Radical SAM Enzyme Research Articles

RSAD2 suppresses viral replication by interacting with the Senecavirus A 2 C protein

Senecavirus A (SVA), an emerging virus that causes blisters on the nose and hooves, reduces the production performance of pigs. RSAD2 is a radical S-adenosylmethionine (SAM) enzyme, and its expression can suppress various viruses due to its broad antiviral activity. However, the regulatory relationship between SVA and RSAD2 and the mechanism of action remain unclear. Here, we demonstrated that SVA infection increased RSAD2 mRNA levels, whereas RSAD2 expression negatively regulated viral replication, as evidenced by decreased viral VP1 protein expression, viral titres, and infected cell numbers. Viral proteins that interact with RSAD2 were screened, and the interaction between the 2 C protein and RSAD2 was found to be stronger than that between other proteins. Additionally, amino acids (aa) 43–70 of RSAD2 were crucial for interacting with the 2 C protein and played an important role in its anti-SVA activity. RSAD2 was induced by type I interferon (IFN-I) via Janus kinase signal transducer and activator of transcription (JAK-STAT), and had antiviral activity. Ruxolitinib, a JAK-STAT pathway inhibitor, and the knockdown of JAK1 expression substantially reduced RSAD2 expression levels and antiviral activity. Taken together, these results revealed that RSAD2 blocked SVA infection by interacting with the viral 2 C protein and provide a strategy for preventing and controlling SVA infection.

Fused radical SAM and αKG-HExxH domain proteins contain a distinct structural fold and catalyse cyclophane formation and β-hydroxylation.

Two of nature's recurring binding motifs in metalloproteins are the CxxxCxxC motif in radical SAM enzymes and the 2-His-1-carboxylate motif found both in zincins and α-ketoglutarate and non-haem iron enzymes. Here we show the confluence of these two domains in a single post-translational modifying enzyme containing an N-terminal radical S-adenosylmethionine domain fused to a C-terminal 2-His-1-carboxylate (HExxH) domain. The radical SAM domain catalyses three-residue cyclophane formation and is the signature modification of triceptides, a class of ribosomally synthesized and post-translationally modified peptides. The HExxH domain is a defining feature of zinc metalloproteases. Yet the HExxH motif-containing domain studied here catalyses β-hydroxylation and is an α-ketoglutarate non-haem iron enzyme. We determined the crystal structure for this HExxH protein at 2.8 Å, unveiling a distinct structural fold, thus expanding the family of α-ketoglutarate non-haem iron enzymes with a class that we propose to name αKG-HExxH. αKG-HExxH proteins represent a unique family of ribosomally synthesized and post-translationally modified peptide modifying enzymes that can furnish opportunities for genome mining, synthetic biology and enzymology.

Chemical Antiquity in Metabolism.

