FDX1 regulates cellular protein lipoylation through direct binding to LIAS.

Ferredoxins are a family of iron-sulfur (Fe-S) cluster proteins that serve as essential electron donors in numerous cellular processes that are conserved through evolution. The promiscuous nature of ferredoxins as electron donors enables them to participate in many metabolic processes including steroid, heme, vitamin D, and Fe-S cluster biosynthesis in different organisms. However, the unique natural function(s) of each of the two human ferredoxins (FDX1 and FDX2) are still poorly characterized. We recently reported that FDX1 is both a crucial regulator of copper ionophore-induced cell death and serves as an upstream regulator of cellular protein lipoylation, a mitochondrial lipid-based post-translational modification naturally occurring on four mitochondrial enzymes that are crucial for TCA cycle function. Here we show that FDX1 directly regulates protein lipoylation by binding the lipoyl synthase (LIAS) enzyme promoting its functional binding to the lipoyl carrier protein GCSH and not through indirect regulation of cellular Fe-S cluster biosynthesis. Metabolite profiling revealed that the predominant cellular metabolic outcome of FDX1 loss of function is manifested through the regulation of the four lipoylation-dependent enzymes ultimately resulting in loss of cellular respiration and sensitivity to mild glucose starvation. Transcriptional profiling established that FDX1 loss-of-function results in the induction of both compensatory metabolism-related genes and the integrated stress response, consistent with our findings that FDX1 loss-of-function is conditionally lethal. Together, our findings establish that FDX1 directly engages with LIAS, promoting its role in cellular protein lipoylation, a process essential in maintaining cell viability under low glucose conditions.

Enzymatic Fluoromethylation Enabled by the S-Adenosylmethionine Analog Te-Adenosyl-L-(fluoromethyl)homotellurocysteine.

Fluoromethyl, difluoromethyl, and trifluoromethyl groups are present in numerous pharmaceuticals and agrochemicals, where they play critical roles in the efficacy and metabolic stability of these molecules. Strategies for late-stage incorporation of fluorine-containing atoms in molecules have become an important area of organic and medicinal chemistry as well as synthetic biology. Herein, we describe the synthesis and use of Te-adenosyl-L-(fluoromethyl)homotellurocysteine (FMeTeSAM), a novel and biologically relevant fluoromethylating agent. FMeTeSAM is structurally and chemically related to the universal cellular methyl donor S-adenosyl-L-methionine (SAM) and supports the robust transfer of fluoromethyl groups to oxygen, nitrogen, sulfur, and some carbon nucleophiles. FMeTeSAM is also used to fluoromethylate precursors to oxaline and daunorubicin, two complex natural products that exhibit antitumor properties.

Functional spectrum and specificity of mitochondrial ferredoxins FDX1 and FDX2.

Ferredoxins comprise a large family of iron-sulfur (Fe-S) proteins that shuttle electrons in diverse biological processes. Human mitochondria contain two isoforms of [2Fe-2S] ferredoxins, FDX1 (aka adrenodoxin) and FDX2, with known functions in cytochrome P450-dependent steroid transformations and Fe-S protein biogenesis. Here, we show that only FDX2, but not FDX1, is involved in Fe-S protein maturation. Vice versa, FDX1 is specific not only for steroidogenesis, but also for heme a and lipoyl cofactor biosyntheses. In the latter pathway, FDX1 provides electrons to kickstart the radical chain reaction catalyzed by lipoyl synthase. We also identified lipoylation as a target of the toxic antitumor copper ionophore elesclomol. Finally, the striking target specificity of each ferredoxin was assigned to small conserved sequence motifs. Swapping these motifs changed the target specificity of these electron donors. Together, our findings identify new biochemical tasks of mitochondrial ferredoxins and provide structural insights into their functional specificity.

Characterization of LipS1 and LipS2 from Thermococcus kodakarensis: Proteins Annotated as Biotin Synthases, which Together Catalyze Formation of the Lipoyl Cofactor.

