S-adenosylmethionine Molecules Research Articles

Theoretical study on the mechanism of tryptophan methylation catalyzed by the cobalamin-dependent radical enzyme TsrM

TsrM is a radical S-adenosylmethionine (SAM) methylase that catalyzes the C2 methylation of L-tryptophan to produce 2-methyltryptophan, an intermediate in the biosynthesis of the antibiotic thiostrepton A. The mechanism of TsrM is investigated through the quantum chemical cluster approach. The calculations suggested an expanded ‘ping-pong’ mechanism that requires the participation of two SAM molecules. In the first half-reaction, one SAM molecule binds to the active site of TsrM and delivers its methyl group to the CoI ion of the cobalamin, leading to the generation of methylcobalamin. With the subsequent dissociation of a SAH molecule, another SAM molecule binds into the active site, which facilitates the transfer of the methyl group from methylcobalamin to the C2 of tryptophan via a non-radical mechanism. The resulting carbon cation intermediate then readily undergoes facile deprotonation to yield the final product 2-methyltryptophan. Our proposed mechanism also rationalizes the reactivity trends observed among four substrates with different substitutions.

Restriction of S-adenosylmethionine conformational freedom by knotted protein binding sites.

S-adenosylmethionine (SAM) is one of the most important enzyme substrates. It is vital for the function of various proteins, including large group of methyltransferases (MTs). Intriguingly, some bacterial and eukaryotic MTs, while catalysing the same reaction, possess significantly different topologies, with the former being a knotted one. Here, we conducted a comprehensive analysis of SAM conformational space and factors that affect its vastness. We investigated SAM in two forms: free in water (via NMR studies and explicit solvent simulations) and bound to proteins (based on all data available in the PDB and on all-atom molecular dynamics simulations in water). We identified structural descriptors-angles which show the major differences in SAM conformation between unknotted and knotted methyltransferases. Moreover, we report that this is caused mainly by a characteristic for knotted MTs compact binding site formed by the knot and the presence of adenine-binding loop. Additionally, we elucidate conformational restrictions imposed on SAM molecules by other protein groups in comparison to conformational space in water.

The interdimeric interface controls function and stability of Ureaplasma urealiticum methionine S-adenosyltransferase

Methionine S-adenosyltransferases (MATs) are predominantly homotetramers, comprised of dimers of dimers. The larger, highly conserved intradimeric interface harbors two active sites, making the dimer the obligatory functional unit. However, functionality of the smaller, more diverged, and recently evolved interdimeric interface is largely unknown. Here, we show that the interdimeric interface of Ureaplasmaurealiticum MAT has evolved to control the catalytic activity and structural integrity of the homotetramer in response to product accumulation. When all four active sites are occupied with the product, S-adenosylmethionine (SAM), binding of four additional SAM molecules to the interdimeric interface prompts a ∼45° shift in the dimer orientation and a concomitant ∼60% increase in the interface area. This rearrangement inhibits the enzymatic activity by locking the flexible active site loops in a closed state and renders the tetramer resistant to proteolytic degradation. Our findings suggest that the interdimeric interface of MATs is subject to rapid evolutionary changes that tailor the molecular properties of the entire homotetramer to the specific needs of the organism.

Paramagnetic 1H NMR Spectroscopy to Investigate the Catalytic Mechanism of Radical S-Adenosylmethionine Enzymes

Iron–sulfur clusters in radical S-adenosylmethionine (SAM) enzymes catalyze an astonishing array of complex and chemically challenging reactions across all domains of life. Here we showed that 1H NMR spectroscopy experiments tailored to reveal hyperfine-shifted signals of metal-ligands is a powerful tool to monitor the binding of SAM and of the octanoyl-peptide substrate to the two [4Fe-4S] clusters of human lipoyl synthase. The paramagnetically shifted signals of the iron-ligands were specifically assigned to each of the two bound [4Fe-4S] clusters, and then used to examine the interaction of SAM and substrate molecules with each of the two [4Fe-4S] clusters of human lipoyl synthase. 1H NMR spectroscopy can therefore contribute to the description of the catalityc mechanism of radical SAM enzymes.

