DOCSLIB.ORG

An Underutilized Tool in the Quest to Elucidate Radical SAM Dynamics

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
An Underutilized Tool in the Quest to Elucidate Radical SAM Dynamics molecules Review Computational Approaches: An Underutilized Tool in the Quest to Elucidate Radical SAM Dynamics Tamra C. Blue and Katherine M. Davis * Department of Chemistry, Emory University, 1515 Dickey Dr, Atlanta, GA 30322, USA; [email protected] * Correspondence: [email protected] Abstract: Enzymes are biological catalysts whose dynamics enable their reactivity. Visualizing conformational changes, in particular, is technically challenging, and little is known about these crucial atomic motions. This is especially problematic for understanding the functional diversity associated with the radical S-adenosyl-L-methionine (SAM) superfamily whose members share a common radical mechanism but ultimately catalyze a broad range of challenging reactions. Compu- tational chemistry approaches provide a readily accessible alternative to exploring the time-resolved behavior of these enzymes that is not limited by experimental logistics. Here, we review the appli- cation of molecular docking, molecular dynamics, and density functional theory, as well as hybrid quantum mechanics/molecular mechanics methods to the study of these enzymes, with a focus on understanding the mechanistic dynamics associated with turnover. Keywords: radical SAM enzymes; dynamics; computational; DFT; MD; docking; QM/MM 1. Introduction Citation: Blue, T.C.; Davis, K.M. Carefully choreographed structural dynamics underlie enzymatic activity. However, Computational Approaches: An Underutilized Tool in the Quest to these motions are often small in magnitude, notoriously difficult to visualize, and remain Elucidate Radical SAM Dynamics. largely unexplored. Short reaction timescales and/or a lack of intermediate state accumula- Molecules 2021, 26, 2590. https:// tion complicate data collection. Furthermore, structural biology methods typically rely on doi.org/10.3390/molecules26092590 cryogenic temperatures that limit study to stable states or those with lifetimes long enough for freeze trapping. Recent advancements in time-resolved crystallography show great Academic Editor: Rafik Karaman promise for monitoring atomic changes as catalysis occurs, but there are limitations [1,2]. Many membrane proteins, as well as large or flexible molecules, for example, are difficult to Received: 30 March 2021 crystallize [3]. Likewise, in order to study dynamical properties, the macromolecule must Accepted: 26 April 2021 be biologically active in the crystalline state, the lattice must support turnover, and near Published: 29 April 2021 simultaneous reaction initiation of all constituent molecules must be achieved. While the conformational dynamics of enzymes that rely on radical-based chemistry are of particular Publisher’s Note: MDPI stays neutral interest due to their ability to catalyze challenging transformations during the biosynthesis with regard to jurisdictional claims in of many natural products and cofactors, these systems are often reactive to molecular published maps and institutional affil- oxygen, which similarly complicates handling. iations. This is especially true for the >300,000 members of the radical S-adenosyl-L-methionine (rSAM) superfamily [4]. These enzymes catalyze a diverse array of reactions that include C–C bond formation, decarboxylation, sulfur insertion, and methylation [5]. They are bioinformatically classified via the presence of a canonical CX3CX2C motif, the thiols Copyright: © 2021 by the authors. of which bind a catalytically-active [4Fe–4S] cluster. Homolytic cleavage of SAM bound Licensee MDPI, Basel, Switzerland. bidentate to the unique iron of this cluster yields methionine and a 50-deoxyadenosyl This article is an open access article radical (dAdo•; Figure1A) [ 5,6]. Most often rSAM enzymes employ this potent radical to distributed under the terms and abstract a substrate hydrogen, thereby enabling the enzymes’ impressive scope of reactivity. conditions of the Creative Commons Although they comprise one of the largest known enzyme superfamilies, characterization Attribution (CC BY) license (https:// of rSAM enzymes has been hindered by their recalcitrant nature. Computational chemistry creativecommons.org/licenses/by/ methods provide a readily accessible alternative to studying the mechanisms and atomic 4.0/). Molecules 2021, 26, 2590. https://doi.org/10.3390/molecules26092590 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 