run in D 2 O led to a mixture of both single and double solvent deuterium incorporation into the product 183. 248These observations implied sequential and reversible deprotonation at C-a and C-b leading to elimination of MTA following SAM-PLP aldimine (167) formation.The unsaturated iminium intermediate, or vinylglycine-PLP aldamine (181), thereby generated then undergoes aza-Michael addition to yield the Nalkylated product 183 (Fig. 36).

Review
Natural Product Reports biosynthetic origin of the C4 moiety on the ve-member ring is likely derived from aspartate and the six-member ring from a nicotinamide containing nucleotide. 256Extensive substrate screening corroborated SAM and b-nicotinamide adenine dinucleotide (b-NAD, 187) as the native substrates of SbzP (Fig. 37). 256Steady-state kinetic analysis implied a ping-pong Bi-Bi mechanism.Upon incubation of SbzP with SAM, MTA is released with concomitant formation of an enzyme-bound intermediate prior to NAD binding and subsequent formation of the annulation product. 256lthough the SbzP catalyzed fragmentation of SAM appears similar to that of Mur24 at the point of generating 168, the second half reactions catalyzed by Mur24 and SbzP are distinct.While the unsaturated iminium (181) generated during the Mur24 catalytic cycle is connected to PLP as an aldamine and undergoes alkylation by an incoming nucleophile, during SbzP catalysis, the vinyl glycine iminium 188 instead links to a quinonoid form of PLP and serves to increase the nucleophilicity of C-g facilitating its addition to the electrophilic nicotinamide ring of NAD (188 / 189) (Fig. 37).Addition of the active site lysine residue covalently links the intermediate 189 with the enzyme as a gem-diamine 190 and facilitates rearrangement to the bicyclic structure 192.Spectroscopic analysis of SbzP revealed that addition of SAM results in a unique absorption at 520 nm consistent with the formation of a b,gunsaturated quinonoid (188) with an extended p-conjugation as predicted. 256An analogous spectroscopic characterization of Mur24 is presently unavailable; however, it would also help provide additional mechanistic insight into how the PLP cofactor participates in reactions with opposite electronic demands.
The enzymes described above employ a SAM-PLP quinonoid intermediate (168) that can react via several different pathways eliminating MTA to either yield an unsaturated iminium or induce an intramolecular cyclization.In the former case, the iminium is susceptible to both electrophilic and nucleophilic addition.Moreover, electrophilic addition can occur at either C-a or C-g, with the latter exemplied by SbzP.In contrast, attack at C-a is operative in the enzyme CqsA.Initial study of CqsA (PDB ID: 2WK8) revealed its structural resemblance with other PLP-dependent enzymes 257,258 and its activity towards different amino acids.However, SAM was later identied as the biosynthetic CqsA substrate to yield EA-CAI-1 (194), which is the precursor to the quorum sensing autoinducer CAI-1 (193)  (Fig. 38). 257CAI-1-type autoinducers are produced by Vibrio cholerae, which is the bacterium that causes the life-threatening disease cholera. 259The net transformation of SAM to EA-CAI-1 (194) catalyzed by CqsA involves C-S bond cleavage at C-g, C-C bond formation at C-a with a fatty acyl-CoA (195) and decarboxylation.The catalytic cycle appears to begin with formation of a PLP-SAM adduct that is converted to a quinonoid intermediate (188) between vinyl glycine and PLP following elimination of MTA as in the case of SbzP.CqsA catalysis then proceeds with nucleophilic addition of the a-carbon to the incoming acyl-CoA substrate (195) before subsequent decarboxylation (196 / 197).The resulting EA-CAI-1 (194) is then converted to CAI-1 (193) by subsequent enzymes in vivo. 260While the aforementioned transformations exploit the nucleofugality of the sulfonium moiety of SAM, they are not direct S N 2-type substitution reactions and may also be considered within the context of exotic PLP chemistry, which has been reviewed recently. 2612 Polyketide synthase 3.2.1 Cyclopropylation.Colibactin (199) is a genotoxic peptide-polyketide metabolite isolated from E. coli featuring a cyclopropane ring, which is believed to be responsible for its DNA alkylation activity.[262][263][264] Unlike plant ACC synthases and GnmY, which catalyze the conversion of SAM to free ACC (46), 235,240 the cyclopropane ring in colibactin is constructed by  Natural Product Reports Review cyclizing SAM as a component residue during peptide-polyketide chain assembly.265 SAM is rst loaded onto a peptidyl carrier protein domain in the NRPS protein ClbH followed by a condensation step, which transfers the acyl chain from the upstream PKS (ClbC, 200) to the amino group of ClbH-SAM (201) to yield intermediate 202 (Fig. 39).265 ClbI, which is annotated as a PKS enzyme, then catalyzes cyclization of the tethered SAM-acyl chain.Sequence alignment reveals that an otherwise conserved cysteine residue in the ketosynthase (KS) domain of ClbI is replaced by a serine (Ser178), and mutation of this residue to alanine abolishes the activity of ClbI.265 It was therefore hypothesized that the serine could act as a general base to deprotonate the a-proton (Fig. 39, mechanism a).265 The generated carbanion can then undergo 3-exo-tet cyclization and eliminate MTA (202 / 203). Alternaively, serine can also serve as a nucleophile to displace MTA thereby forming an enzymesubstrate adduct 204 (Fig. 39, mechanism b) prior to deprotonation induced cyclization.265 As the crystal structure of ClbI or ClbI with SAM-loaded ClbH is currently unavailable, the precise role of the Ser178 and how the non-acidic proton at C-a in 202 is deprotonated is presently uncertain.