ConspectusLife is an exergonic chemical reaction. The same was true when the very first cells emerged at life's origin. In order to live, all cells need a source of carbon, energy, and electrons to drive their overall reaction network (metabolism). In most cells, these are separate pathways. There is only one biochemical pathway that serves all three needs simultaneously: the acetyl-CoA pathway of CO2 fixation. In the acetyl-CoA pathway, electrons from H2 reduce CO2 to pyruvate for carbon supply, while methane or acetate synthesis are coupled to energy conservation as ATP. This simplicity and thermodynamic favorability prompted Georg Fuchs and Erhard Stupperich to propose in 1985 that the acetyl-CoA pathway might mark the origin of metabolism, at the same time that Steve Ragsdale and Harland Wood were uncovering catalytic roles for Fe, Co, and Ni in the enzymes of the pathway. Subsequent work has provided strong support for those proposals.In the presence of Fe, Co, and Ni in their native metallic state as catalysts, aqueous H2 and CO2 react specifically to formate, acetate, methane, and pyruvate overnight at 100 °C. These metals (and their alloys) thus replace the function of over 120 enzymes required for the conversion of H2 and CO2 to pyruvate via the pathway and its cofactors, an unprecedented set of findings in the study of biochemical evolution. The reactions require alkaline conditions, which promote hydrogen oxidation by proton removal and are naturally generated in serpentinizing (H2-producing) hydrothermal vents. Serpentinizing hydrothermal vents furthermore produce natural deposits of native Fe, Co, Ni, and their alloys. These are precisely the metals that reduce CO2 with H2 in the laboratory; they are also the metals found at the active sites of enzymes in the acetyl-CoA pathway. Iron, cobalt and nickel are relicts of the environments in which metabolism arose, environments that still harbor ancient methane- and acetate-producing autotrophs today. This convergence indicates bedrock-level antiquity for the acetyl-CoA pathway. In acetogens and methanogens growing on H2 as reductant, the acetyl-CoA pathway requires flavin-based electron bifurcation as a source of reduced ferredoxin (a 4Fe4S cluster-containing protein) in order to function. Recent findings show that H2 can reduce the 4Fe4S clusters of ferredoxin in the presence of native iron, uncovering an evolutionary precursor of flavin-based electron bifurcation and suggesting an origin of FeS-dependent electron transfer in proteins. Traditionally discussed as catalysts in early evolution, the most common function of FeS clusters in metabolism is one-electron transfer, also in radical SAM enzymes, a large and ancient enzyme family. The cofactors and active sites in enzymes of the acetyl-CoA pathway uncover chemical antiquity in metabolism involving metals, methyl groups, methyl transfer reactions, cobamides, pterins, GTP, S-adenosylmethionine, radical SAM enzymes, and carbon-metal bonds. The reaction sequence from H2 and CO2 to pyruvate on naturally deposited native metals is maximally simple. It requires neither nitrogen, sulfur, phosphorus, RNA, ion gradients, nor light. Solid-state metal catalysts tether the origin of metabolism to a H2-producing, serpentinizing hydrothermal vent.

Arsinothricin Biosynthesis Involving a Radical SAM Enzyme for Noncanonical SAM Cleavage and C-As Bond Formation.

Arsinothricin is a potent antibiotic secreted by soil bacteria. The biosynthesis of arsinothricin was proposed to involve a C-As bond formation between trivalent As and the 3-amino-3-carboxypropyl (ACP) group of S-adenosyl-l-methionine (SAM), which is catalyzed by the protein ArsL. However, ArsL has not been characterized in detail. Interestingly, ArsL contains a CxxxCxxC motif and thus belongs to the radical SAM enzyme superfamily, the members of which cleave SAM and generate a 5'-deoxyadenosyl radical. Here, we found that ArsL cleaves the Cγ,Met-S bond of SAM and generates an ACP radical that resembles Dph2, a noncanonical radical SAM enzyme involved in diphthamid biosynthesis. As Dph2 does not contain the CxxxCxxC motif, ArsL is a unique radical SAM enzyme that contains this motif but generates a noncanonical ACP radical. Together with the methyltransferase ArsM, we successfully reconstituted arsinothricin biosynthesis in vitro. ArsL has a conserved RCCLKC motif in the C-terminal sequence and belongs to the RCCLKC-tail radical SAM protein subfamily. By truncation and mutagenesis, we showed that this motif plays an important role in binding to the substrate arsenite and is highly important for its activity. Our results suggested that ArsL has a canonical radical SAM enzyme motif but catalyzes a noncanonical radical SAM reaction, implying that more noncanonical radical SAM chemistry may exist within the radical SAM enzyme superfamily.

The H-cluster of [FeFe] Hydrogenases: Its Enzymatic Synthesis and Parallel Inorganic Semisynthesis.

