Cleavage Of S-adenosylmethionine Research Articles

DPH1 Gene Mutations Identify a Candidate SAM Pocket in Radical Enzyme Dph1•Dph2 for Diphthamide Synthesis on EF2.

In eukaryotes, the Dph1•Dph2 dimer is a non-canonical radical SAM enzyme. Using iron-sulfur (FeS) clusters, it cleaves the cosubstrate S-adenosyl-methionine (SAM) to form a 3-amino-3-carboxy-propyl (ACP) radical for the synthesis of diphthamide. The latter decorates a histidine residue on elongation factor 2 (EF2) conserved from archaea to yeast and humans and is important for accurate mRNA translation and protein synthesis. Guided by evidence from archaeal orthologues, we searched for a putative SAM-binding pocket in Dph1•Dph2 from Saccharomyces cerevisiae. We predict an SAM-binding pocket near the FeS cluster domain that is conserved across eukaryotes in Dph1 but not Dph2. Site-directed DPH1 mutagenesis and functional characterization through assay diagnostics for the loss of diphthamide reveal that the SAM pocket is essential for synthesis of the décor on EF2 in vivo. Further evidence from structural modeling suggests particularly critical residues close to the methionine moiety of SAM. Presumably, they facilitate a geometry specific for SAM cleavage and ACP radical formation that distinguishes Dph1•Dph2 from classical radical SAM enzymes, which generate canonical 5'-deoxyadenosyl (dAdo) radicals.

Phage T3 overcomes the BREX defense through SAM cleavage and inhibition of SAM synthesis by SAM lyase

Bacteriophage T3 encodes a SAMase that, through cleavage of S-adenosyl methionine (SAM), circumvents the SAM-dependent type I restriction-modification (R-M) defense. We show that SAMase also allows T3 to evade the BREX defense. Although SAM depletion weakly affects BREX methylation, it completely inhibits the defensive function of BREX, suggesting that SAM could be a co-factor for BREX-mediated exclusion of phage DNA, similar to its anti-defense role in type I R-M. The anti-BREX activity of T3 SAMase is mediated not just by enzymatic degradation of SAM but also by direct inhibition of MetK, the host SAM synthase. We present a 2.8Å cryoelectron microscopy (cryo-EM) structure of the eight-subunit T3 SAMase-MetK complex. Structure-guided mutagenesis reveals that this interaction stabilizes T3 SAMase invivo, further stimulating its anti-BREX activity. This work provides insights in the versatility of bacteriophage counterdefense mechanisms and highlights the role of SAM as a co-factor of diverse bacterial immunity systems.

Computational Description of Alkylated Iron-Sulfur Organometallic Clusters.

The radical S-adenosyl methionine (SAM) enzyme superfamily has widespread roles in hydrogen atom abstraction reactions of crucial biological importance. In these enzymes, reductive cleavage of SAM bound to a [4Fe-4S]1+ cluster generates the 5'-deoxyadenosyl radical (5'-dAdo•) which ultimately abstracts an H atom from the substrate. However, overwhelming experimental evidence has surprisingly revealed an obligatory organometallic intermediate Ω exhibiting an Fe-C5'-adenosyl bond, whose properties are the target of this theoretical investigation. We report a readily applied, two-configuration version of broken symmetry DFT, denoted 2C-DFT, designed to allow the accurate description of the hyperfine coupling constants and g-tensors of an alkyl group bound to a multimetallic iron-sulfur cluster. This approach has been validated by the excellent agreement of its results both with those of multiconfigurational complete active space self-consistent field computations for a series of model complexes and with the results from electron nuclear double-resonance/electron paramagnetic resonance spectroscopic studies for the crystallographically characterized complex, M-CH3, a [4Fe-4S] cluster with a Fe-CH3 bond. The likewise excellent agreement between spectroscopic results and 2C-DFT computations for Ω confirm its identity as an organometallic complex with a bond between an Fe of the [4Fe-4S] cluster and C5' of the deoxyadenosyl moiety, as first proposed.

