RESUMEN
Each catalytic cycle of type IA topoisomerases has been proposed to comprise multistep reactions. The capture of the transport-segment DNA (T-segment) into the central cavity of the N-terminal toroidal structure is an important action, which is preceded by transient gate-segment (G-segment) cleavage and succeeded by G-segment religation for the relaxation of negatively supercoiled DNA and decatenation of DNA. The T-segment passage in and out of the central cavity requires significant domain-domain rearrangements, including the movement of D3 relative to D1 and D4 for the opening and closing of the gate towards the central cavity. Here we report a direct observation of the interaction of a duplex DNA in the central cavity of a type IA topoisomerase and its associated domain-domain conformational changes in a crystal structure of a Mycobacterium tuberculosis topoisomerase I complex that also has a bound G-segment. The duplex DNA within the central cavity illustrates the non-sequence-specific interplay between the T-segment DNA and the enzyme. The rich structural information revealed from the novel topoisomerase-DNA complex, in combination with targeted mutagenesis studies, provides new insights into the mechanism of the topoisomerase IA catalytic cycle.
Asunto(s)
ADN-Topoisomerasas de Tipo I , ADN , Mycobacterium tuberculosis , ADN/química , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/enzimologíaRESUMEN
Human topoisomerase III beta (hTOP3B) is the only topoisomerase in the human cell that can act on both DNA and RNA substrates. Recent findings have emphasized the physiological importance of hTOP3B and consolidated it as a valuable drug target for antiviral and anticancer therapeutics. Although type IA topoisomerases of different organisms have been studied over the years, the step-by-step interaction of hTOP3B and nucleic acid substrates is still not well understood. Due to the lack of hTOP3B-RNA structures as well as DNA/RNA covalent complexes, computational investigations have been limited. In our study, we utilized molecular dynamics (MD) simulations to study the interactions between hTOP3B and nucleic acids to get a closer look into the residues that play a role in binding DNA or RNA and facilitate catalysis, along with the differences and similarities when hTOP3B interacts with DNA compared to RNA. For this, we generated multiple models of hTOP3B complexed with DNA and RNA sequences using the hTOP3B crystal structure and 8-mer single-stranded DNA and RNA sequences. These models include both covalent and noncovalent complexes, which are then subjected to MD simulations and analyzed. Our findings highlight the complexes' stability, sequence preference, and interactions of the binding pocket residues with different nucleotides. Our work demonstrates that hTOP3B forms stable complexes with both DNA and RNA and provides a better understanding of the enzyme's interaction with different nucleic acid substrate sequences.
Asunto(s)
ADN , Simulación de Dinámica Molecular , ARN , Humanos , ARN/metabolismo , ARN/química , ADN/metabolismo , ADN/química , Unión Proteica , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo I/química , Conformación de Ácido NucleicoRESUMEN
Endophytic microorganisms associated with medicinal plants are of particular interest as they are a potential source of new bioactive chemicals effective against novel emerging and drug-resistant pathogens. Agave americana is a tropical medicinal plant with antibacterial, antifungal, and anticancer properties. We studied the biodiversity of fungal endophytes of A. americana and their antimicrobial production potential. Isolated endophytic fungi were classified into 32 morphotypes (15 from stem and 17 from leaf) based on their cultural and morphological characteristics. Among the fungal crude extracts tested, 82% of isolates from the leaves and 80% of the isolates from the stem showed antibacterial activity against the bacterial strains (Escherichia coli ATTC 25902, Staphylococcus aureus ATTC 14775, and Bacillus subtilis NRRL 5109) tested. Extracts from four fungal isolates from leaves showed antifungal activity against at least one of the fungal strains (Candida albicans ATTC 10231 and Aspergillus fumigatus NRRL 5109) tested. Crude extracts of seven fungal isolates showed a zone of inhibition of more than 11 mm at 10 mgml-1 against both Gram-positive and Gram-negative bacteria tested. Penicillium, Colletotrichum, Curvularia, Pleosporales, Dothideomycetes, and Pleurotus are the main endophytes responsible for bioactive potential. These results indicate that A. americana harbors endophytes capable of producing antimicrobial metabolites.