Lipoic acid is an eight-carbon sulfur-containing biomolecule that functions primarily as a cofactor in several multienzyme complexes. It is biosynthesized as an attachment to a specific lysyl residue on one of the subunits of these multienzyme complexes. In Escherichia coli and many other organisms, this biosynthetic pathway involves two dedicated proteins: octanoyltransferase (LipB) and lipoyl synthase (LipA). LipB transfers an n-octanoyl chain from the octanoyl-acyl carrier protein to the target lysyl residue, and then, LipA attaches two sulfur atoms (one at C6 and one at C8) to give the final lipoyl cofactor. All classical lipoyl synthases (LSs) are radical S-adenosylmethionine (SAM) enzymes, which use an [Fe4S4] cluster to reductively cleave SAM to generate a 5'-deoxyadenosyl 5'-radical. Classical LSs also contain a second [Fe4S4] cluster that serves as the source of both appended sulfur atoms. Recently, a novel pathway for generating the lipoyl cofactor was reported. This pathway replaces the canonical LS with two proteins, LipS1 and LipS2, which act together to catalyze formation of the lipoyl cofactor. In this work, we further characterize LipS1 and LipS2 biochemically and spectroscopically. Although LipS1 and LipS2 were previously annotated as biotin synthases, we show that both proteins, unlike E. coli biotin synthase, contain two [Fe4S4] clusters. We identify the cluster ligands to both iron-sulfur clusters in both proteins and show that LipS2 acts only on an octanoyl-containing substrate, while LipS1 acts only on an 8-mercaptooctanoyl-containing substrate. Therefore, similarly to E. coli biotin synthase and in contrast to E. coli LipA, sulfur attachment takes place initially at the terminal carbon (C8) and then at the C6 methylene carbon.

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.

Discovery, structure and mechanism of a tetraether lipid synthase.

Archaea synthesize isoprenoid-based ether-linked membrane lipids, which enable them to withstand extreme environmental conditions, such as high temperatures, high salinity, and low or high pH values1-5. In some archaea, such as Methanocaldococcus jannaschii, these lipids are further modified by forming carbon-carbon bonds between the termini of two lipid tails within one glycerophospholipid to generate the macrocyclic archaeol or forming two carbon-carbon bonds between the termini of two lipid tails from two glycerophospholipids to generate the macrocycle glycerol dibiphytanyl glycerol tetraether (GDGT)1,2. GDGT contains two 40-carbon lipid chains (biphytanyl chains) that span both leaflets of the membrane, providing enhanced stability to extreme conditions. How these specialized lipids are formed has puzzled scientists for decades. The reaction necessitates the coupling of two completely inert sp3-hybridized carbon centres, which, to our knowledge, has not been observed in nature. Here we show that the gene product of mj0619 from M. jannaschii, which encodes a radical S-adenosylmethionine enzyme, is responsible for biphytanyl chain formation during synthesis of both the macrocyclic archaeol and GDGT membrane lipids6. Structures of the enzyme show the presence of four metallocofactors: three [Fe4S4] clusters and one mononuclear rubredoxin-like iron ion. In vitro mechanistic studies show that Csp3-Csp3 bond formation takes place on fully saturated archaeal lipid substrates and involves an intermediate bond between the substrate carbon and a sulfur of one of the [Fe4S4] clusters. Our results not only establish the biosynthetic route for tetraether formation but also improve the use of GDGT in GDGT-based paleoclimatology indices7-10.

Structural characterization of cobalamin-dependent radical S-adenosylmethionine methylases.

Cobalamin-dependent radical S-adenosylmethionine (SAM) methylases catalyze key steps in the biosynthesis of numerous biomolecules, including protein cofactors, antibiotics, herbicides, and other natural products, but have remained a relatively understudied subclass of radical SAM enzymes due to their inherent insolubility upon overproduction in Escherichia coli. These enzymes contain two cofactors: a [4Fe-4S] cluster that is ligated by three cysteine residues, and a cobalamin cofactor typically bound by residues in the N-terminal portion of the enzyme. Recent advances in the expression and purification of these enzymes in their active states and with both cofactors present has allowed for more detailed biochemical studies as well as structure determination by X-ray crystallography. Herein, we use KsTsrM and TokK to highlight methods for the structural characterization of cobalamin-dependent radical SAM (RS) enzymes and describe recent advances in in the overproduction and purification of these enzymes.

Using peptide substrate analogs to characterize a radical intermediate in NosN catalysis.

Nosiheptide is a ribosomally produced and post-translationally modified thiopeptide antibiotic that displays potent antibacterial activity in vitro, especially against Gram-positive pathogens. It comprises a core peptide macrocycle that contains multiple thiazole rings, dehydrated serine and threonine residues, a tri-substituted 3-hydroxypyridine ring and several other modifications. Among these additional modifications includes a 3,4-dimethyl-2-indolic acid (DMIA) moiety that bridges Glu6 and Cys8 of the core peptide to form a second smaller ring system. This side-ring system is formed by the action of NosN, a radical S-adenosylmethionine (SAM) enzyme that falls within the class C radical SAM methylase (RSMT) family. However, the true function of NosN is to transfer a methylene group from the methyl moiety of SAM to C4 of 3-methylindolic acid (MIA) attached in a thioester linkage to Cys8 of the core peptide to set up a highly electrophilic species. This species is then trapped by the side chain of Glu6, resulting in formation of a lactone and the side-ring system. The NosN reaction requires two simultaneously bound molecules of SAM. The first, SAMI, is cleaved to generate a 5'-deoxyadenosyl 5'-radical, which abstracts a hydrogen atom from the methyl group of the second molecule of SAM, SAMII. The resulting SAMII radical is believed to add to C4 of MIA, affording a radical intermediate on the MIA substrate. Herein we describe synthetic approaches that allow detection of this radical by electron paramagnetic resonance (EPR) spectroscopy.