Conclusions
SAM is ranked among the most frequently observed substrates in enzymatic transformations. 18The majority of known SAM dependent biological reactions involve methylation; however, regioselective transfer of all three alkyl substituents of the SAM sulfonium have been reported (Sections 2.1 and 2.2) and may represent components of more complex biosynthetic cascades (Section 2.3).In addition to alkyl transfer, several other types of SAM-dependent reactions catalyzed by enzymes have been established including a-deprotonation with the potential for ylide formation (Section 2.5) as well as b-elimination concomitant with elimination of a neutral disulde (Section 3).In all cases, these enzyme catalyzed reactions conform to the well-known chemistry of the sulfonium cation, such that the role of the enzyme is to selectively stabilize those transition states representing one mode of reaction over another.Structural investigations of these enzymes have been critical in dening these constraints thereby explaining both the modes of catalysis as well as the observed regiochemistry underlying these various enzyme catalyzed reactions.Moreover, the principles underlying sulfonium enzymology are not limited to SAM-dependent enzymes alone and have also been appreciated among the dimethylsulfoniopropionate (DMSP) lyases found in marine bacteria 266 as well as BurG during the biosynthesis of malleicyprols. 267,268any aspects of SAM enzymology, however, remain to be explored.In particular, several enzymes as described in Section 2.4 require SAM even though the catalytic cycles of these enzymes do not appear to involve any sort of covalent rearrangement or fragmentation of SAM itself.In these instances, SAM may be required for proper folding and structural integrity of the enzyme, 269 or it may play a more direct role in catalysis by modifying the structural and electrostatic features of the active site. 270,271ny of the SAM-dependent enzymes also exhibit structural features that imply homology with the SAM methyltransferases, whereas others do not, suggesting different evolutionary lineages.Consequently, how these enzymes diversied in some cases from a common evolutionary origin to exhibit the broad range of biochemical activities and unique catalytic roles of SAM seen today is yet another question that remains open to investigation.In any case, SAM has become a focal point of modern enzymology that will likely bring many new discoveries and surprises in the years to come.
This journal is © The Royal Society of Chemistry 2023 Natural Product Reports Review Ren was born in Nanjing, China.He received his bachelor's degree from Nanjing University in 2016.He then joined Prof. Hung-wen Liu's group at the University of Texas at Austin as a graduate student.His graduate research has focused on the biosynthesis of Cnucleoside natural products and elucidating the mechanisms of enzymatic reactions.Hung-wen (Ben) Liu was born in Taipei (Taiwan).He completed his PhD degree with Prof. Koji Nakanishi from Columbia University in natural product chemistry and continued his postdoctoral research with Prof. Christopher Walsh at MIT.He started his independent career in the chemistry department at the University of Minnesota in 1984 and moved to University of Texas at Austin in 2000.His most recent research focuses on atypical sugar biosynthesis, radical SAM enzymology and mechanistic investigations of enzyme catalyzed transformations during natural product biosynthesis.

