Twenty Years of Radical SAM! The Genesis of the Superfamily.
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ABSTRACT:
SUBMITTER: Booker SJ
PROVIDER: S-EPMC10114671 | biostudies-literature | 2022 Dec
REPOSITORIES: biostudies-literature
Publications
Twenty Years of Radical SAM! The Genesis of the Superfamily.
Booker Squire J SJ Lloyd Cody T CT
ACS bio & med chem Au 20221205 6
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Project description: Not available
| S-EPMC5930932 | biostudies-literature
Project description:4-Amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase catalyzes a complex rearrangement of 5-aminoimidazole ribonucleotide (AIR) to form HMP-P, the pyrimidine moiety of thiamine phosphate. We determined the three-dimensional structures of HMP-P synthase and its complexes with the product HMP-P and a substrate analog imidazole ribotide. The structure of HMP-P synthase reveals a homodimer in which each protomer comprises three domains: an N-terminal domain with a novel fold, a central (betaalpha)(8) barrel and a disordered C-terminal domain that contains a conserved CX(2)CX(4)C motif, which is suggestive of a [4Fe-4S] cluster. Biochemical studies have confirmed that HMP-P synthase is iron sulfur cluster-dependent, that it is a new member of the radical SAM superfamily and that HMP-P and 5'-deoxyadenosine are products of the reaction. Mössbauer and EPR spectroscopy confirm the presence of one [4Fe-4S] cluster. Structural comparisons reveal that HMP-P synthase is homologous to a group of adenosylcobalamin radical enzymes. This similarity supports an evolutionary relationship between these two superfamilies.
| S-EPMC2587053 | biostudies-literature
Project description:The radical SAM superfamily contains over 100,000 homologous enzymes that catalyze a remarkably broad range of reactions required for life, including metabolism, nucleic acid modification, and biogenesis of cofactors. While the highly conserved SAM-binding motif responsible for formation of the key 5'-deoxyadenosyl radical intermediate is a key structural feature that simplifies identification of superfamily members, our understanding of their structure-function relationships is complicated by the modular nature of their structures, which exhibit varied and complex domain architectures. To gain new insight about these relationships, we classified the entire set of sequences into similarity-based subgroups that could be visualized using sequence similarity networks. This superfamily-wide analysis reveals important features that had not previously been appreciated from studies focused on one or a few members. Functional information mapped to the networks indicates which members have been experimentally or structurally characterized, their known reaction types, and their phylogenetic distribution. Despite the biological importance of radical SAM chemistry, the vast majority of superfamily members have never been experimentally characterized in any way, suggesting that many new reactions remain to be discovered. In addition to 20 subgroups with at least one known function, we identified additional subgroups made up entirely of sequences of unknown function. Importantly, our results indicate that even general reaction types fail to track well with our sequence similarity-based subgroupings, raising major challenges for function prediction for currently identified and new members that continue to be discovered. Interactive similarity networks and other data from this analysis are available from the Structure-Function Linkage Database.
| S-EPMC6445391 | biostudies-literature
Project description:RimO, encoded by the yliG gene in Escherichia coli, has been recently identified in vivo as the enzyme responsible for the attachment of a methylthio group on the beta-carbon of Asp88 of the small ribosomal protein S12 [Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 1826-1831]. To date, it is the only enzyme known to catalyze methylthiolation of a protein substrate; the four other naturally occurring methylthio modifications have been observed on tRNA. All members of the methylthiotransferase (MTTase) family, to which RimO belongs, have been shown to contain the canonical CxxxCxxC motif in their primary structures that is typical of the radical S-adenosylmethionine (SAM) family of proteins. MiaB, the only characterized MTTase, and the enzyme experimentally shown to be responsible for methylthiolation of N(6)-isopentenyladenosine of tRNA in E. coli and Thermotoga maritima, has been demonstrated to harbor two distinct [4Fe-4S] clusters. Herein, we report in vitro biochemical and spectroscopic characterization of RimO. We show by analytical and spectroscopic methods that RimO, overproduced in E. coli in the presence of iron-sulfur cluster biosynthesis proteins from Azotobacter vinelandii, contains one [4Fe-4S](2+) cluster. Reconstitution of this form of RimO (RimO(rcn)) with (57)Fe and sodium sulfide results in a protein that contains two [4Fe-4S](2+) clusters, similar to MiaB. We also show by mass spectrometry that RimO(rcn) catalyzes the attachment of a methylthio group to a peptide substrate analogue that mimics the loop structure bearing aspartyl 88 of the S12 ribosomal protein from E. coli. Kinetic analysis of this reaction shows that the activity of RimO(rcn) in the presence of the substrate analogue does not support a complete turnover. We discuss the possible requirement for an assembled ribosome for fully active RimO in vitro. Our findings are consistent with those of other enzymes that catalyze sulfur insertion, such as biotin synthase, lipoyl synthase, and MiaB.