New mechanism for a methylating enzyme

A team of researchers in China has found a reaction mechanism unprecedented for the many enzymes that add methyl groups to biomolecules. Most of these so-called methyltransferases depend on S-adenosylmethionine (SAM) as a methyl donor, plucking off the molecule’s methyl group and relocating it either through an SN2 mechanism or through a radical mechanism. Many scientists thought this type of SAM-dependent enzyme exclusively produced S-adenosylhomocysteine as a coproduct during the transfer. A team led by Qi Zhang of Fudan University reports that’s not always the case. NosN, a class C radical SAM methyltransferase involved in the biosynthesis of a thiopeptide antibiotic, uses a different methyl donor and produces thioadenosine as a coproduct (Angew. Chem. Int. Ed. 2017, DOI: 10.1002/anie.201609948). Knowledge of this mechanism could help researchers reengineer the enzyme to produce other compounds, Zhang says. Although NosN uses two SAM molecules, neither of them serves directly as the methyl donor.

Immunoassay of S-adenosylmethionine and S-adenosylhomocysteine: the methylation index as a biomarker for disease and health status.

Background S-Adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are relevant to a variety of diseases. Previous reports that quantified SAM and SAH were based on HPLC or LC–MS/MS. No antibody against SAM has been generated, and the antibody against SAH cannot be used with blood samples. Immunoassays have not been used to measure SAM and SAH. In this study, ELISA was used to measure blood SAM and SAH levels.ResultsSpecific antibodies against SAM were produced for the first time using a stable analog as the antigen. The monoclonal antibodies against SAM and SAH were characterized. No cross-reactivity was detected for the analyzed analogs. For the anti-SAM antibodies, the ELISA sensitivity was ~2 nM, and the affinity was 7.29 × 1010 L/mol. For the anti-SAH antibodies, the sensitivity was ~15 nM, and the affinity was 2.79 × 108 L/mol. Using high-quality antibodies against SAM and SAH, immunoassays for the detection of SAM and SAH levels in blood and tissue samples were developed. Clinical investigations using immunoassays to measure SAM, SAH and the methylation index (MI) in normal and diseased samples indicated that (1) the SAM level is age and gender dependent; (2) the SAM level is associated with the severity of liver diseases, inflammatory reactions and other diseases; and (3) the methylation index (MI) is significantly reduced in many diseases and may serve as a screening biomarker to identify potentially unfavorable health conditions.ConclusionIt is possible to generate antibodies against active small biomolecules with weak immunogenicity, such as SAM and SAH, using traditional hybridoma technology. The antigens and antibodies described here will contribute to the development of immunoassays to measure SAM, SAH and related molecules. These assays enable the MI to be measured specifically, accurately, easily and quickly without costly equipment. This preliminary study indicates that the MI could be an effective indicator of general health, except under conditions that may alter the value of the MI, such as special diets and medications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-2296-8) contains supplementary material, which is available to authorized users.

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.