ConspectusNature's prototypical hydrogen-forming catalysts─hydrogenases─have attracted much attention because they catalyze hydrogen evolution at near zero overpotential and ambient conditions. Beyond any possible applications in the energy sphere, the hydrogenases feature complicated active sites, which implies novel biosynthetic pathways. In terms of the variety of cofactors, the [FeFe]-hydrogenase is among the most complex.For more than a decade, we have worked on the biosynthesis of the active site of [FeFe] hydrogenases. This site, the H-cluster, is a six-iron ensemble consisting of a [4Fe-4S]H cluster linked to a [2Fe]H cluster that is coordinated to CO, cyanide, and a unique organic azadithiolate ligand. Many years ago, three enzymes, namely, HydG, HydE, and HydF, were shown to be required for the biosynthesis and the in vitro maturation of [FeFe] hydrogenases. The structures of the maturases were determined crystallographically, but still little progress was made on the biosynthetic pathway. As described in this Account, the elucidation of the biosynthetic pathway began in earnest with the identification of a molecular iron-cysteinate complex produced within HydG.In this Account, we present our most recent progress toward the molecular mechanism of [2Fe]H biosynthesis using a collaborative approach involving cell-free biosynthesis, isotope and element-sensitive spectroscopies, as well as inorganic synthesis of purported biosynthetic intermediates. Our study starts from the radical SAM enzyme HydG that lyses tyrosine into CO and cyanide and forms an Fe(CO)2(CN)-containing species. Crystallographic identification of a unique auxiliary 5Fe-4S cluster in HydG leads to a proposed catalytic cycle in which a free cysteine-chelated "dangler" Fe serves as the platform for the stepwise formation of a [4Fe-4S][Fe(CO)(CN)(cysteinate)] intermediate, which releases the [Fe(CO)2(CN)(cysteinate)] product, Complex B. Since Complex B is unstable, we applied synthetic organometallic chemistry to make an analogue, syn-B, and showed that it fully replaces HydG in the in vitro maturation of the H-cluster. Syn-B serves as the substrate for the next radical SAM enzyme HydE, where the low-spin Fe(II) center is activated by 5'-dAdo• to form an adenosylated Fe(I) intermediate. We propose that this Fe(I) species strips the carbon backbone and dimerizes in HydE to form a [Fe2(SH)2(CO)4(CN)2]2- product. This mechanistic scenario is supported by the use of a synthetic version of this dimer complex, syn-dimer, which allows for the formation of active hydrogenase with only the HydF maturase. Further application of this semisynthesis strategy shows that an [Fe2(SCH2NH2)2(CO)4(CN)2]2- complex can activate the apo hydrogenase, marking it as the last biosynthetic intermediate en route to the H-cluster. This combined enzymatic and semisynthetic approach greatly accelerates our understanding of H-cluster biosynthesis. We anticipate additional mechanistic details regarding H-cluster biosynthesis to be gleaned, and this methodology may be further applied in the study of other complex metallocofactors.

Biosynthesis of GMGT lipids by a radical SAM enzyme associated with anaerobic archaea and oxygen-deficient environments

Archaea possess characteristic membrane-spanning lipids that are thought to contribute to the adaptation to extreme environments. However, the biosynthesis of these lipids is poorly understood. Here, we identify a radical S-adenosyl-L-methionine (SAM) enzyme that synthesizes glycerol monoalkyl glycerol tetraethers (GMGTs). The enzyme, which we name GMGT synthase (Gms), catalyzes the formation of a C(sp3)–C(sp3) linkage between the two isoprenoid chains of glycerol dialkyl glycerol tetraethers (GDGTs). This conclusion is supported by heterologous expression of gene gms from a GMGT-producing species in a methanogen, as well as demonstration of in vitro activity using purified Gms enzyme. Additionally, we show that genes encoding putative Gms homologs are present in obligate anaerobic archaea and in metagenomes obtained from oxygen-deficient environments, and appear to be absent in metagenomes from oxic settings.

Functional Integrity of Radical SAM Enzyme Dph1•Dph2 Requires Non-Canonical Cofactor Motifs with Tandem Cysteines.

The Dph1•Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cys residue in each motif is found next to another ill-defined cysteine that we show is conserved across eukaryotes. As judged from structural modeling, the orientation of these tandem cysteine motifs (TCMs) suggests a candidate Fe-S cluster ligand role. Hence, we generated, by site-directed DPH1 and DPH2 mutagenesis, Dph1•Dph2 variants with cysteines from each TCM replaced individually or in combination by serines. Assays diagnostic for diphthamide formation in vivo reveal that while single substitutions in the TCM of Dph2 cause mild defects, double mutations almost entirely inactivate the RS enzyme. Based on enhanced Dph1 and Dph2 subunit instability in response to cycloheximide chases, the variants with Cys substitutions in their cofactor motifs are particularly prone to protein degradation. In sum, we identify a fourth functionally cooperative Cys residue within the Fe-S motif of Dph2 and show that the Cys-based cofactor binding motifs in Dph1 and Dph2 are critical for the structural integrity of the dimeric RS enzyme in vivo.