In Vitro Demonstration of Human Lipoyl Synthase Catalytic Activity in the Presence of NFU1.

Lipoyl synthase (LS) catalyzes the last step in the biosynthesis of the lipoyl cofactor, which is the attachment of sulfur atoms at C6 and C8 of an n-octanoyllysyl side chain of a lipoyl carrier protein (LCP). The protein is a member of the radical S-adenosylmethionine (SAM) superfamily of enzymes, which use SAM as a precursor to a 5′-deoxyadenosyl 5′-radical (5′-dA·). The role of the 5′-dA· in the LS reaction is to abstract hydrogen atoms from C6 and C8 of the octanoyl moiety of the substrate to initiate subsequent sulfur attachment. All radical SAM enzymes have at least one [4Fe–4S] cluster that is used in the reductive cleavage of SAM to generate the 5′-dA·; however, LSs contain an additional auxiliary [4Fe–4S] cluster from which sulfur atoms are extracted during turnover, leading to degradation of the cluster. Therefore, these enzymes catalyze only 1 turnover in the absence of a system that restores the auxiliary cluster. In Escherichia coli, the auxiliary cluster of LS can be regenerated by the iron–sulfur (Fe–S) cluster carrier protein NfuA as fast as catalysis takes place, and less efficiently by IscU. NFU1 is the human ortholog of E. coli NfuA and has been shown to interact directly with human LS (i.e., LIAS) in yeast two-hybrid analyses. Herein, we show that NFU1 and LIAS form a tight complex in vitro and that NFU1 can efficiently restore the auxiliary cluster of LIAS during turnover. We also show that BOLA3, previously identified as being critical in the biosynthesis of the lipoyl cofactor in humans and Saccharomyces cerevisiae, has no direct effect on Fe–S cluster transfer from NFU1 or GLRX5 to LIAS. Further, we show that ISCA1 and ISCA2 can enhance LIAS turnover, but only slightly.

The biosynthesis of lipoic acid: A saga of death, destruction, and rebirth

Lipoyl synthase (LipA) catalyzes the terminal step in the biosynthesis of the lipoyl cofactor, which is the attachment of sulfur atoms at C6 and C8 of an n‐octanoyl chain connected in an amide linkage to a specific lysine residue on a lipoyl carrying protein. The protein belongs to the radical S‐adenosylmethionine (SAM) superfamily of enzymes, which use a [4Fe–4S] cluster cofactor to catalyze a reductive cleavage of SAM to generate a 5’‐deoxyadenosyl 5’‐radical (5’‐dA•). The 5’‐dA• removes the C6 and C8 hydrogen atoms for subsequent sulfur attachment; however, the identity of the immediate sulfur source has been controversial. LipA contains a second [4Fe–4S] cluster, which has been proposed to provide the sulfur atoms during the reaction, limiting the enzyme just to one turnover due to destruction of a required cofactor. In this lecture, biochemical, spectroscopic, and structural evidence for this unique sacrificial role in catalysis will be provided. Moreover, evidence that the cluster can be reconstructed after each turnover will be presented.

Viperin Catalyzes the Conversion of Cytidine Triphosphate (CTP) to 3′‐deoxy‐3′,4′‐didehydro‐CTP by Radical S‐adenosylmethionine