The Biosynthesis of the Indolic Acid Moiety from the Antibiotic Nosiheptide

Nosiheptide (NOS) is a highly modified thiopeptide natural product produced by Streptomyces actuosus that belongs to a unique class of thiazole‐containing peptide antibiotics. Recent studies have shown that NOS is as an effective antibacterial agent against a number of pathogenic bacteria, such as methicillin resistant Staphylococcus aureus, penicillin‐resistant Streptococcus pneumoniae, and vancomycin‐resistant enterococci. One key structural feature of NOS essential for its antibacterial properties is an indolic acid ring system characteristic of e‐series thiopeptides. The indolic acid moiety is derived from the amino acid tryptophan. In the first step of the proposed pathway, catalyzed by NosL, tryptophan is converted into 3‐methyl indolic acid (MIA). The remaining steps and enzymes involved in the formation and installation of MIA into the NOS core have not yet been definitively determined, but are believed to be catalyzed by the action of four enzymes: NosI, NosJ, NosK, and NosN. The research herein uses isothermal titration calorimetry (ITC), high‐pressure liquid chromatography (HPLC), mass spectrometry, and bioinformatics to delineate the exact roles of each of the enzymes in the biosynthesis of the indolic acid moiety. We show that NosJ is an acyl carrier protein that is post‐translationally modified with a 4′‐phosphopantetheine group, and that NosI catalyzes the ATP‐dependent adenylation of MIA. Upon incubation of NosI with ATP and MIA in the presence of NosJ, we observe transfer of MIA to NosJ. We believe that NosK then catalyzes the transfer of MIA from NosJ to the side‐chain of a cysteinyl residue of NOS. NosN, annotated as a radical S‐adenosylmethionine (SAM) methylase, subsequently attaches a C1 unit—suggested to be a methyl group—to C4 of the indolic acid moiety and then connects the C1 unit to Glu 43 of the macrocyclic core, completing the synthesis of NOS. We are particularly interested in understanding how NosN carries out this reaction. Preliminary ITC experiments show that two SAM molecules bind to the enzyme simultaneously. We suggest that one SAM molecule is converted into a 5′‐deoxyadenosyl 5′‐radical that initiates a novel methylation reaction by abstracting a hydrogen atom from the methyl moiety of the second SAM molecule. These studies shed new light on novel mechanisms for methylating unactivated carbon centers.Support or Funding InformationThis work was supported by NIH (GM103268), the President's Undergraduate Research Fund (Penn State), and the Rodney Erickson Discovery Grant (Penn State).

Characterisation of Desulfovibrio vulgaris haem b synthase, a radical SAM family member

An alternative route for haem b biosynthesis is operative in sulfate-reducing bacteria of the Desulfovibrio genus and in methanogenic Archaea. This pathway diverges from the canonical one at the level of uroporphyrinogen III and progresses via a distinct branch, where sirohaem acts as an intermediate precursor being converted into haem b by a set of novel enzymes, named the alternative haem biosynthetic proteins (Ahb). In this work, we report the biochemical characterisation of the Desulfovibrio vulgaris AhbD enzyme that catalyses the last step of the pathway. Mass spectrometry analysis showed that AhbD promotes the cleavage of S-adenosylmethionine (SAM) and converts iron-coproporphyrin III via two oxidative decarboxylations to yield haem b, methionine and the 5′-deoxyadenosyl radical. Electron paramagnetic resonance spectroscopy studies demonstrated that AhbD contains two [4Fe–4S]2+/1+ centres and that binding of the substrates S-adenosylmethionine and iron-coproporphyrin III induces conformational modifications in both centres. Amino acid sequence comparisons indicated that D. vulgaris AhbD belongs to the radical SAM protein superfamily, with a GGE-like motif and two cysteine-rich sequences typical for ligation of SAM molecules and iron-sulfur clusters, respectively. A structural model of D. vulgaris AhbD with putative binding pockets for the iron-sulfur centres and the substrates SAM and iron-coproporphyrin III is discussed.

Quantum chemistry studies of adenosine 2503 methylation by S-adenosylmethionine-dependent enzymes

Ribosome methylation is important for life processes and is mainly catalyzed by radical S-Adenosylmethionine (SAM) enzymes. Two SAM molecules serve as the cofactor by providing the 5 ′-deoxyadenosyl radical for substrate activation and the methyl. Recently, Booker and coworkers (Science 2011, 332, 604) proposed an alternative mechanism for a pair of radical SAM enzymes, RlmN and Cfr, which respectively methylate the C2 and C8 of adenosine 2503. Their deuterium labeling experiments reveal that methyl group does not transfer directly from SAM to adenosine, instead it passes to Cys355 first, then onto adenosine. In this article, this new reaction mechanism is studied using density functional theory with B3LYP hybrid functional. The reaction system is simulated using small model compounds in the gas phase, and the protein environment is approximated using polarizable continuum model. The structures of the transition states and the intermediates are identified, and their free energies are calculated. The activation barriers indicate that the proposed reaction mechanism is plausible. The formation of a disulfide bond is found to be the rate-limiting step. © 2012 Wiley Periodicals, Inc.