Radical SAM Enzyme PylB Generates a Lysyl Radical Intermediate in the Biosynthesis of Pyrrolysine by Using SAM as a Cofactor.

Pyrrolysine, the 22nd amino acid encoded by the natural genetic code, is essential for methanogenic archaea to catabolize methylamines into methane. The structure of pyrrolysine consists of a methylated pyrroline carboxylate that is linked to the ε-amino group of the l-lysine via an amide bond. The biosynthesis of pyrrolysine requires three enzymes: PylB, PylC, and PylD. PylB is a radical S-adenosyl-l-methionine (SAM) enzyme and catalyzes the first biosynthetic step, the isomerization of l-lysine into methylornithine. PylC catalyzes an ATP-dependent ligation of methylornithine and a second l-lysine to form l-lysine-Nε-methylornithine. The last biosynthetic step is catalyzed by PylD via oxidation of the PylC product to form pyrrolysine. While enzymatic reactions of PylC and PylD have been well characterized by X-ray crystallography and in vitro studies, mechanistic understanding of PylB is still relatively limited. Here, we report the first in vitro activity of PylB to form methylornithine via the isomerization of l-lysine. We also identify a lysyl C4 radical intermediate that is trapped, with its electronic structure and geometric structure well characterized by EPR and ENDOR spectroscopy. In addition, we demonstrate that SAM functions as a catalytic cofactor in PylB catalysis rather than canonically as a cosubstrate. This work provides detailed mechanistic evidence for elucidating the carbon backbone rearrangement reaction catalyzed by PylB during the biosynthesis of pyrrolysine.

Expanding the Landscape of Noncanonical Amino Acids in RiPP Biosynthesis.

Advancements in DNA sequencing technologies and bioinformatics have enabled the discovery of new metabolic reactions from overlooked microbial species and metagenomic sequences. Using a bioinformatic co-occurrence strategy, we previously generated a network of ∼600 uncharacterized quorum-sensing-regulated biosynthetic gene clusters that code for ribosomally synthesized and post-translationally modified peptide (RiPP) natural products and are tailored by radical S-adenosylmethionine (RaS) enzymes in streptococci. The most complex of these is the GRC subfamily, named after a conserved motif in the precursor peptide and found exclusively in Streptococcus pneumoniae, the causative agent of bacterial pneumonia. In this study, using both in vivo and in vitro approaches, we have elucidated the modifications installed by the grc biosynthetic enzymes, including a ThiF-like adenylyltransferase/cyclase that generates a C-terminal Glu-to-Cys thiolactone macrocycle, and two RaS enzymes, which selectively epimerize the β-carbon of threonine and desaturate histidine to generate the first instances of l-allo-Thr and didehydrohistidine in RiPP biosynthesis. RaS-RiPPs that have been discovered thus far have stood out for their exotic macrocycles. The product of the grc cluster breaks this trend by generating two noncanonical residues rather than an unusual macrocycle in the peptide substrate. These modifications expand the landscape of nonproteinogenic amino acids in RiPP natural product biosynthesis and motivate downstream biocatalytic applications of the corresponding enzymes.

ENDOR Spectroscopy Reveals the "Free" 5'-Deoxyadenosyl Radical in a Radical SAM Enzyme Active Site Actually is Chaperoned by Close Interaction with the Methionine-Bound [4Fe-4S]2+ Cluster.