Virus inhibitory protein, endoplasmic reticulum‐associated, interferon‐inducible (viperin) is a member of the radical S‐adenosylmethionine (SAM) superfamily of enzymes. Its expression is induced by interferon, and it inhibits a variety of DNA and RNA viruses such as influenza, HIV, dengue virus, West Nile virus, hepatitis C virus, and rabies virus. For flaviviridae especially, it catalyzes the conversion of cytidine triphosphate (CTP) to 3′‐deoxy‐3′,4′‐didehydro‐CTP (ddhCTP). When ddhCTP is incorporated by the viral RNA‐dependent RNA polymerase, RNA polymerization stops because the growing RNA strand lacks a 3’‐hydroxyl group. For other virus families, viperin has been found to interfere with mitochondrial metabolism and interact with a variety of cellular proteins. The Summit Country Day School MSOE Center for BioMolecular Modeling MAPS Team used 3D modeling and printing technology to examine structure‐function relationships of Mus musculus viperin bound with CTP. Viperin is a globular protein that has been shown to be highly conserved and is made up of three regions: N‐terminal extension, central domain, and the C‐terminal extension. The N‐terminal extension localizes viperin to the endoplasmic reticulum and lipid droplets where flaviviruses assemble. The central binding domain contains four sequence motifs associated with the radical SAM superfamily, including the tricysteine motif (CX3CX2C), responsible for binding the catalytic [4Fe‐4S] cluster. The C‐terminal extension binds to CTP and is involved in the installation of the [4Fe‐4S] cluster by the iron‐sulfur cluster–installing protein CIAO1. Binding of CTP to viperin results in conformational changes such as ordering of the C‐terminal extension and formation of an eight residue β‐hairpin loop that binds the γ‐phosphate of CTP. When bound, the 4’‐hydrogen of CTP is ideally positioned for abstraction by the 5′‐deoxyadenosyl radical generated by the reductive cleavage of SAM in the central domain. This is followed by the loss of the 3’‐hydroxyl group leading to the formation of the chain terminator, ddhCTP. The structural insights of CTP‐bound viperin provides an understanding of the role of the C‐terminal extension and its importance as an anti‐viral protein.

Structural basis for tRNA methylthiolation by the radical SAM enzyme MiaB.

Numerous post-transcriptional modifications of transfer RNAs have vital roles in translation. The 2-methylthio-N6-isopentenyladenosine(ms2i6A) modification occurs at position 37 (A37) in transfer RNAs that contain adenine in position 36 of the anticodon, and serves to promote efficient A:U codon-anticodon base-pairing and to prevent unintended basepairing by near cognates, thus enhancing translational fidelity1-4. The ms2i6A modification is installed onto isopentenyladenosine (i6A) by MiaB, a radical S-adenosylmethionine (SAM) methylthiotransferase. As a radical SAM protein, MiaB contains one [Fe4S4]RS cluster used in the reductive cleavage of SAM to form a 5'-deoxyadenosyl 5'-radical, which is responsible for removing the C2 hydrogen of the substrate5. MiaB also contains an auxiliary [Fe4S4]aux cluster, which has been implicated6-9 in sulfur transfer to C2 of i6A37. How this transfer takes place is largely unknown. Here we present several structures of MiaB from Bacteroides uniformis. These structures are consistent with a two-step mechanism, in which one molecule of SAM is first used to methylate a bridging µ-sulfido ion of the auxiliary cluster. In the second step, a second SAM molecule is cleaved to a 5'-deoxyadenosyl 5'-radical, which abstracts the C2 hydrogen of the substrate but only after C2 has undergone rehybridization from sp2 to sp3. This work advances our understanding of how enzymes functionalize inert C-H bonds with sulfur.

Biochemical Characterization of the Radical SAM Methylase Involved in Tetrahydromethanopterin Biosynthesis

Radical S-adenosyl methionine (SAM) enzymes utilize SAM and a [4Fe-4S] cluster to catalyze complex radical reactions throughout various biochemical processes. Methylases are one of the largest subgroups of radical SAM enzymes, which add a methyl group to otherwise unreactive substrates. Our previous work identified the radical SAM methylase, MjMptM, from Methanocaldococcus jannaschii, which likely catalyzes the addition of methyl groups to the C7 and C9 positions of tetrahydromethanopterin, a central C1-carrier cofactor in methanogenic archaea. MjMptM is a unique methylase since it contains multiple [4Fe-4S] clusters and our current experimental evidence indicates that it does not utilize SAM as the methyl group donor. Here, we heterologously expressed and purified MjMptM for in vitro enzymatic and spectroscopic studies. We demonstrated that the enzyme catalyzes two methylation reactions to produce a dimethylated folate species likely using methylenetetrahydrofolate as the C1 source for the methylation reactions. Site-directed mutagenesis and UV-Vis spectroscopic analysis indicate that MjMpt harbors three [4Fe-4S] clusters, two of which reside in the two canonical CX3CX2C radical SAM motifs in the N-terminus. Interestingly, our results indicate that both of the [4Fe-4S] clusters within radical SAM motifs catalyze the reductive cleavage of SAM to produce 5’deoxyadenosine, the key first step in radical SAM enzyme catalysis. The exact location and function of the third auxiliary cluster is yet to be determined, but we propose it is bound by conserved cysteine residues in the C-terminus. Current work is focused on understanding the role of this auxiliary cluster as well as details of the methylation reaction mechanism.

