Computational Approaches: An Underutilized Tool in the Quest to Elucidate Radical SAM Dynamics.
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ABSTRACT:
SUBMITTER: Blue TC
PROVIDER: S-EPMC8124187 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Posttranslational modification of ribosomally synthesized peptides provides an elegant means for the production of biologically active molecules known as RiPPs (ribosomally synthesized and posttranslationally modified peptides). Although the leader sequence of the precursor peptide is often required for turnover, the exact mode of recognition by the modifying enzymes remains unclear for many members of this class of natural products. Here, we have used X-ray crystallography and computational modeling to examine the role of the leader peptide in the biosynthesis of a homolog of streptide, a recently identified peptide natural product with an intramolecular lysine-tryptophan cross-link, which is installed by the radical S-adenosylmethionine (SAM) enzyme, StrB. We present crystal structures of SuiB, a close ortholog of StrB, in various forms, including apo SuiB, SAM-bound SuiB, and a complex of SuiB with SAM and its peptide substrate, SuiA. Although the N-terminal domain of SuiB adopts a typical RRE (RiPP recognition element) motif, which has been implicated in precursor peptide recognition, we observe binding of the leader peptide in the catalytic barrel rather than the N-terminal domain. Computational simulations support a mechanism in which the leader peptide guides posttranslational modification by positioning the cross-linking residues of the precursor peptide within the active site. Together the results shed light onto binding of the precursor peptide and the associated conformational changes needed for the formation of the unique carbon-carbon cross-link in the streptide family of natural products.
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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.
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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.
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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.
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Project description:Carbonic anhydrase IX is an important biomarker to fight hypoxic tumours in both initial and metastatic stages of many forms of cancer. Overexpression of hCA IX in the hypoxic environment, has an active role in pH maintenance and makes the hCA IX a better target for the inhibitors targeting specific types of cancer stages. Being a member of the carbonic anhydrase family and having sixteen isoforms, it is important to have a selective inhibition of hCA IX to limit the disruption in the biological and metabolic pathways where other isoforms of hCA are localised and to avoid the other toxicity and adverse effects we try to find selective hCA IX inhibitors from a natural derivative. In the process of finding selective hCA inhibitors we developed a pharmacophore model based on existing inhibitors with IC50 values of less than 50 nm, which is then validated with the external decoy set and used for database searching followed by virtual screening to identify the hits based on the pharmacophore fit score and RMSD. Molecular docking studies were performed to identify protein ligand interaction and molecular dynamics simulation studies to analyse the stability of the complex and DFT studies were carried out. The initial screening yielded 43 hits with the RMSD value less than 1, which when subjected to docking exhibited very good interaction with key residues ZN301, HIS94, HIS96 and HIS119. The top 4 compounds in the molecular dynamics simulation studies for 100 ns provided useful insights on the stability of the complex and the DFT studies confirmed the energy variation between HOMO and LUMO is within an acceptable range. An average binding score of -7.8 Kcal mol-1 for the lead compounds and high stability margin in the dynamics study concludes that these lead compounds demonstrated outstanding potential for hCA IX inhibitory action theoretically and that further experimental studies for selective inhibition are inevitable.
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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.
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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.
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Project description:Enzymes of the radical SAM (RS) superfamily catalyze a diverse assortment of reactions that proceed via intermediates containing unpaired electrons. The radical initiator is the common metabolite S-adenosyl-l-methionine (SAM), which is reductively cleaved to generate a 5'-deoxyadenosyl 5'-radical, a universal and obligate intermediate among enzymes within this class. A bioinformatics study that appeared in 2001 indicated that this superfamily contained over 600 members, many catalyzing reactions that were rich in novel chemical transformations. Since that seminal study, the RS superfamily has grown immensely, and new details about the scope of reactions and biochemical pathways in which its members participate have emerged. This review will highlight only a few of the most significant findings from the past 2-3 years, focusing primarily on: RS enzymes involved in complex metallocofactor maturation; characterized RS enzymes that lack the canonical CxxxCxxC motif; RS enzymes containing multiple iron-sulfur clusters; RS enzymes catalyzing reactions with compelling medical implications; and the energetics and mechanism of generating the 5'-deoxyadenosyl radical. A number of significant studies of RS enzymes will unfortunately be omitted, and it is hoped that the reader will access the relevant literature - particularly a number of superb review articles recently written on the subject - to acquire a deeper appreciation of this class of enzymes.
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Project description:Nikkomycins and polyoxins are antifungal peptidylnucleoside antibiotics active against human and plant pathogens. Here we report that during peptidylnucleoside biosynthesis in Streptomyces cacaoi and S. tendae, the C5' extension of the nucleoside essential for downstream structural diversification is catalyzed by a conserved radical S-adenosyl-L-methionine (SAM) enzyme, PolH or NikJ. This is distinct from the nucleophilic mechanism reported for antibacterial nucleosides and represents a new mechanism of nucleoside natural product biosynthesis.
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Project description:Radical S-adenosylmethionine (SAM) enzymes exist in organisms from all kingdoms of life, and all of these proteins generate an adenosyl radical via the homolytic cleavage of the S-C(5') bond of SAM. Of particular interest are radical SAM enzymes, such as heme chaperones, that insert heme into respiratory enzymes. For example, heme chaperones insert heme into target proteins but have been studied only for the formation of cytochrome c-type hemoproteins. Here, we report that a radical SAM protein, the heme chaperone HemW from bacteria, is required for the insertion of heme b into respiratory chain enzymes. As other radical SAM proteins, HemW contains three cysteines and one SAM coordinating an [4Fe-4S] cluster, and we observed one heme per subunit of HemW. We found that an intact iron-sulfur cluster was required for HemW dimerization and HemW-catalyzed heme transfer but not for stable heme binding. A bacterial two-hybrid system screen identified bacterioferritins and the heme-containing subunit NarI of the respiratory nitrate reductase NarGHI as proteins that interact with HemW. We also noted that the bacterioferritins potentially serve as heme donors for HemW. Of note, heme that was covalently bound to HemW was actively transferred to a heme-depleted, catalytically inactive nitrate reductase, restoring its nitrate-reducing enzyme activity. Finally, the human HemW orthologue radical SAM domain-containing 1 (RSAD1) stably bound heme. In conclusion, our findings indicate that the radical SAM protein family HemW/RSAD1 is a heme chaperone catalyzing the insertion of heme into hemoproteins.
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