15 Moleculescharacterization2021, 26, 2590 of rSAM enzymes has been hindered by their recalcitrant nature. Com‐2 of 14 putational chemistry methods provide a readily accessible alternative to studying the mechanisms and atomic motions that facilitate rSAM catalysis while overcoming the tech‐ motions that facilitate rSAM catalysis while overcoming the technical limitations associated nical limitations associated with time‐resolved structure determination of these enzymes. with time-resolved structure determination of these enzymes. Figure 1. Substrate binding in TsrM. (A) Shared reaction of rSAM enzymes. (B) Reaction catalyzed by TsrM, which while Figure 1. Substrate binding in TsrM. (A) Shared reaction of rSAM enzymes. (B) Reaction catalyzed annotated as a rSAM enzyme, does not appear to generate dAdo•.(C) The active site structure of TsrM (PDB accession code: • 6WTF)by TsrM, depicts which the target while carbon annotated in an unproductive as a rSAM position. enzyme, Docking does simulations not appear suggest to generate an alternate dAdo flipped. (C) binding The modeactive for Trp site in structure the presence of of TsrM MeCbl. (PDB This accession proposal helps code: to rationalize 6WTF) depicts observed the reorientation target carbon of a nearby in an Tyr.unpro‐ ductive position. Docking simulations suggest an alternate flipped binding mode for Trp in the presence of MeCbl. This proposalComputational helps to approaches rationalize have observed played reorientation an integral role of ina nearby defining Tyr. the rSAM su- perfamily, since its classification in 2001, when bioinformatics was used to survey and Computationalcategorize approaches the original have played ~600 members an integral [7]. Computational role in defining chemistry the rSAM methods super that lever-‐ age our understanding of classical and quantum mechanics to analyze the fundamental family, since its classificationproperties of atoms,in 2001, molecules, when bioinformatics and chemical reactions, was used however, to survey have been and sorely cate under-‐ gorize the original utilized~600 members in the study [7]. of Computational these fascinating enzymes. chemistry Here, methods we summarize that leverage work that hasour been understanding of classicalperformed and to apply quantum both ab mechanics initio and semi-empirical to analyze the calculations fundamental to the study properties of rSAM en- of atoms, molecules,zyme and dynamics chemical with reactions, a focus on dockinghowever, (D), have molecular been dynamics sorely (MD),underutilized density functional in theory (DFT) and hybrid quantum mechanics/molecular mechanics (QM/MM) methods the study of these(Table fascinating1). Areas enzymes. of untapped Here, potential we are summarize highlighted alongwork with that potential has been pitfalls. per‐ formed to apply both ab initio and semi‐empirical calculations to the study of rSAM en‐ zyme dynamics with2. Molecular a focus Dockingon docking (D), molecular dynamics (MD), density func‐ tional theory (DFT) andSome hybrid of the most quantum critical conformational mechanics/molecular changes that anmechanics enzyme undergoes (QM/MM) are those methods (Table 1).associated Areas of with untapped substrate potential binding. Accordingly,are highlighted visualization along with of reactant potential complexes pit‐ can provide unprecedented insight into the structural basis for reactivity. Binding of a peptidyl falls. analog for the protein substrate pyruvate formate-lyase (PFL) by the radical SAM activating enzyme (PFL-AE) is accompanied by rearrangement of an active site beta strand, which 2. Molecular Dockingpartially loses secondary structure to provide contacts with the peptide [8]. Complexation with the PFL fragment further induces changes to side chain conformations that stabilize Some of the mostthe cosubstrate critical conformational SAM, a correlation changes that may that help an to enzyme prevent unproductive undergoes cleavage.are those More associated with substrategenerally, binding. loop motions Accordingly, that occlude solventvisualization access to theof reactant active site complexes appear to be relativelycan provide unprecedentedwidespread insight amongst into membersthe structural of the superfamily basis for [reactivity.9,10]. Despite Binding a shared mechanism,of a pep‐ the tidyl analog for thevast protein substrate substrate diversity pyruvate of rSAM enzymes formate precludes‐lyase (PFL) a common by substrate-bindingthe radical SAM motif, and conformational changes associated with ligand binding are often subtle. The structural activating enzyme (PFL‐AE) is accompanied by rearrangement of an active site beta strand, which partially loses secondary structure to provide contacts with the peptide [8]. Complexation with the PFL fragment further induces changes to side chain conformations that stabilize the cosubstrate SAM, a correlation that may help to prevent unproductive cleavage. More generally, loop motions that occlude solvent access to the active site ap‐ pear to be relatively widespread amongst members of the superfamily [9,10]. Despite a shared mechanism, the