[FeFe]-Hydrogenase: Defined Lysate-Free Maturation Reveals a Key Role for Lipoyl-H-Protein in DTMA Ligand Biosynthesis.

Maturation of [FeFe]-hydrogenase (HydA) involves synthesis of a CO, CN- , and dithiomethylamine (DTMA)-coordinated 2Fe subcluster that is inserted into HydA to make the active hydrogenase. This process requires three maturation enzymes: the radical S-adenosyl-l-methionine (SAM) enzymes HydE and HydG, and the GTPase HydF. In vitro maturation with purified maturation enzymes has been possible only when clarified cell lysate was added, with the lysate presumably providing essential components for DTMA synthesis and delivery. Here we report maturation of [FeFe]-hydrogenase using a fully defined system that includes components of the glycine cleavage system (GCS), but no cell lysate. Our results reveal for the first time an essential role for the aminomethyl-lipoyl-H-protein of the GCS in hydrogenase maturation and the synthesis of the DTMA ligand of the H-cluster. In addition, we show that ammonia is the source of the bridgehead nitrogen of DTMA.

Structure of a B12-dependent radical SAM enzyme in carbapenem biosynthesis.

Carbapenems are antibiotics of last resort in the clinic. Owing to their potency and broad-spectrum activity, they are an important part of the antibiotic arsenal. The vital role of carbapenems is exemplified by the approval acquired by Merck from the US Food and Drug Administration (FDA) for the use of an imipenem combination therapy to treat the increased levels of hospital-acquired and ventilator-associated bacterial pneumonia that have occurred during the COVID-19 pandemic1. The C6 hydroxyethyl side chain distinguishes the clinically used carbapenems from the other classes of ß-lactam antibiotics and is responsible for their low susceptibility to inactivation by occluding water from the ß-lactamase active site2. The construction of the C6 hydroxyethyl side chain is mediated by cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes3. These radical SAM methylases (RSMTs) assemble the alkyl backbone by sequential methylation reactions, and thereby underlie the therapeutic usefulness of clinically used carbapenems. Here we present X-ray crystal structures of TokK, a B12-dependent RSMT that catalyses three-sequential methylations during the biosynthesis of asparenomycin A. These structures, which contain the two metallocofactors of the enzyme and were determined in the presence and absence of a carbapenam substrate, provide a visualization of a B12-dependent RSMT that uses the radical mechanism that is shared by most of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggest that substrate positioning governs the rate of each methylation event.

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 base pairing 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 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 basis for non-radical catalysis by TsrM, a radical SAM methylase.

Tryptophan 2C methyltransferase (TsrM) methylates C2 of the indole ring of L-tryptophan during biosynthesis of the quinaldic acid moiety of thiostrepton. TsrM is annotated as a cobalamin-dependent radical S-adenosylmethionine (SAM) methylase; however, TsrM does not reductively cleave SAM to the universal 5'-deoxyadenosyl 5'-radical intermediate, a hallmark of radical SAM (RS) enzymes. Herein, we report structures of TsrM from Kitasatospora setae, which are the first structures of a cobalamin-dependent radical SAM methylase. Unexpectedly, the structures show an essential arginine residue that resides in the proximal coordination sphere of the cobalamin cofactor, and a [4Fe-4S] cluster that is ligated by a glutamyl residue and three cysteines in a canonical CXXXCXXC RS motif. Structures in the presence of substrates suggest a substrate-assisted mechanism of catalysis, wherein the carboxylate group of SAM serves as a general base to deprotonate N1 of the tryptophan substrate, facilitating the formation of a C2 carbanion.

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.

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.

Analysis of RNA Methylation by Phylogenetically Diverse Cfr Radical S-Adenosylmethionine Enzymes Reveals an Iron-Binding Accessory Domain in a Clostridial Enzyme.