Biochemical Approaches to Probe the Role of the Auxiliary Iron-Sulfur Cluster of Lipoyl Synthase from Mycobacterium Tuberculosis.

Lipoic acid is an essential sulfur-containing cofactor used by several multienzyme complexes involved in energy metabolism and the breakdown of certain amino acids. It is composed of n-octanoic acid with sulfur atoms appended at C6 and C8. Lipoic acid is biosynthesized de novo in its cofactor form, in which it is covalently bound in an amide linkage to a target lysyl residue on a lipoyl carrier protein (LCP). The n-octanoyl moiety of the cofactor is derived from type 2 fatty acid biosynthesis and is transferred to an LCP to afford an octanoyllysyl amino acid. Next, lipoyl synthase (LipA in bacteria) catalyzes the attachment of the two sulfur atoms to afford the intact cofactor. LipA is a radical S-adenosylmethionine (SAM) enzyme that contains two [4Fe-4S] clusters. One [4Fe-4S] cluster is used to facilitate a reductive cleavage of SAM to render the highly oxidizing 5'-deoxyadenosyl 5'-radical needed to abstract C6 and C8 hydrogen atoms to allow for sulfur attachment. By contrast, the second cluster is the sulfur source, necessitating its destruction during turnover. In Escherichia coli, this auxiliary cluster can be restored after each turnover by NfuA or IscU, which are two iron-sulfur cluster carrier proteins that are implicated in iron-sulfur cluster biogenesis. In this chapter, we describe methods for purifying and characterizing LipA and NfuA from Mycobacterium tuberculosis, a human pathogen for which endogenously synthesized lipoic acid is essential. These studies provide the foundation for assessing lipoic acid biosynthesis as a potential target for the design of novel antituberculosis agents.

Structural Properties and Catalytic Implications of the SPASM Domain Iron-Sulfur Clusters in Methylorubrum extorquens PqqE.

Understanding the relationship between the metallocofactor and its protein environment is the key to uncovering the mechanism of metalloenzymes. PqqE, a radical S-adenosylmethionine enzyme in pyrroloquinoline quinone (PQQ) biosynthesis, contains three iron-sulfur cluster binding sites. Two auxiliary iron-sulfur cluster binding sites, designated as AuxI and AuxII, use distinctive ligands compared to other proteins in the family while their functions remain unclear. Here, we investigate the electronic properties of these iron-sulfur clusters and compare the catalytic efficiency of wild-type (WT) Methylorubrum extorquens AM1 PqqE to a range of mutated constructs. Using native mass spectrometry, protein film electrochemistry, and electron paramagnetic resonance spectroscopy, we confirm the previously proposed incorporation of a mixture of [2Fe-2S] and [4Fe-4S] clusters at the AuxI site and are able to assign redox potentials to each of the three iron-sulfur clusters. Significantly, a conservative mutation at AuxI, C268H, shown to selectively incorporate a [4Fe-4S] cluster, catalyzes an enhancement of uncoupled S-adenosylmethionine cleavage relative to WT, together with the elimination of detectable peptide cross-linked product. While a [4Fe-4S] cluster can be tolerated at the AuxI site, the aggregate findings suggest a functional [2Fe-2S] configuration within the AuxI site. PqqE variants with nondestructive ligand replacements at AuxII also show that the reduction potential at this site can be manipulated by changing the electronegativity of the unique aspartate ligand. A number of novel mechanistic features are proposed based on the kinetic and spectroscopic data. Additionally, bioinformatic analyses suggest that the unique ligand environment of PqqE may be relevant to its role in PQQ biosynthesis within an oxygen-dependent biosynthetic pathway.