Eco-Catalysis Leads the Way to Green Synthetic Chemistry

Methylation is an essential and ubiquitous reaction that plays an important role in a wide range of biological processes. Most biological methylations use S-adenosylmethionine (SAM) as the methyl donor and proceed via an S(N)2 displacement mechanism. However, researchers have discovered an increasing number of methylations that involve radical chemistry. The enzymes known to catalyze these reactions all belong to the radical SAM superfamily. This family of enzymes utilizes a specialized [4Fe-4S] cluster for reductive cleavage of SAM to yield a highly reactive 5'-deoxyadenosyl (dAdo) radical. Radical chemistry is then imposed on a variety of organic substrates, leading to a diverse array of transformations. Until recently, researchers had not fully understood how these enzymes employ radical chemistry to mediate a methyl transfer reaction. Sequence analyses reveal that the currently identified radical SAM methyltransferases (RSMTs) can be grouped into three classes, which appear distinct in protein architecture and mechanism. Class A RSMTs mainly include the rRNA methyltransferases RImN and Or from various origins. As exemplified by Escherichia coil RImN, these proteins have a single canonical radical SAM core domain that includes an (beta alpha)(6) partial barrel most similar to that of pyruvate formate lyase-activase. The exciting recent studies on RImN and Cfr are beginning to provide insights into the intriguing chemistry of class A RSMTs. These enzymes utilize a methylene radical generated on a unique methylated cysteine residue. However, based on the variety of substrates used by the other classes of RSMTs, alternative mechanisms are likely to be discovered. Class B RSMTs contain a proposed N-terminal cobalamin binding domain in addition to a radical SAM domain at the C-terminus. This class of proteins methylates diverse substrates at inert sp(3) carbons, aromatic heterocycles, and phosphinates, possibly involving a cobalamin-mediated methyl transfer process. Class C RSMTs share significant sequence similarity with coproporphyrinogen III oxidase HemN. Despite methylating similar substrates (aromatic heterocycles), class C RSMTs likely employ a mechanism distinct from that of class A because two conserved cysteines that are required for class A are typically not found in class C RSMTs. Class A and class B enzymes probably share the use of two molecules of SAM: one to generate a dAdo radical and one to provide the methyl group to the substrate. In class A, a cysteine would act as a conduit of the methyl group whereas In class B cobalamin may serve this purpose. Currently no clues are available regarding the mechanism of class C RSMTs, but the sequence similarities between its members and HemN and the observation that HemN binds two SAM molecules suggest that class C enzymes could use two SAM molecules for catalysis. The diverse strategies for using two SAM molecules reflect the rich chemistry of radical-mediated methylation reactions and ...