1/2H and 13C hyperfine coupling constants to 5'-deoxyadenosyl (5'-dAdo•) radical trapped within the active site of the radical S-adenosyl-l-methionine (SAM) enzyme, pyruvate formate lyase-activating enzyme (PFL-AE), both in the absence of substrate and the presence of a reactive peptide-model of the PFL substrate, are completely characteristic of a classical organic free radical whose unpaired electron is localized in the 2pπ orbital of the sp2 C5'-carbon (J. Am. Chem. Soc. 2019, 141, 12139-12146). However, prior electron-nuclear double resonance (ENDOR) measurements had indicated that this 5'-dAdo• free radical is never truly "free": tight van der Waals contact with its target partners and active-site residues guide it in carrying out the exquisitely precise, regioselective reactions that are hallmarks of RS enzymes. Here, our understanding of how the active site chaperones 5'-dAdo• is extended through the finding that this apparently unexceptional organic free radical has an anomalous g-tensor and exhibits significant 57Fe, 13C, 15N, and 2H hyperfine couplings to the adjacent, isotopically labeled, methionine-bound [4Fe-4S]2+ cluster cogenerated with 5'-dAdo• during homolytic cleavage of cluster-bound SAM. The origin of the 57Fe couplings through nonbonded radical-cluster contact is illuminated by a formal exchange-coupling model and broken symmetry-density functional theory computations. Incorporation of ENDOR-derived distances from C5'(dAdo•) to labeled-methionine as structural constraints yields a model for active-site positioning of 5'-dAdo• with a short, nonbonded C5'-Fe distance (∼3 Å). This distance involves substantial motion of 5'-dAdo• toward the unique Fe of the [4Fe-4S]2+ cluster upon S-C(5') bond-cleavage, plausibly an initial step toward formation of the Fe-C5' bond of the organometallic complex, Ω, the central intermediate in catalysis by radical-SAM enzymes.

Structural, Biochemical, and Bioinformatic Basis for Identifying Radical SAM Cyclopropyl Synthases.

The importance of radical S-adenosyl-l-methionine (RS) enzymes in the maturation of ribosomally synthesized and post-translationally modified peptides (RiPPs) continues to expand, specifically for the RS-SPASM subfamily. We recently discovered an RS-SPASM enzyme that installs a carbon-carbon bond between the geminal methyls of valine residues, resulting in the formation of cyclopropylglycine (CPG). Here, we sought to define the family of cyclopropyl (CP) synthases because of the importance of cyclopropane scaffolds in pharmaceutical development. Using RadicalSAM.org, we bioinformatically expanded the family of CP synthases and assigned unique peptide sequences to each subclade. We identified a unique RiPP biosynthetic pathway that encodes a precursor peptide, TigB, with a repeating TIGSVS motif. Using LCMS and NMR techniques, we show that the RS enzyme associated with the pathway, TigE, catalyzes the formation of a methyl-CPG from the conserved isoleucine residing in the repeating motif of TigB. Furthermore, we obtained a crystal structure of TigE, which reveals an unusual tyrosyl ligation to the auxiliary I [4Fe-4S] cluster, provided by a glycine-tyrosine-tryptophan motif unique to all CP synthases. Further, we show that this unique tyrosyl ligation is absolutely required for TigE activity. Together, our results provide insight into how CP synthases perform this unique reaction.

Hydrogen-Deuterium Exchange Mass Spectrometry Identifies Local and Long-Distance Interactions within the Multicomponent Radical SAM Enzyme, PqqE.

Interactions among proteins and peptides are essential for many biological activities including the tailoring of peptide substrates to produce natural products. The first step in the production of the bacterial redox cofactor pyrroloquinoline quinone (PQQ) from its peptide precursor is catalyzed by a radical SAM (rSAM) enzyme, PqqE. We describe the use of hydrogen-deuterium exchange mass spectrometry (HDX-MS) to characterize the structure and conformational dynamics in the protein-protein and protein-peptide complexes necessary for PqqE function. HDX-MS-identified hotspots can be discerned in binary and ternary complex structures composed of the peptide PqqA, the peptide-binding chaperone PqqD, and PqqE. Structural conclusions are supported by size-exclusion chromatography coupled to small-angle X-ray scattering (SEC-SAXS). HDX-MS further identifies reciprocal changes upon the binding of substrate peptide and S-adenosylmethionine (SAM) to the PqqE/PqqD complex: long-range conformational alterations have been detected upon the formation of a quaternary complex composed of PqqA/PqqD/PqqE and SAM, spanning nearly 40 Å, from the PqqA binding site in PqqD to the PqqE active site Fe4S4. Interactions among the various regions are concluded to arise from both direct contact and distal communication. The described experimental approach can be readily applied to the investigation of protein conformational communication among a large family of peptide-modifying rSAM enzymes.