Crystallographic snapshots of TsrM, a radical S‐adenosylmethionine enzyme whose reaction is not so radical

TsrM methylates C2 of the indole ring of L‐tryptophan (Trp) as the first step in the biosynthesis of the quinaldic acid moiety of thiostrepton, a ribosomally synthesized and post‐translationally modified thiazolyl peptide natural product that exhibits potent in vitro activity against many Gram‐positive bacteria of urgent clinical relevance. TsrM is annotated as a member of the radical S‐adenosylmethionine (SAM) superfamily and exhibits hallmarks of these enzymes, such as a [4Fe–4S] cluster ligated by cysteines residing in a CxxxCxxC motif and a binding site for S‐adenosylmethionine. However, unlike all other characterized members of the radical SAM (RS) superfamily, TsrM does not catalyze a reductive cleavage of SAM to the almost universal 5′‐deoxyadenosyl 5′‐radical intermediate. Instead, recent studies suggest that it catalyzes its reaction via two polar SN2 displacements, an attack on the methyl group of SAM by a cob(I)alamin species and a subsequent attack of C2 of Trp onto methylcobalamin. Herein we report the first crystal structures of a class B RS methylase. The structure contains both the cobalamin and [4Fe‐4S] cluster cofactors bound and show the presence of unique ligands to each. We additionally have solved a structure with substrate analogs bound, which reveals a novel active site, suggesting that TsrM is only masquerading as a radical SAM enzyme.

First Step in Catalysis of the Radical S-Adenosylmethionine Methylthiotransferase MiaB Yields an Intermediate with a [3Fe-4S]0-Like Auxiliary Cluster.

The enzyme MiaB catalyzes the attachment of a methylthio (-SCH3) group at the C2 position of N6-(isopentenyl)adenosine (i6A) in the final step of the biosynthesis of the hypermodified tRNA nucleotide 2-methythio-N6-(isopentenyl)adenosine (ms2i6A). MiaB belongs to the expanding subgroup of enzymes of the radical S-adenosylmethionine (SAM) superfamily that harbor one or more auxiliary [4Fe-4S] clusters in addition to the [4Fe-4S] cluster that all family members require for the reductive cleavage of SAM to afford the common 5'-deoxyadenosyl 5'-radical (5'-dA•) intermediate. While the role of the radical SAM cluster in generating the 5'-dA• is well understood, the detailed role of the auxiliary cluster, which is essential for MiaB catalysis, remains unclear. It has been proposed that the auxiliary cluster may serve as a coordination site for exogenously derived sulfur destined for attachment to the substrate or that the cluster itself provides the sulfur atom and is sacrificed during turnover. In this work, we report spectroscopic and biochemical evidence that the auxiliary [4Fe-4S]2+ cluster in Bacteroides thetaiotaomicron (Bt) MiaB is converted to a [3Fe-4S]0-like cluster during the methylation step of catalysis. Mössbauer characterization of the MiaB [3Fe-4S]0-like cluster revealed unusual spectroscopic properties compared to those of other well-characterized cuboidal [3Fe-4S]0 clusters. Specifically, the Fe sites of the mixed-valent moiety do not have identical Mössbauer parameters. Our results support a mechanism where the auxiliary [4Fe-4S] cluster is the direct sulfur source during catalysis.

An in vitro DNA phosphorothioate modification reaction.