Load more

Recommended publications
  • Noncanonical Coproporphyrin-Dependent Bacterial Heme Biosynthesis Pathway That Does Not Use Protoporphyrin
    Noncanonical coproporphyrin-dependent bacterial heme biosynthesis pathway that does not use protoporphyrin Harry A. Daileya,b,c,1, Svetlana Gerdesd, Tamara A. Daileya,b,c, Joseph S. Burcha, and John D. Phillipse aBiomedical and Health Sciences Institute and Departments of bMicrobiology and cBiochemistry and Molecular Biology, University of Georgia, Athens, GA 30602; dMathematics and Computer Science Division, Argonne National Laboratory, Argonne, IL 60439; and eDivision of Hematology, Department of Medicine, University of Utah School of Medicine, Salt Lake City, UT 84132 Edited by J. Clark Lagarias, University of California, Davis, CA, and approved January 12, 2015 (received for review August 25, 2014) It has been generally accepted that biosynthesis of protoheme of a “primitive” pathway in Desulfovibrio vulgaris (13). This path- (heme) uses a common set of core metabolic intermediates that way, named the “alternative heme biosynthesis” path (or ahb), has includes protoporphyrin. Herein, we show that the Actinobacteria now been characterized by Warren and coworkers (15) in sulfate- and Firmicutes (high-GC and low-GC Gram-positive bacteria) are reducing bacteria. In the ahb pathway, siroheme, synthesized unable to synthesize protoporphyrin. Instead, they oxidize copro- from uroporphyrinogen III, can be further metabolized by suc- porphyrinogen to coproporphyrin, insert ferrous iron to make Fe- cessive demethylation and decarboxylation to yield protoheme (14, coproporphyrin (coproheme), and then decarboxylate coproheme 15) (Fig. 1 and Fig. S1). A similar pathway exists for protoheme- to generate protoheme. This pathway is specified by three genes containing archaea (15, 16). named hemY, hemH, and hemQ. The analysis of 982 representa- Current gene annotations suggest that all enzymes for pro- tive prokaryotic genomes is consistent with this pathway being karyotic heme synthetic pathways are now identified.
    [Show full text]
  • The Crystal Structure of Biotin Synthase, an S-Adenosylmethionine-Dependent Radical Enzyme F
    2-74 LIFE SCIENCES SCIENCE HIGHLIGHTS 2-75 The Crystal Structure of Biotin Synthase, an S-Adenosylmethionine-Dependent Radical Enzyme F. Berkovitch1, Y. Nicolet1, J.T. Wan2, J.T. Jarrett2, and C.L. Drennan1 1Department of Chemistry, Massachusetts Institute of Technology; 2Johnson Research Foundation and Department of Biochemistry and Biophysics, University of Pennsylvania BEAMLINE X25 The crystal structure of biotin synthase addresses how “AdoMet radical” enzymes, also called “Radical SAM” enzymes, use an Fe S cluster and S-adenosyl-L-methionine to 4 4 Funding generate organic radicals. Biotin synthase catalyzes the radical-mediated insertion of National Institutes of Health; Searle Scholars Program; sulfur into dethiobiotin (DTB) to form biotin (vitamin B8). The structure places the substrates, i.e. DTB and AdoMet, between the Fe S cluster (essential for radical gen- Cecil and Ida Green Career 4 4 Development Fund; Lester eration) and the Fe2S2 cluster (postulated to be the source of sulfur), with both clusters Wolfe Predoctoral Fellowship; in unprecedented coordination environments. Cellular, Biochemical, and Molecular Sciences training Biotin is an essential vitamin that plays a ubiquitous role in human growth and grant; U.S. Department of Energy; National Institute of metabolism. Biotin deficiency results in skin lesions, abnormal fat distribution, General Medical Sciences neurological symptoms, and immunodeficiency. A low biotin level has also been correlated to an increased incidence of type II diabetes mellitus. Biotin is a valuable Publication commercial commodity, used as an additive in food, health, and cosmetic prod- F. Berkovitch, Y. Nicolet, J.T. Wan, J.T. Jarrett, and ucts, and as a research tool in the biochemical sciences.