Cfr is a radical S-adenosylmethionine (SAM) RNA methylase linked to multidrug antibiotic resistance in bacterial pathogens. It catalyzes a chemically challenging C-C bond-forming reaction to methylate C8 of A2503 (Escherichia coli numbering) of 23S rRNA during ribosome assembly. The cfr gene has been identified as a mobile genetic element in diverse bacteria and in the genome of select Bacillales and Clostridiales species. Despite the importance of Cfr, few representatives have been purified and characterized in vitro. Here we show that Cfr homologues from Bacillus amyloliquefaciens, Enterococcus faecalis, Paenibacillus lautus, and Clostridioides difficile act as C8 adenine RNA methylases in biochemical assays. C. difficile Cfr contains an additional Cys-rich C-terminal domain that binds a mononuclear Fe2+ ion in a rubredoxin-type Cys4 motif. The C-terminal domain can be truncated with minimal impact on C. difficile Cfr activity, but the rate of turnover is decreased upon disruption of the Fe2+-binding site by Zn2+ substitution or ligand mutation. These findings indicate an important purpose for the observed C-terminal iron in the native fusion protein. Bioinformatic analysis of the C. difficile Cfr Cys-rich domain shows that it is widespread (∼1400 homologues) as a stand-alone gene in pathogenic or commensal Bacilli and Clostridia, with >10% encoded adjacent to a predicted radical SAM RNA methylase. Although the domain is not essential for in vitro C. difficile Cfr activity, the genomic co-occurrence and high abundance in the human microbiome suggest a possible functional role for a specialized rubredoxin in certain radical SAM RNA methylases that are relevant to human health.

Methanogenesis marker protein 10 (Mmp10) from Methanosarcina acetivorans is a radical S-adenosylmethionine methylase that unexpectedly requires cobalamin.

Methyl coenzyme M reductase (MCR) catalyzes the last step in the biological production of methane by methanogenic archaea, as well as the first step in the anaerobic oxidation of methane to methanol by methanotrophic archaea. MCR contains a number of unique post-translational modifications in its α subunit, including thioglycine, 1-N-methylhistidine, S-methylcysteine, 5-C-(S)-methylarginine, and 2-C-(S)-methylglutamine. Recently, genes responsible for the thioglycine and methylarginine modifications have been identified in bioinformatics studies and in vivo complementation of select mutants; however, none of these reactions has been verified in vitro Herein, we purified and biochemically characterized the radical S-adenosylmethionine (SAM) protein MaMmp10, the product of the methanogenesis marker protein 10 gene in the methane-producing archaea Methanosarcina acetivorans Using an array of approaches, including kinetic assays, LC-MS-based quantification, and MALDI TOF-TOF MS analyses, we found that MaMmp10 catalyzes the methylation of the equivalent of Arg285 in a peptide substrate surrogate, but only in the presence of cobalamin. We noted that the methyl group derives from SAM, with cobalamin acting as an intermediate carrier, and that MaMmp10 contains a C-terminal cobalamin-binding domain. Given that Mmp10 has not been annotated as a cobalamin-binding protein, these findings suggest that cobalamin-dependent radical SAM proteins are more prevalent than previously thought.

Capturing Intermediates in the Reaction Catalyzed by NosN, a Class C Radical S-Adenosylmethionine Methylase Involved in the Biosynthesis of the Nosiheptide Side-Ring System.

Nosiheptide is a ribosomally synthesized and post-translationally modified thiopeptide natural product that possesses antibacterial, anticancer, and immunosuppressive properties. It contains a bicyclic structure composed of a large macrocycle and a unique side-ring system containing a 3,4-dimethylindolic acid bridge connected to the side chains of Glu6 and Cys8 of the core peptide via ester and thioester linkages, respectively. In addition to the structural peptide, encoded by the nosM gene, the biosynthesis of the side-ring structure requires the actions of NosI, -J, -K, -L, and -N. NosN is annotated as a class C radical S-adenosylmethionine (SAM) methylase, but its true function is to transfer a C1 unit from SAM to C4 of 3-methyl-2-indolic acid (MIA) with concomitant formation of a bond between the carboxylate of Glu6 of the core peptide and the nascent C1 unit. However, exactly when NosN performs its function during the biosynthesis of nosiheptide is unknown. Herein, we report the syntheses and use of three peptide mimics as potential substrates designed to address the timing of NosN's function. Our results show that NosN clearly closes the side ring before NosO forms the pyridine ring and most likely before NosD/E catalyzes formation of the dehydrated amino acids, although the possibility of a more random process (i.e., NosN acting after NosD/E) cannot be ruled out. Using a substrate mimic containing a rigid structure, we also identify and characterize two reaction-based adducts containing SAM fused to C4 of MIA. The two SAM adducts are derived from a consensus radical-containing species proposed to be the key intermediate-or a derivative of the key intermediate-in our proposed catalytic mechanism of NosN.

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.