Asunto(s)
Agave , Antiinfecciosos , Ascomicetos , Plantas Medicinales , Antifúngicos/farmacología , Antifúngicos/metabolismo , Antibacterianos/farmacología , Plantas Medicinales/microbiología , Bacterias Gramnegativas , Pruebas de Sensibilidad Microbiana , Bacterias Grampositivas , Hongos , Antiinfecciosos/farmacología , Antiinfecciosos/metabolismo , Endófitos , Mezclas Complejas/metabolismo , Mezclas Complejas/farmacologíaRESUMEN
Mycobacterial infectious diseases, including tuberculosis (TB), severely threaten global public health. Nonreplicating Mycobacterium tuberculosis (Mtb) is extremely difficult to eradicate using current TB drugs that primarily act on replicating cells. Novel TB drugs acting on unconventional targets are needed to combat TB efficiently. Although membrane-disrupting antimicrobial peptides and their synthetic mimics exhibit the potential to kill persisters, the lack of microbe selectivity, especially toward mycobacteria, has been a concern. Here, we report that the recently developed poly(guanylurea)-piperazine (PGU-P) shows fast and selective mycobactericidal effects. Using a nonpathogenic model organism, Mycobacterium smegmatis (Msm), we have found that the mycobactericidal effects of PGU-P are correlated to the disruption of the mycobacterial membrane potential and bioenergetics. Accordingly, PGU-P also potentiates bedaquiline, an oxidative phosphorylation-targeting TB drug disturbing mycobacterial bioenergetics. Importantly, PGU-P also exhibits a promising activity against pathogenic Mtb with a minimum inhibitory concentration of 37 µg/mL. Our results support that PGU-P is a novel class of antimycobacterial biomaterial, and the unique structural feature can contribute to developing novel antimycobacterial drugs.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Humanos , Antituberculosos/farmacología , Fuerza Protón-Motriz , Polímeros/farmacología , Tuberculosis/tratamiento farmacológico , Pruebas de Sensibilidad MicrobianaRESUMEN
Type IA topoisomerases interact with G-strand and T-strand ssDNA to regulate DNA topology. However, simultaneous binding of two ssDNA segments to a type IA topoisomerase has not been observed previously. We report here the crystal structure of a type IA topoisomerase with ssDNA segments bound in opposite polarity to the N- and C-terminal domains. Titration of small ssDNA oligonucleotides to Mycobacterium smegmatis topoisomerase I with progressive C-terminal deletions showed that the C-terminal region has higher affinity for ssDNA than the N-terminal active site. This allows the C-terminal domains to capture one strand of underwound negatively supercoiled DNA substrate first and position the N-terminal domains to bind and cleave the opposite strand in the relaxation reaction. Efficiency of negative supercoiling relaxation increases with the number of domains that bind ssDNA primarily with conserved aromatic residues and possibly with assistance from polar/basic residues. A comparison of bacterial topoisomerase I structures showed that a conserved transesterification unit (N-terminal toroid structure) for cutting and rejoining of a ssDNA strand can be combined with two different types of C-terminal ssDNA binding domains to form diverse bacterial topoisomerase I enzymes that are highly efficient in their physiological role of preventing excess negative supercoiling in the genome.
Asunto(s)
ADN-Topoisomerasas de Tipo I/química , ADN de Cadena Simple/metabolismo , Mycobacterium smegmatis/enzimología , Cristalografía por Rayos X , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Modelos Moleculares , Dominios Proteicos , Eliminación de SecuenciaRESUMEN
Type IA topoisomerases have highly conserved catalytic N-terminal domains for the cleaving and rejoining of a single DNA/RNA strand that have been extensively characterized. In contrast, the C-terminal region has been less covered. Two major types of small tandem C-terminal domains, Topo_C_ZnRpt (containing C4 zinc finger) and Topo_C_Rpt (without cysteines) were initially identified in Escherichia coli and Mycobacterium tuberculosis topoisomerase I, respectively. Their structures and interaction with DNA oligonucleotides have been revealed in structural studies. Here, we first present the diverse distribution and combinations of these two structural elements in various bacterial topoisomerase I (TopA). Previously, zinc fingers have not been seen in type IA topoisomerases from well-studied fungal species within the phylum Ascomycota. In our extended studies of C-terminal DNA-binding domains, the presence of zf-GRF and zf-CCHC types of zinc fingers in topoisomerase III (Top3) from fungi species in many phyla other than Ascomycota has drawn our attention. We secondly analyze the distribution and combination of these fungal zf-GRF- and zf-CCHC-containing domains. Their potential structures and DNA-binding mechanism are evaluated. The highly diverse arrangements and combinations of these DNA/RNA-binding domains in microbial type IA topoisomerase C-terminal regions have important implications for their interactions with nucleic acids and protein partners as part of their physiological functions.