Phosphorothioation (PT) involves the replacement of a nonbridging phosphate oxygen on the DNA backbone with sulfur. In bacteria, the procedure is both sequence- and stereo-specific. We reconstituted the PT reaction using purified DndCDE from Salmonella enterica and IscS from Escherichia coli. We determined that the in vitro process of PT was oxygen sensitive. Only one strand on a double-stranded (ds) DNA substrate was modified in the reaction. The modification was dominant between G and A in the GAAC/GTTC conserved sequence. The modification between G and T required the presence of PT between G and A on the opposite strand. Cysteine, S-adenosyl methionine (SAM) and the formation of an iron-sulfur cluster in DndCDE (DndCDE-FeS) were essential for the process. Results from SAM cleavage reactions support the supposition that PT is a radical SAM reaction. Adenosine triphosphate (ATP) promoted the reaction but was not essential. The data and conclusions presented suggest that the PT reaction in bacteria involves three steps. The first step is the binding of DndCDE-FeS to DNA and searching for the modification sequence, possibly with the help of ATP. Cysteine locks DndCDE-FeS to the modification site with an appropriate protein conformation. SAM triggers the radical SAM reaction to complete the oxygen-sulfur swapping.

Understanding the role of electron donors in the reaction catalyzed by Tsrm, a cobalamin-dependent radical S-adenosylmethionine methylase.

The cobalamin-dependent radical S-adenosylmethionine (SAM) enzyme TsrM catalyzes the methylation of C2 of L-tryptophan to form 2-methyltryptophan during the biosynthesis of thiostrepton A. Although TsrM is a member of the radical SAM superfamily, unlike all other annotated members, it does not catalyze a reductive cleavage of SAM to a 5'-deoxyadenosyl 5'-radical intermediate. In fact, it has been proposed that TsrM catalyzes its reaction through two polar nucleophilic displacements, with its cobalamin cofactor cycling directly between methylcobalamin (MeCbl) and cob(I)alamin. Nevertheless, the enzyme has been stated to require the action of a reductant, which can be satisfied by dithiothreitol. By contrast, all other annotated RS enzymes require a reductant that exhibits a much lower reduction potential, which is necessary for the reductive cleavage of SAM. Herein, we show that TsrM can catalyze multiple turnovers in the absence of any reducing agent, but only when it is pre-loaded with MeCbl. When hydroxocobalamin (OHCbl) or cob(II)alamin is bound to TsrM, a reductant is required to convert it to cob(I)alamin, which can acquire a methyl group directly from SAM. Our studies suggest that TsrM uses an external reductant to prime its reaction by converting bound OHCbl or cob(II)alamin to MeCbl, and to regenerate the MeCbl form of the cofactor upon adventitious oxidation of the cob(I)alamin intermediate to cob(II)alamin.

Ferredoxins as interchangeable redox components in support of MiaB, a radical S-adenosylmethionine methylthiotransferase.

MiaB is a member of the methylthiotransferase subclass of the radical S-adenosylmethionine (SAM) superfamily of enzymes, catalyzing the methylthiolation of C2 of adenosines bearing an N6 -isopentenyl (i6 A) group found at position 37 in several tRNAs to afford 2-methylthio-N6 -(isopentenyl)adenosine (ms2 i6 A). MiaB uses a reduced [4Fe-4S]+ cluster to catalyze a reductive cleavage of SAM to generate a 5'-deoxyadenosyl 5'-radical (5'-dA•)-a required intermediate in its reaction-as well as an additional [4Fe-4S]2+ auxiliary cluster. In Escherichia coli and many other organisms, re-reduction of the [4Fe-4S]2+ cluster to the [4Fe-4S]+ state is accomplished by the flavodoxin reducing system. Most mechanistic studies of MiaBs have been carried out on the enzyme from Thermotoga maritima (Tm), which lacks the flavodoxin reducing system, and which is not activated by E. coli flavodoxin. However, the genome of this organism encodes five ferredoxins (TM0927, TM1175, TM1289, TM1533, and TM1815), each of which might donate the requisite electron to MiaB and perhaps to other radical SAM enzymes. The genes encoding each of these ferredoxins were cloned, and the associated proteins were isolated and shown to support turnover by Tm MiaB. In addition, TM1639, the ferredoxin-NADP+ oxidoreductase subunit α (NfnA) from Tm was overproduced and isolated and shown to provide electrons to the Tm ferredoxins during Tm MiaB turnover. The resulting reactions demonstrate improved coupling between formation of the 5'-dA• and ms2 i6 A production, indicating that only one hydrogen atom abstraction is required for the reaction.