Radical-Mediated Enzymatic Methylation: A Tale of Two SAMS

Methylation is an essential and ubiquitous reaction that plays an important role in a wide range of biological processes. Most biological methylations use S-adenosylmethionine (SAM) as the methyl donor and proceed via an SN2 displacement mechanism. However, researchers have discovered an increasing number of methylations that involve radical chemistry. The enzymes known to catalyze these reactions all belong to the radical SAM superfamily. This family of enzymes utilizes a specialized [4Fe-4S] cluster for reductive cleavage of SAM to yield a highly reactive 5'-deoxyadenosyl (dAdo) radical. Radical chemistry is then imposed on a variety of organic substrates, leading to a diverse array of transformations. Until recently, researchers had not fully understood how these enzymes employ radical chemistry to mediate a methyl transfer reaction.Sequence analyses reveal that the currently identified radical SAM methyltransferases (RSMTs) can be grouped into three classes, which appear distinct in protein architecture and mechanism. Class A RSMTs mainly include the rRNA methyltransferases RlmN and Cfr from various origins. As exemplified by Escherichia coli RlmN, these proteins have a single canonical radical SAM core domain that includes an (βα)6 partial barrel most similar to that of pyruvate formate lyase-activase. The exciting recent studies on RlmN and Cfr are beginning to provide insights into the intriguing chemistry of class A RSMTs. These enzymes utilize a methylene radical generated on a unique methylated cysteine residue. However, based on the variety of substrates used by the other classes of RSMTs, alternative mechanisms are likely to be discovered. Class B RSMTs contain a proposed N-terminal cobalamin binding domain in addition to a radical SAM domain at the C-terminus. This class of proteins methylates diverse substrates at inert sp3 carbons, aromatic heterocycles, and phosphinates, possibly involving a cobalamin-mediated methyl transfer process. Class C RSMTs share significant sequence similarity with coproporphyrinogen III oxidase HemN. Despite methylating similar substrates (aromatic heterocycles), class C RSMTs likely employ a mechanism distinct from that of class A because two conserved cysteines that are required for class A are typically not found in class C RSMTs.Class A and class B enzymes probably share the use of two molecules of SAM: one to generate a dAdo radical and one to provide the methyl group to the substrate. In class A, a cysteine would act as a conduit of the methyl group whereas in class B cobalamin may serve this purpose. Currently no clues are available regarding the mechanism of class C RSMTs, but the sequence similarities between its members and HemN and the observation that HemN binds two SAM molecules suggest that class C enzymes could use two SAM molecules for catalysis. The diverse strategies for using two SAM molecules reflect the rich chemistry of radical-mediated methylation reactions and the remarkable versatility of the radical SAM superfamily.

Allosteric activation of Arabidopsis threonine synthase by S-adenosylmethionine.

Plant threonine synthase, in contrast to its bacterial counterpart, is strongly stimulated by S-adenosylmethionine via a noncovalent interaction [Giovanelli et al. (1984) Plant. Physiol. 76, 285-292]. The mechanism of activation remained, however, largely unknown. To further characterize this unusual role for S-adenosylmethionine, the Arabidopsis thaliana threonine synthase was overexpressed in Escherichia coli, purified to homogeneity, and then used for kinetic and enzyme-bound pyridoxal 5'-phosphate fluorescence equilibrium-binding experiments. We observed that the activating effect of S-adenosylmethionine results from an 8-fold increase in the rate of catalysis and from a 25-fold decrease in the Km value for the O-phosphohomoserine substrate. The data can be well fitted by a kinetic model assuming binding of two S-adenosylmethionine molecules on the native enzyme. We suggest that the dramatic modifications of the enzyme kinetic properties originate most presumably from an allosteric and cooperative transition induced by S-adenosylmethionine. This transition occurs at a much faster rate in the presence of the substrate than in its absence.

The EcoDXX1 restriction and modification system of Escherichia coli ET7. Purification, subunit structure and properties of the restriction endonuclease.

The Escherichia coli plasmid pDXX1 codes for a new restriction-modification system. The specific restriction endonuclease coded by this system has been purified by a procedure that includes phosphocellulose and heparin-agarose chromatography. Sedimentation on glycerol gradients showed one peak of activity with a value of about 12 S. The highly purified enzyme require ATP and Mg2+ for activity as well as S-adenosylmethionine, although some S-adenosylmethionine molecules are probably bound to the enzyme. The enzyme does not cleave lambda DNA at well-defined sites and has a strong non-modified DNA-dependent ATPase activity. The enzyme has also methylase activity acting against non-modified DNA.

Direct proof of propionate involvement in choline synthesis in rats

Choline, which belongs to the vitamins group, is a biologically important substance found in animals [5, 6]. Endogenous choline is formed in animals by the transfer of methyl groups from three S-adenosylmethionine molecules to the amino group of the ethanolamine residue in the structure of phosphatidylethanolamine. As a result of this reaction phosphatidylcholine is formed. However, the endogenous pathway of choline synthesis is insufficient to supply the physiological needs of the body, and for that reason choline intake in the diet is essential.