    [Show full text]
  • Radical SAM Enzymes in the Biosynthesis of Ribosomally Synthesized and Post-Translationally Modified Peptides (Ripps) Alhosna Benjdia, Clémence Balty, Olivier Berteau
    Radical SAM enzymes in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs) Alhosna Benjdia, Clémence Balty, Olivier Berteau To cite this version: Alhosna Benjdia, Clémence Balty, Olivier Berteau. Radical SAM enzymes in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs). Frontiers in Chemistry, Frontiers Media, 2017, 5, 10.3389/fchem.2017.00087. hal-02627786 HAL Id: hal-02627786 https://hal.inrae.fr/hal-02627786 Submitted on 26 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License REVIEW published: 08 November 2017 doi: 10.3389/fchem.2017.00087 Radical SAM Enzymes in the Biosynthesis of Ribosomally Synthesized and Post-translationally Modified Peptides (RiPPs) Alhosna Benjdia*, Clémence Balty and Olivier Berteau* Micalis Institute, ChemSyBio, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large and diverse family of natural products. They possess interesting biological properties such as antibiotic or anticancer activities, making them attractive for therapeutic applications. In contrast to polyketides and non-ribosomal peptides, RiPPs derive from ribosomal peptides and are post-translationally modified by diverse enzyme families.
    [Show full text]
  • Structural Insights Into Recognition and Repair of UV-DNA Damage by Spore Photoproduct Lyase, a Radical SAM Enzyme Alhosna Benjdia1,*, Korbinian Heil2, Thomas R
    9308–9318 Nucleic Acids Research, 2012, Vol. 40, No. 18 Published online 2 July 2012 doi:10.1093/nar/gks603 Structural insights into recognition and repair of UV-DNA damage by Spore Photoproduct Lyase, a radical SAM enzyme Alhosna Benjdia1,*, Korbinian Heil2, Thomas R. M. Barends1, Thomas Carell2,* and Ilme Schlichting1,* 1Department of Biomolecular Mechanisms, Max-Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg and 2Department of Chemistry, Center for Integrated Protein Science (CiPSM), Ludwig-Maximilians University, Butenandtstrasse 5-13, 81377 Munich, Germany Received April 2, 2012; Revised May 26, 2012; Accepted May 29, 2012 ABSTRACT INTRODUCTION Bacterial spores possess an enormous resistance Given the myriad of DNA-damaging agents, bacteria have to ultraviolet (UV) radiation. This is largely due to a evolved diverse DNA repair pathways to resist and survive unique DNA repair enzyme, Spore Photoproduct up to thousands of years in the extreme case of spores. Lyase (SP lyase) that repairs a specific UV-induced This tremendous resistance is achieved by a combination DNA lesion, the spore photoproduct (SP), through of several factors including DNA packing assisted by an unprecedented radical-based mechanism. small acid-soluble spore proteins, dehydration and the Unlike DNA photolyases, SP lyase belongs to the high content of dipicolinic acid (2,6-pyridinedicarboxylic emerging superfamily of radical S-adenosyl-L-me- acid) (1). Under these conditions, ultraviolet (UV) radi- thionine (SAM) enzymes and uses a [4Fe–4S]1+ ation results in the formation of one major DNA lesion, cluster and SAM to initiate the repair reaction. We the so-called spore photoproduct (SP or 5-(R-thyminyl)- report here the first crystal structure of this enig- 5,6-dihydrothymine) (2,3).
    [Show full text]
  • “Metalloproteins at the Crossroads of Design & Nature”
    Virtual Symposium “Metalloproteins at the crossroads of design & nature” 27 January 2021 Keynote Lecture Prof. Hannah Shafaat Ohio State University Emergent Complexity: Reconstructing primordial energy conversion processes in model metalloproteins Abstract: Nature has evolved diverse systems to carry out energy conversion reactions. Metalloenzymes such as hydrogenase, carbon monoxide dehydrogenase, acetyl coenzyme A synthase, and methyl coenzyme M reductase use earth- abundant transition metals such as nickel and iron to generate and oxidize small-molecule fuels such as hydrogen, carbon monoxide, acetate, thioesters, and methane. These reactions are highly valuable to understand and harness in the context of the impending global energy and climate crisis. However, the native enzymes require complex multimetallic cofactors, are costly to isolate, highly sensitive to external