Fig. 1
Fig. 1 Distinct uses of SAM in enzymatic reactions.
Two enzymes have been proposed to catalyze aminopropylation including a SAM-dependent methyltransferase MccD (PDB ID: 5FCD) and the N-terminal pyridoxal 5 ′ -phosphate (PLP)-dependent decarboxylase domain of MccE (Fig. 5, route a). 56In vitro characterization of the two enzymes initially suggested that aminopropylation may proceed with ACP transfer to McC 1120 (16) catalyzed by MccD followed by decarboxylation of the alkylated intermediate McC 1221 (17) catalyzed by MccE. 57However, an alternative route similar to polyamine biosynthesis, where decarboxy-SAM (6) is the aminopropyl donor such that MccE and MccD operate in the reversed order (Fig. 5, route b), has not been completely ruled out.Moreover, the activity of

Fig. 5
Fig. 5 Aminopropylation during maturation of microcin C (15).MccD can catalyze transfer of an ACP group onto McC 1120 (16), which can further be decarboxylated in a reaction catalyzed by MccE (route a).An alternative pathway starts with the formation of dc-SAM catalyzed by MccE followed by MccD mediated aminopropyl transfer (route b).

Fig. 6
Fig. 6 Other natural products with ACP groups derived from SAM.

Fig. 7
Fig. 7 Enzymatic functions of (A) Tsr3 and (B) its closest homolog Trm10.Tsr3 catalyzes ACP modification of a pseudouridine residue whereas Trm10 catalyzes methylation of a guanosine or adenosine residue.(C) Structure of wybutosine highlighting the ACP moiety transferred under the action of Tyw2.Solvent accessible area of (D) Tsr3 (PDB ID: 5APG) and (E) Trm10 (PDB ID: 4JWF) is shown colored with the most hydrophilic residues in cyan to the most hydrophobic in gold.In Tsr3, the ACP side chain of Se-SAM (pink) is exposed to solvent and stabilized by hydrophilic interactions.Asp70, Ser72 and Trp73 in the DTW domain are in purple.In contrast, the ACP side chain of SAH (pink) in Trm10 is buried, and the entrance to the binding cavity is lined with hydrophobic residues.(F) Enzymatic function of TapT.(G) Sequence alignment of Tsr3 and TapT at the DTW domain (gray) (Ec: Escherichia coli; Vp: Variovorax paradoxus; Cs: Clostridium saccharoperbutylacetonicum; Vd: Vulcanisaeta distributa; Ss: Sulfolobus solfataricus; Hs: Homo sapiens; Sc: Saccharomyces cerevisiae).

Fig. 8 (
Fig. 8 (A) Enzymatic functions of CmoA and CmoB.Two CmoA homologs TglE and MccS Bam also catalyze the formation of 29.(B) Crystal structure of Cx-SAM bound to CmoB (PDB ID: 4QNV).The carboxymethyl moiety of Cx-SAM is coordinated by Tyr200, Lys91 and Arg315.The unique 100 residues at the N-terminus of CmoB are shown in green.

Fig. 14
Fig. 14 The proposed two enzyme cascade for C10P and C10Q catalyzed formation of the cyclopropane ring during biosynthesis of CC-1065 (51) and related compounds (52 & 53).