Stereochemical and Mechanistic Investigation of the Reaction Catalyzed by Fom3 from Streptomyces fradiae, a Cobalamin-Dependent Radical S-Adenosylmethionine Methylase.

Fom3, a cobalamin-dependent radical S-adenosylmethionine (SAM) methylase, has recently been shown to catalyze the methylation of carbon 2″ of cytidylyl-2-hydroxyethylphosphonate (HEP-CMP) to form cytidylyl-2-hydroxypropylphosphonate (HPP-CMP) during the biosynthesis of fosfomycin, a broad-spectrum antibiotic. It has been hypothesized that a 5'-deoxyadenosyl 5'-radical (5'-dA•) generated from the reductive cleavage of SAM abstracts a hydrogen atom from HEP-CMP to prime the substrate for addition of a methyl group from methylcobalamin (MeCbl); however, the mechanistic details of this reaction remain elusive. Moreover, it has been reported that Fom3 catalyzes the methylation of HEP-CMP to give a mixture of the ( S)-HPP and ( R)-HPP stereoisomers, which is rare for an enzyme-catalyzed reaction. Herein, we describe a detailed biochemical investigation of a Fom3 that is purified with 1 equiv of its cobalamin cofactor bound, which is almost exclusively in the form of MeCbl. Electron paramagnetic resonance and Mössbauer spectroscopies confirm that Fom3 contains one [4Fe-4S] cluster. Using deuterated enantiomers of HEP-CMP, we demonstrate that the 5'-dA• generated by Fom3 abstracts the C2″- pro-R hydrogen of HEP-CMP and that methyl addition takes place with inversion of configuration to yield solely ( S)-HPP-CMP. Fom3 also sluggishly converts cytidylyl-ethylphosphonate to the corresponding methylated product but more readily acts on cytidylyl-2-fluoroethylphosphonate, which exhibits a lower C2″ homolytic bond-dissociation energy. Our studies suggest a mechanism in which the substrate C2″ radical, generated upon hydrogen atom abstraction by the 5'-dA•, directly attacks MeCbl to transfer a methyl radical (CH3•) rather than a methyl cation (CH3+), directly forming cob(II)alamin in the process.

Structural studies of viperin, an antiviral radical SAM enzyme

Viperin is an IFN-inducible radical S-adenosylmethionine (SAM) enzyme that inhibits viral replication. We determined crystal structures of an anaerobically prepared fragment of mouse viperin (residues 45-362) complexed with S-adenosylhomocysteine (SAH) or 5'-deoxyadenosine (5'-dAdo) and l-methionine (l-Met). Viperin contains a partial (βα)6-barrel fold with a disordered N-terminal extension (residues 45-74) and a partially ordered C-terminal extension (residues 285-362) that bridges the partial barrel to form an overall closed barrel structure. Cys84, Cys88, and Cys91 located after the first β-strand bind a [4Fe-4S] cluster. The active site architecture of viperin with bound SAH (a SAM analog) or 5'-dAdo and l-Met (SAM cleavage products) is consistent with the canonical mechanism of 5'-deoxyadenosyl radical generation. The viperin structure, together with sequence alignments, suggests that vertebrate viperins are highly conserved and that fungi contain a viperin-like ortholog. Many bacteria and archaebacteria also express viperin-like enzymes with conserved active site residues. Structural alignments show that viperin is similar to several other radical SAM enzymes, including the molybdenum cofactor biosynthetic enzyme MoaA and the RNA methyltransferase RlmN, which methylates specific nucleotides in rRNA and tRNA. The viperin putative active site contains several conserved positively charged residues, and a portion of the active site shows structural similarity to the GTP-binding site of MoaA, suggesting that the viperin substrate may be a nucleoside triphosphate of some type.