| S-EPMC2952840 | biostudies-other
Project description:Enzymes in the radical SAM (RS) superfamily catalyze a wide variety of reactions through unique radical chemistry. The characteristic markers of the superfamily include a [4Fe-4S] cluster coordinated to the protein via a cysteine triad motif, typically CX(3)CX(2)C, with the fourth iron coordinated by S-adenosylmethionine (SAM). The SAM serves as a precursor for a 5'-deoxyadenosyl radical, the central intermediate in nearly all RS enzymes studied to date. The SAM-bound [4Fe-4S] cluster is located within a partial or full triosephosphate isomerase (TIM) barrel where the radical chemistry occurs protected from the surroundings. In addition to the TIM barrel and a RS [4Fe-4S] cluster, many members of the superfamily contain additional domains and/or additional Fe-S clusters. Recently characterized superfamily members are providing new examples of the remarkable range of reactions that can be catalyzed, as well as new structural and mechanistic insights into these fascinating reactions.
| S-EPMC4083504 | biostudies-literature
Project description:About two decades ago, cloning of the autoimmune regulator (AIRE) gene materialized one of the most important actors on the scene of self-tolerance. Thymic transcription of genes encoding tissue-specific antigens (ts-ags) is activated by AIRE protein and embodies the essence of thymic self-representation. Pathogenic AIRE variants cause the autoimmune polyglandular syndrome type 1, which is a rare and complex disease that is gaining attention in research on autoimmunity. The animal models of disease, although not identically reproducing the human picture, supply fundamental information on mechanisms and extent of AIRE action: thanks to its multidomain structure, AIRE localizes to chromatin enclosing the target genes, binds to histones, and offers an anchorage to multimolecular complexes involved in initiation and post-initiation events of gene transcription. In addition, AIRE enhances mRNA diversity by favoring alternative mRNA splicing. Once synthesized, ts-ags are presented to, and cause deletion of the self-reactive thymocyte clones. However, AIRE function is not restricted to the activation of gene transcription. AIRE would control presentation and transfer of self-antigens for thymic cellular interplay: such mechanism is aimed at increasing the likelihood of engagement of the thymocytes that carry the corresponding T-cell receptors. Another fundamental role of AIRE in promoting self-tolerance is related to the development of thymocyte anergy, as thymic self-representation shapes at the same time the repertoire of regulatory T cells. Finally, AIRE seems to replicate its action in the secondary lymphoid organs, albeit the cell lineage detaining such property has not been fully characterized. Delineation of AIRE functions adds interesting data to the knowledge of the mechanisms of self-tolerance and introduces exciting perspectives of therapeutic interventions against the related diseases.
| S-EPMC5816566 | biostudies-literature
Project description:TYW1 is a radical S-adenosyl-l-methionine (SAM) enzyme that catalyzes the condensation of pyruvate and N-methylguanosine-containing tRNAPhe, forming 4-demethylwyosine-containing tRNAPhe. Homologues of TYW1 are found in both archaea and eukarya; archaeal homologues consist of a single domain, while eukaryal homologues contain a flavin binding domain in addition to the radical SAM domain shared with archaeal homologues. In this study, TYW1 from Saccharomyces cerevisiae (ScTYW1) was heterologously expressed in Escherichia coli and purified to homogeneity. ScTYW1 is purified with 0.54 ± 0.07 and 4.2 ± 1.9 equiv of flavin mononucleotide (FMN) and iron, respectively, per mole of protein, suggesting the protein is ∼50% replete with Fe-S clusters and FMN. While both NADPH and NADH are sufficient for activity, significantly more product is observed when used in combination with flavin nucleotides. ScTYW1 is the first example of a radical SAM flavoenzyme that is active with NAD(P)H alone.
| S-EPMC8941971 | biostudies-literature
Project description:Radical S-adenosyl-l-methionine (SAM) enzymes are a large and diverse superfamily with functions ranging from enzyme activation through a single H atom abstraction to complex organic and metal cofactor synthesis involving a series of steps. Though these enzymes carry out a variety of functions, they share common structural and mechanistic characteristics. All of them contain a site-differentiated [4Fe-4S] cluster, ligated by a CX(3)CX(2)C or similar motif, which binds SAM at the unique iron. The [4Fe-4S](1+) state of the cluster reductively cleaves SAM to produce a 5'-deoxyadenosyl radical, which serves to initiate the diverse reactions catalyzed by these enzymes. Recent highlights in the understanding of radical SAM enzymes will be presented, with a particular emphasis on enzymes catalyzing methylation and methythiolation reactions.
| S-EPMC4012327 | biostudies-literature
Project description: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.
| S-EPMC7990684 | biostudies-literature
Project description:DesII is a member of the radical SAM family of enzymes that catalyzes radical-mediated transformations of TDP-4-amino-4,6-didexoy-D-glucose as well as other sugar nucleotide diphosphates. Like nearly all radical SAM enzymes, the reactions begin with the reductive homolysis of SAM to produce a 5'-deoxyadenosyl radical which is followed by regiospecific hydrogen atom abstraction from the substrate. What happens next, however, depends on the nature of the substrate radical so produced. In the case of the biosynthetically relevant substrate, a radical-mediated deamination ensues; however, when this amino group is replaced with a hydroxyl, one instead observes dehydrogenation. The factors that govern the fate of the initially generated substrate radical as well as the mechanistic details underlying these transformations have been a key focus of research into the chemistry of DesII. This review will discuss recent discoveries pertaining to the enzymology of DesII, how it may relate to understanding other radical-mediated lyases and dehydrogenases and the working hypotheses currently being investigated regarding the mechanism of DesII catalysis.
| S-EPMC5021314 | biostudies-literature