conditions, and generally poorly suited for large- scale application. We are applying metalloprotein engineering approaches in order to better understand the mechanisms of the native enzymes and develop robust functional models with potential for implementation in anthropogenic energy conversion. Robust metalloprotein scaffolds such as rubredoxin, ferredoxin, and azurin and have been converted into model systems that mimic the structure and function of complex nickel metalloenzymes. By introducing non-native metals and redesigning the primary and secondary coordination spheres, we have installed novel activity into these simple electron transfer proteins, including
    [Show full text]
  • The Requirement of Inorganic Fe-S Clusters for the Biosynthesis of the Organometallic Molybdenum Cofactor
    inorganics Review The Requirement of Inorganic Fe-S Clusters for the Biosynthesis of the Organometallic Molybdenum Cofactor Ralf R. Mendel 1, Thomas W. Hercher 1, Arkadiusz Zupok 2, Muhammad A. Hasnat 2 and Silke Leimkühler 2,* 1 Institute of Plant Biology, Braunschweig University of Technology, Humboldtstr. 1, 38106 Braunschweig, Germany; [email protected] (R.R.M.); [email protected] (T.W.H.) 2 Department of Molecular Enzymology, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany; [email protected] (A.Z.); [email protected] (M.A.H.) * Correspondence: [email protected]; Tel.: +49-331-977-5603; Fax: +49-331-977-5128 Received: 18 June 2020; Accepted: 14 July 2020; Published: 16 July 2020 Abstract: Iron-sulfur (Fe-S) clusters are essential protein cofactors. In enzymes, they are present either in the rhombic [2Fe-2S] or the cubic [4Fe-4S] form, where they are involved in catalysis and electron transfer and in the biosynthesis of metal-containing prosthetic groups like the molybdenum cofactor (Moco). Here, we give an overview of the assembly of Fe-S clusters in bacteria and humans and present their connection to the Moco biosynthesis pathway. In all organisms, Fe-S cluster assembly starts with the abstraction of sulfur from l-cysteine and its transfer to a scaffold protein. After formation, Fe-S clusters are transferred to carrier proteins that insert them into recipient apo-proteins. In eukaryotes like humans and plants, Fe-S cluster assembly takes place both in mitochondria and in the cytosol.
    [Show full text]
  • Radical SAM-Mediated Methylation Reactions 1,2
    Available online at www.sciencedirect.com Radical SAM-mediated methylation reactions 1,2 Danica Galonic´ Fujimori A subset of enzymes that belong to the radical S- the [4Fe–4S] cluster in these proteins [3]. A subset of adenosylmethionine (SAM) superfamily is able to catalyze radical SAM enzymes has been demonstrated, or is pre- methylation reactions. Substrates of these enzymes are distinct dicted, to catalyze methylation on a diverse set of bio- from the nucleophilic substrates that undergo methylation by a molecules, some with electronic properties distinct from polar mechanism. Recently, activities of several radical SAM conventional nucleophilic methylation substrates. Akin methylating enzymes have been reconstituted in vitro and their to the profound biological roles of methyltransferses that mechanisms of catalysis investigated. The RNA modifying use polar mechanisms [4], radical SAM methylating enzymes RlmN and Cfr catalyze methylation via a methyl enzymes influence important cellular functions, such synthase mechanism. These enzymes use SAM in two distinct as regulation of translation, susceptibility to antibiotics, roles: as a source of a methyl group transferred to a conserved and maintenance of bacterial intracytoplasmic mem- 0 0 cysteine and as a source of 5 -deoxyadenosyl radical (5 -dA ). branes [5–9]. Recently, significant progress has been Hydrogen atom abstraction by this species generates a made in the reconstitution and mechanistic analysis of thiomethylene radical which adds into the RNA substrate, radical SAM methylating enzymes. This work has forming an enzyme-substrate covalent adduct. In another expanded the repertoire of known biological methylation recent study, methylation of the indole moiety of tryptophan by catalysts, as well as the scope of substrates that can the radical SAM and cobalamin-binding domain enzyme TsrM undergo enzymatic methylation.