Bacterial cyclophane-containing RiPPs from radical SAM enzymes.

Covering: 2016 to 2023Ribosomally synthesized and posttranslationally modified peptides (RiPPs) continue to be a rich source of chemically diverse and bioactive peptide natural products. In recent years, cyclophane-containing RiPP natural products and their biosynthetic pathways have been more frequently encountered. This highlight will focus on bacterial monoaryl cyclophane-containing RiPPs. This class of RiPPs is produced by radical SAM/SPASM enzymes that form a crosslink between the aromatic ring and sidechain of two amino acid residues of the precursor peptide. Selected natural products from these pathways exhibit specific antibacterial activity against gram-negative pathogens. The approaches used to discover these pathways and products will be described and categorized as natural product-first or enzyme-first. The breadth of ring systems formed by the enzymes, enzyme mechanism, and recent reports of synthetic methods for constructing these ring systems will also be presented. Bacterial cyclophane-containing RiPPs and their biosynthetic enzymes represent an untapped source of scaffolds for drug discovery and tools for synthetic biology.

Elucidation of factors shaping reactivity of 5'-deoxyadenosyl - a prominent organic radical in biology.

This study investigates the factors modulating the reactivity of 5'-deoxyadenosyl (5'dAdo˙) radical, a potent hydrogen atom abstractor that forms in the active sites of radical SAM enzymes and that otherwise undergoes a rapid self-decay in aqueous solution. Here, we compare hydrogen atom abstraction (HAA) reactions between native substrates of radical SAM enzymes and 5'dAdo˙ in aqueous solution and in two enzymatic microenvironments. With that we reveal that HAA efficiency of 5'dAdo˙ is due to (i) the in situ formation of 5'dAdo˙ in a pre-ordered complex with a substrate, which attenuates the unfavorable effect of substrate:5'dAdo˙ complex formation, and (ii) the prevention of the conformational changes associated with self-decay by a tight active-site cavity. The enzymatic cavity, however, does not have a strong effect on the HAA activity of 5'dAdo˙. Thus, we performed an analysis of in-water HAA performed by 5'dAdo˙ based on a three-component thermodynamic model incorporating the diagonal effect of the free energy of reaction, and the off-diagonal effect of asynchronicity and frustration. To this aim, we took advantage of the straightforward relationship between the off-diagonal thermodynamic effects and the electronic-structure descriptor - the redistribution of charge between the reactants during the reaction. It allows to access HAA-competent redox and acidobasic properties of 5'dAdo˙ that are otherwise unavailable due to its instability upon one-electron reduction and protonation. The results show that all reactions feature a favourable thermodynamic driving force and tunneling, the latter of which lowers systematically barriers by ∼2 kcal mol-1. In addition, most of the reactions experience a favourable off-diagonal thermodynamic contribution. In HAA reactions, 5'dAdo˙ acts as a weak oxidant as well as a base, also 5'dAdo˙-promoted HAA reactions proceed with a quite low degree of asynchronicity of proton and electron transfer. Finally, the study elucidates the crucial and dual role of asynchronicity. It directly lowers the barrier as a part of the off-diagonal thermodynamic contribution, but also indirectly increases the non-thermodynamic part of the barrier by presumably controlling the adiabatic coupling between proton and electron transfer. The latter signals that the reaction proceeds as a hydrogen atom transfer rather than a proton-coupled electron transfer.

A continuous fluorescence assay to measure nicotianamine synthase activity.

S-adenosylmethionine (SAM) is most widely known as the biological methylating agent of methyltransferases and for generation of radicals by the iron-sulfur dependent Radical SAM enzymes. SAM also serves as a substrate in biosynthetic reactions that harvest the aminobutyrate moiety of the methionine, producing methylthioadenosine as a co-product. These reactions are found in the production of polyamines such as spermine, siderophores derived from nicotianamine, and opine metallophores staphylopine and pseudopaline, among others. This procedure defines a highly sensitive, continuous fluorescence assay for the determination of steady state kinetic parameters for enzymes that generate the co-product methylthioadenosine.

Trapping the substrate radical of heme synthase AhbD.