Fig. 18 (
Fig. 18 (A) Mechanism of Ecm18 catalyzed rearrangement of a disulfide bridge to a thioacetal.(B) Hydrophobic surface of the Ecm18 substrate binding pocket (PDB ID: 4NEC) colored according to most hydrophobic (gold) and most hydrophilic (cyan) residues.(C) Ecm18/ 76/SAH ternary complex.The distance between the sulfur of SAH and the methyl group in 76 is consistent with the proposed methyltransfer reaction.The basic nitrogen on His115 (cyan) is near the disulfide bridge and could facilitate ylide 80 formation via deprotonation of the methylated sulfonium intermediate 79.
Fig.20(A) LepI catalyzed dehydration-cyclization cascade during leporin C biosynthesis.Although the stereochemistry at C7 in 91 is currently unclear, it has been shown that the diastereomer of 91 is not accepted by LepI.Comparison of crystal structures of (B) LepI/SAM/89 complex (PDB ID: 6IX9) and (C) OxaC/SFG/95 complex (PDB ID: 5W7S).In LepI, SAM is too far from the product (89, in tan) for methyl transfer; however, in the methyltransferase OxaC, the substrate meleagrin (95) (tan) is located in between SFG and the His313-Glu369 dyad.In contrast, the catalytic histidine is substituted with Arg295 in LepI such that deprotonation cannot occur.Green ribbons in LepI are residues from the second monomeric peptide.(D) OxaC catalyzed methylation during formation of oxaline(96).(E) Structure of sinefungin(94).(F) Cyclization of 98 catalyzed by FinI.(G) Sequence alignment of LepI, FinI and OxaC highlighting the conserved SAM binding arginine residue (Arg310) that interacts with SAM, the catalytic base (His313) in OxaC (in gray boxes) and the essential arginine (Arg295/Arg313) in the pericyclases in red.

Fig. 21
Fig. 21 Crystal structures of (A) LepI (PDB ID: 6IX5), (B) SpnF (PDB ID: 4PNE) and (C) SpnL (PDB ID: 7V6H).Each enzyme contains a Cterminal Rossmann fold catalytic domain containing seven b-strands (cyan) surrounded by five a-helices (blue), while the N-terminal domains differ between each protein.The cofactor, SAM (1) for LepI and SAH (3) for SpnF and SpnL (pink with a gray surface), binds similarly at the C-terminal domain of all three enzymes.The substrate analog (90) bound in the LepI active site is shown with a green surface.

Fig. 22
Fig. 22 Biosynthesis of spinosyn A highlighting the [4 + 2] cycloaddition catalyzed by SpnF and two proposed mechanisms for the transannulation catalyzed by SpnL.

Fig. 23 (
Fig. 23 (A) C13-fluorinated substrate (109) leads to covalent modification of SpnL.(B) Crystal structure of SpnL/SAH binary complex (PDB ID: 7V6H) highlighting the SAH (3) binding site and the catalytic Cys60 (carbons as cyan and sulfur as yellow spheres), which is buried in the protein interior.

Fig. 25
Fig. 25 SlnM catalyzes spirocyclization during salinomycin biosynthesis.SAM is proposed to stabilize an anionic aspartate residue via electrostatic interactions to facilitate substrate protonation.

Fig. 27 (
Fig. 27 (A) Comparison of the binding of SAM (light green)/131 ′ (green) in RdmB and SAH (salmon)/136 (pink) in DnrK.(B) Sequence alignment of DnrK and RdmB at the a-helix that contains the aromatic residue, highlighting the position of serine insertion in DnrK-Ser.The orientation of Phe296 in DnrK or Phe300 in RdmB dictates the catalytic activity being methylation or hydroxylation.(C) Clip view of the RdmB/ SAM/131 ′ complex (PDB ID: 1XDS).(D) Clip view of the DnrK/SAH/136 complex (PDB ID: 1TW2).The channel to the bulk solvent is blocked in RdmB due to the presence of Phe300 (cyan), whereas it is opened in DnrK.

Fig. 28 Fig. 29
Fig. 28 NDUFAF5 catalyzes modification of the substrate protein NDUFS7 at Arg73.The precise site of hydroxylation is unknown.

Fig. 32 (
Fig. 32 (A) Transamination of SAM catalyzed by BioA.(B) Sequence alignment of RquA with related functional methyltransferases.RquA has two essential aspartate residues (Asp118 and Asp143, numbers in blue) for SAM binding whereas the DXXXGXG (red box) motif typically found in Rossmann fold MTs is missing in RquA.(C and D) Two proposed mechanisms for RquA catalyzed transamination during the biosynthesis of rhodoquinone (159).

Fig. 35
Fig. 35 Natural products that contain the ADR-GlyU disaccharide.The bold alkyl chain indicates the putative methionine-derived aminopropyl moiety.

Fig. 39
Fig. 39 Proposed mechanism for the ClbI catalyzed cyclopropylation during the formation of colibactin biosynthetic intermediate 203.