    [Show full text]
  • Radical-Sam Enzymes with Two Iron-Sulfur Clusters: Cofactor
    RADICAL-SAM ENZYMES WITH TWO IRON-SULFUR CLUSTERS: COFACTOR COMPOSITION AND SPECTROSCOPIC STUDIES OF ESCHERICHIA COLI AND BACILLUS SUBTILIS BIOTIN SYNTHASE, HUMAN MOCS1A, AND THERMOTOGA MARITIMA MIAB by HEATHER LOUISE HERNÁNDEZ (Under the Direction of Michael Kenneth Johnson) ABSTRACT A new class of Fe-S proteins, termed radical-SAM enzymes, catalyzes radical reactions in a variety of biosynthetic processes. These [4Fe-4S]2+,+ cluster-containing enzymes initiate radical enzymatic reactions via reductive cleavage of S-adenosyl-L-methionine (SAM) to yield methionine and an extremely reactive 5'-deoxyadenosyl radical. A growing number of radical-SAM enzymes have recently been discovered to contain a second Fe-S cluster of unknown function, although possible roles include acting as sacrificial S-donor or anchoring and possibly activating the substrate. The combination of analytical and spectroscopic studies, including EPR, Mössbauer, UV-visible absorption/circular dichroism/variable temperature magnetic circular dichroism, and resonance Raman, have been used to investigate the cofactor composition and properties of the two cluster containing radical-SAM enzymes Escherichia coli and Bacillus subtilis biotin synthase (BioB), human MOCS1A, and Thermotoga maritima MiaB. These enzymes are involved in crucial steps in the biosynthesis of biotin and molybdopterin, and in the thiomethylation of tRNA. E. coli and B. subtilis BioB are shown to house a radical-SAM [4Fe-4S] cluster and a [2Fe-2S] cluster in separate binding sites. The function and relevance of the [2Fe-2S] cluster, which has been suggested to be the S-donor to biotin, is addressed. In E. coli BioB, the most active form of the enzyme contains a 1:1 ratio of [2Fe-2S]/[4Fe-4S] clusters and the [2Fe-2S] cluster degrades during turnover.
    [Show full text]
  • The Radical SAM Enzyme Spore Photoproduct Lyase Employs A
    ChemComm View Article Online COMMUNICATION View Journal | View Issue The radical SAM enzyme spore photoproduct lyase Cite this: Chem. Commun., 2013, employs a tyrosyl radical for DNA repair† 49, 722 Andrea Christa Kneuttinger, Korbinian Heil, Gengo Kashiwazaki and Received 24th October 2012, Thomas Carell* Accepted 27th November 2012 DOI: 10.1039/c2cc37735g www.rsc.org/chemcomm The spore photoproduct lyase is a radical SAM enzyme, which repairs by the Atta12 and Li laboratories13 showed that the allyl reducing 5-(a-thyminyl)-5,6-dihydrothymidine. Here we show that the enzyme moiety is a conserved cysteine. A recent crystal structure of the SPL establishes a complex radical transfer cascade and creates a cysteine from Geobacillus thermodenitrificans14 in complex with the lesion15 and a tyrosyl radical dyade to establish repair. This allows the enzyme (Fig. 1B) shows the topological problem associated with closing the to solve topological and energetic problems associated with the radical catalytic cycle. While the allyl radical is situated and reduced at the based repair reaction. 30-side, transfer of the radical center back to the 50-dAdoH requires movingtheradicalbacktothe50-part in the active side over a UV irradiation causes the formation of a variety of dinucleotide distance of roughly 10 Å. This creates next to a topological problem lesions, which are typically formed between two pyrimidines.1,2 also an energetic obstacle, because regeneration of the adenosyl In cellular DNA, UV irradiation causes the formation of the well radical by the thiyl radical would be endothermic by 62 kJ molÀ1.16 studied cyclobutane pyrimidine dimers (CPDs), pyrimidine Here we provide first evidence that the enzyme uses a (6-4)-pyrimidone photoproducts (6-4PPs), and their Dewar-valence further tyrosyl radical intermediate to solve the energetic and isomers.1 In bacterial endospores, in contrast, these bipyrimidine topological problem.
    [Show full text]
  • Radical New Paradigm for Heme Degradation in Escherichia Coli O157:H7
    Radical new paradigm for heme degradation in Escherichia coli O157:H7 Joseph W. LaMattinaa,b, David B. Nixa,c, and William Nicholas Lanzilottaa,b,1 aDepartment of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602; bCenter for Metalloenzyme Studies, University of Georgia, Athens, GA 30602; and cComplex Carbohydrate Research Center, University of Georgia, Athens, GA 30602 Edited by Iqbal Hamza, University of Maryland, College Park, MD, and accepted by Editorial Board Member Michael A. Marletta September 7, 2016 (received for review February 25, 2016) All of the heme-degrading enzymes that have been characterized Vibrio cholerae and Escherichia coli O157:H7 are hemolytic en- to date require molecular oxygen as a cosubstrate. Escherichia coli teric pathogens that colonize the nonsterile region of the lower O157:H7 has been shown to express heme uptake and transport intestines. Despite their ability to use heme as an iron source, a proteins, as well as use heme as an iron source. This enteric pathogen bona fide heme oxygenase has not been characterized for either colonizes the anaerobic space of the lower intestine in mammals, yet organism. However, both organisms contain a heme uptake operon no mechanism for anaerobic heme degradation has been reported. that is up-regulated during iron duress (14, 15). The operon en- Herein we provide evidence for an oxygen-independent heme- codes for the heme uptake and transport machinery, as well as degradation pathway. Specifically, we demonstrate that ChuW is three additional genes, chuW, chuX,andchuY. (Fig. 1A)(16). a radical S-adenosylmethionine methyltransferase that catalyzes a ChuW is annotated as a member of the radical S-adenosylme- radical-mediated mechanism facilitating iron liberation and the thionine (SAM) superfamily, a large class of enzymes that perform “ ” production of the tetrapyrrole product we termed anaerobilin.