The heme synthase AhbD catalyzes the last step of the siroheme-dependent heme biosynthesis pathway, which is operative in archaea and sulfate-reducing bacteria. The AhbD-catalyzed reaction consists of the oxidative decarboxylation of two propionate side chains of iron-coproporphyrin III to the corresponding vinyl groups of heme b. AhbD is a Radical SAM enzyme employing radical chemistry to achieve the decarboxylation reaction. Previously, it was proposed that the central iron ion of the substrate iron-coproporphyrin III participates in the reaction by enabling electron transfer from the initially formed substrate radical to an iron-sulfur cluster in AhbD. In this study, we investigated the substrate radical that is formed during AhbD catalysis. While the iron-coproporphyrinyl radical was not detected by electron paramagnetic resonance (EPR) spectroscopy, trapping and visualization of the substrate radical was successful by employing substrate analogs such as coproporphyrin III and zinc-coproporphyrin III. The radical signals detected by EPR were analyzed by simulations based on density functional theory (DFT) calculations. The observed radical species on the substrate analogs indicate that hydrogen atom abstraction takes place at the β-position of the propionate side chain and that an electron donating ligand is located in proximity to the central metal ion of the porphyrin.

Unusual cysteine modifications in natural product biosynthesis.

l-Cysteine is a highly reactive amino acid that is modified into a variety of chemical structures, including cysteine sulfinic acid in human metabolic pathways, and sulfur-containing scaffolds of amino acids, alkaloids, and peptides in natural product biosynthesis. Among the modification enzymes responsible for these cysteine-derived compounds, metalloenzymes constitute an important family of enzymes that catalyze a wide variety of reactions. Therefore, understanding their reaction mechanisms is important for the biosynthetic production of cysteine-derived natural products. This review mainly summarizes recent mechanistic investigations of metalloenzymes, with a particular focus on recently discovered mononuclear non-heme iron (NHI) enzymes, dinuclear NHI enzymes, and radical-SAM enzymes involved in unusual cysteine modifications in natural product biosynthesis.

Structural and mechanistic basis for RiPP epimerization by a radical SAM enzyme.

D-Amino acid residues, found in countless peptides and natural products including ribosomally synthesized and post-translationally modified peptides (RiPPs), are critical for the bioactivity of several antibiotics and toxins. Recently, radical S-adenosyl-L-methionine (SAM) enzymes have emerged as the only biocatalysts capable of installing direct and irreversible epimerization in RiPPs. However, the mechanism underpinning this biochemical process is ill-understood and the structural basis for this post-translational modification remains unknown. Here we report an atomic-resolution crystal structure of a RiPP-modifying radical SAM enzyme in complex with its substrate properly positioned in the active site. Crystallographic snapshots, size-exclusion chromatography-small-angle x-ray scattering, electron paramagnetic resonance spectroscopy and biochemical analyses reveal how epimerizations are installed in RiPPs and support an unprecedented enzyme mechanism for peptide epimerization. Collectively, our study brings unique perspectives on how radical SAM enzymes interact with RiPPs and catalyze post-translational modifications in natural products.

Structural Insights and Mechanistic Understanding of Iron-Molybdenum Cofactor Biosynthesis by NifB in Nitrogenase Assembly Process.

NifB, a radical S-adenosylmethionine (SAM) enzyme, is pivotal in the biosynthesis of the iron-molybdenum cofactor (FeMo-co), commonly referred to as the M-cluster. This cofactor, located within the active site of nitrogenase, is essential for the conversion of dinitrogen (N2) to NH3. Recognized as the most intricate metallocluster in nature, FeMo-co biosynthesis involves multiple proteins and a sequence of steps. Of particular significance, NifB directs the fusion of two [Fe4S4] clusters to assemble the 8Fe core, while also incorporating an interstitial carbide. Although NifB has been extensively studied, its molecular mechanisms remain elusive. In this review, we explore recent structural analyses of NifB and provide a comprehensive overview of the established catalytic mechanisms. We propose prospective directions for future research, emphasizing the relevance to biochemistry, agriculture, and environmental science. The goal of this review is to lay a solid foundation for future endeavors aimed at elucidating the atomic details of FeMo-co biosynthesis.

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.