Insights into the catalysis of a lysine-tryptophan bond in bacterial peptides by a SPASM domain radical S-adenosylmethionine (SAM) peptide cyclase

Radical S-adenosylmethionine (SAM) enzymes are emerging as a major superfamily of biological catalysts involved in the biosynthesis of the broad family of bioactive peptides called ribosomally synthesized and post-translationally modified peptides (RiPPs). These enzymes have been shown to catalyze unconventional reactions, such as methyl transfer to electrophilic carbon atoms, sulfur to Cα atom thioether bonds, or carbon-carbon bond formation. Recently, a novel radical SAM enzyme catalyzing the formation of a lysine-tryptophan bond has been identified in Streptococcus thermophilus, and a reaction mechanism has been proposed. By combining site-directed mutagenesis, biochemical assays, and spectroscopic analyses, we show here that this enzyme, belonging to the emerging family of SPASM domain radical SAM enzymes, likely contains three [4Fe-4S] clusters. Notably, our data support that the seven conserved cysteine residues, present within the SPASM domain, are critical for enzyme activity. In addition, we uncovered the minimum substrate requirements and demonstrate that KW cyclic peptides are more widespread than anticipated, notably in pathogenic bacteria. Finally, we show a strict specificity of the enzyme for lysine and tryptophan residues and the dependence of an eight-amino acid leader peptide for activity. Altogether, our study suggests novel mechanistic links among SPASM domain radical SAM enzymes and supports the involvement of non-cysteinyl ligands in the coordination of auxiliary clusters.

Redox Behavior of the S-Adenosylmethionine (SAM)-Binding Fe-S Cluster in Methylthiotransferase RimO, toward Understanding Dual SAM Activity.

RimO, a radical-S-adenosylmethionine (SAM) enzyme, catalyzes the specific C3 methylthiolation of the D89 residue in the ribosomal S12 protein. Two intact iron-sulfur clusters and two SAM cofactors both are required for catalysis. By using electron paramagnetic resonance, Mössbauer spectroscopies, and site-directed mutagenesis, we show how two SAM molecules sequentially bind to the unique iron site of the radical-SAM cluster for two distinct chemical reactions in RimO. Our data establish that the two SAM molecules bind the radical-SAM cluster to the unique iron site, and spectroscopic evidence obtained under strongly reducing conditions supports a mechanism in which the first molecule of SAM causes the reoxidation of the reduced radical-SAM cluster, impeding reductive cleavage of SAM to occur and allowing SAM to methylate a HS- ligand bound to the additional cluster. Furthermore, by using density functional theory-based methods, we provide a description of the reaction mechanism that predicts the attack of the carbon radical substrate on the methylthio group attached to the additional [4Fe-4S] cluster.

Characterization of a Radical Intermediate in Lipoyl Cofactor Biosynthesis.

Lipoyl synthase (LipA) catalyzes the final step in the biosynthesis of the lipoyl cofactor, the insertion of two sulfur atoms at C6 and C8 of an n-octanoyl chain. LipA is a member of the radical S-adenosylmethionine (SAM) superfamily of enzymes and uses two [4Fe-4S] clusters to catalyze its transformation. One cluster binds in contact with SAM and donates the requisite electron for the reductive cleavage of SAM to generate two 5'-deoxyadenosyl 5'-radicals, which abstract hydrogen atoms from C6 and C8 of the substrate. By contrast, the second, auxiliary [4Fe-4S] cluster, has been hypothesized to serve as the sulfur donor in the reaction. Such a sacrificial role for an iron-sulfur cluster during catalysis has not been universally accepted. Use of a conjugated 2,4-hexadienoyl-containing substrate analogue has allowed the substrate radical to be trapped and characterized by continuous-wave and pulsed electron paramagnetic resonance methods. Here we report the observation of a (57)Fe hyperfine coupling interaction with the paramagnetic signal, which indicates that the iron-sulfur cluster of LipA and its substrate are within bonding distance.