    [Show full text]
  • Analysis of RNA Modifications by Second
    G C A T T A C G G C A T genes Review Analysis of RNA Modifications by Second- and Third-Generation Deep Sequencing: 2020 Update Yuri Motorin 1,* and Virginie Marchand 2,* 1 Université de Lorraine, CNRS, IMoPA (UMR7365), F54000 Nancy, France 2 Université de Lorraine, CNRS, INSERM, IBSLor (UMS2008/US40), Epitranscriptomics and RNA Sequencing Core Facility, F54000 Nancy, France * Correspondence: [email protected] (Y.M.); [email protected] (V.M.); Tel.: +33-3-7274-6629 (Y.M.); +33-3-7274-6669 (V.M.) Abstract: The precise mapping and quantification of the numerous RNA modifications that are present in tRNAs, rRNAs, ncRNAs/miRNAs, and mRNAs remain a major challenge and a top priority of the epitranscriptomics field. After the keystone discoveries of massive m6A methylation in mRNAs, dozens of deep sequencing-based methods and protocols were proposed for the analysis of various RNA modifications, allowing us to considerably extend the list of detectable modified residues. Many of the currently used methods rely on the particular reverse transcription signatures left by RNA modifications in cDNA; these signatures may be naturally present or induced by an appropriate enzymatic or chemical treatment. The newest approaches also include labeling at RNA abasic sites that result from the selective removal of RNA modification or the enhanced cleavage of the RNA ribose-phosphate chain (perhaps also protection from cleavage), followed by specific adapter ligation. Classical affinity/immunoprecipitation-based protocols use either antibodies against modified RNA bases or proteins/enzymes, recognizing RNA modifications. In this survey, we review the most recent achievements in this highly dynamic field, including promising attempts to map RNA modifications by the direct single-molecule sequencing of RNA by nanopores.
    [Show full text]
  • The Antiviral Protein Viperin Suppresses T7 Promoter Dependent
    www.nature.com/scientificreports OPEN The antiviral protein Viperin suppresses T7 promoter dependent RNA synthesis–possible Received: 17 November 2017 Accepted: 11 May 2018 implications for its antiviral activity Published: xx xx xxxx Anna Dukhovny1, Amir Shlomai2,3 & Ella H. Sklan1 Viperin is a multifunctional interferon-inducible broad-spectrum antiviral protein. Viperin belongs to the S-Adenosylmethionine (SAM) superfamily of enzymes known to catalyze a wide variety of radical- mediated reactions. However, the exact mechanism by which viperin exerts its functions is still unclear. Interestingly, for many RNA viruses viperin was shown to inhibit viral RNA accumulation by interacting with diferent viral non-structural proteins. Here, we show that viperin inhibits RNA synthesis by bacteriophage T7 polymerase in mammalian cells. This inhibition is specifc and occurs at the RNA level. Viperin expression signifcantly reduced T7-mediated cytoplasmic RNA levels. The data showing that viperin inhibits the bacteriophage T7 polymerase supports the conservation of viperin’s antiviral activity between species. These results highlight the possibility that viperin might utilize a broader mechanism of inhibition. Accordingly, our results suggest a novel mechanism involving polymerase inhibition and provides a tractable system for future mechanistic studies of viperin. Te interferon (IFN) response is one of the frst lines of host innate defense against viral infections. Pattern recog- nition sensors in the infected host cell recognize the incoming pathogens and initiate signal transduction cascades that culminate in the host cell’s nucleus. Tese signaling cascades activate type I IFN production and the induc- tion of interferon-stimulated genes (ISGs) aimed to prevent infection in an autocrine and paracrine manner1.
    [Show full text]