Asunto(s)
ADN-Topoisomerasas de Tipo I , ADN , ADN-Topoisomerasas de Tipo I/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Dominios Proteicos , Dedos de ZincRESUMEN
The structural elucidation of native macromolecular assemblies has been a subject of considerable interest in native mass spectrometry (MS), and more recently in tandem with ion mobility spectrometry (IMS-MS), for a better understanding of their biochemical and biophysical functions. In the present work, we describe a new generation trapped ion mobility spectrometer (TIMS), with extended mobility range (K0 = 0.185-1.84 cm2·V-1·s-1), capable of trapping high-molecular-weight (MW) macromolecular assemblies. This compact 4 cm long TIMS analyzer utilizes a convex electrode, quadrupolar geometry with increased pseudopotential penetration in the radial dimension, extending the mobility trapping to high-MW species under native state (i.e., lower charge states). The TIMS capabilities to perform variable scan rate (Sr) mobility measurements over short time (100-500 ms), high-mobility resolution, and ion-neutral collision cross-section (CCSN2) measurements are presented. The trapping capabilities of the convex electrode TIMS geometry and ease of operation over a wide gas flow, rf range, and electric field trapping range are illustrated for the first time using a comprehensive list of standards varying from CsI clusters (n = 6-73), Tuning Mix oligomers (n = 1-5), common proteins (e.g., ubiquitin, cytochrome C, lysozyme, concanavalin (n = 1-4), carbonic anhydrase, ß clamp (n = 1-4), topoisomerase IB, bovine serum albumin (n = 1-3), topoisomerase IA, alcohol dehydrogenase), IgG antibody (e.g., avastin), protein-DNA complexes, and macromolecular assemblies (e.g., GroEL and RNA polymerase (n = 1-2)) covering a wide mass (up to m/z 19â¯000) and CCS range (up to 22â¯000 Å2 with <0.6% relative standard deviation (RSD)).
Asunto(s)
Espectrometría de Movilidad Iónica , Proteínas , Iones , Espectrometría de Masas , UbiquitinaRESUMEN
We have obtained new crystal structures of Mycobacterium tuberculosis topoisomerase I, including structures with ssDNA substrate bound to the active site, with and without Mg2+ ion present. Significant enzyme conformational changes upon DNA binding place the catalytic tyrosine in a pre-transition state position for cleavage of a specific phosphodiester linkage. Meanwhile, the enzyme/DNA complex with bound Mg2+ ion may represent the post-transition state for religation in the enzyme's multiple-step DNA relaxation catalytic cycle. The first observation of Mg2+ ion coordinated with the TOPRIM residues and DNA phosphate in a type IA topoisomerase active site allows assignment of likely catalytic role for the metal and draws a comparison to the proposed mechanism for type IIA topoisomerases. The critical function of a strictly conserved glutamic acid in the DNA cleavage step was assessed through site-directed mutagenesis. The functions assigned to the observed Mg2+ ion can account for the metal requirement for DNA rejoining but not DNA cleavage by type IA topoisomerases. This work provides new structural insights into a more stringent requirement for DNA rejoining versus cleavage in the catalytic cycle of this essential enzyme, and further establishes the potential for selective interference of DNA rejoining by this validated TB drug target.
Asunto(s)
Proteínas Bacterianas/química , Dominio Catalítico , ADN-Topoisomerasas de Tipo I/química , ADN Bacteriano/química , Metales/química , Mycobacterium tuberculosis/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Biocatálisis , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Magnesio/química , Magnesio/metabolismo , Metales/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mycobacterium tuberculosis/genética , Conformación de Ácido Nucleico , Conformación Proteica , Especificidad por SustratoRESUMEN
Topoisomerases in the type IA subfamily can catalyze change in topology for both DNA and RNA substrates. A type IA topoisomerase may have been present in a last universal common ancestor (LUCA) with an RNA genome. Type IA topoisomerases have since evolved to catalyze the resolution of topological barriers encountered by genomes that require the passing of nucleic acid strand(s) through a break on a single DNA or RNA strand. Here, based on available structural and biochemical data, we discuss how a type IA topoisomerase may recognize and bind single-stranded DNA or RNA to initiate its required catalytic function. Active site residues assist in the nucleophilic attack of a phosphodiester bond between two nucleotides to form a covalent intermediate with a 5'-phosphotyrosine linkage to the cleaved nucleic acid. A divalent ion interaction helps to position the 3'-hydroxyl group at the precise location required for the cleaved phosphodiester bond to be rejoined following the passage of another nucleic acid strand through the break. In addition to type IA topoisomerase structures observed by X-ray crystallography, we now have evidence from biophysical studies for the dynamic conformations that are required for type IA topoisomerases to catalyze the change in the topology of the nucleic acid substrates.
Asunto(s)
ADN-Topoisomerasas de Tipo I/genética , ADN de Cadena Simple/genética , Conformación Proteica , ARN/genética , Catálisis , Dominio Catalítico/genética , Cristalografía por Rayos X , ADN-Topoisomerasas de Tipo I/química , ADN-Topoisomerasas de Tipo I/ultraestructura , ADN de Cadena Simple/ultraestructura , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Genoma/genética , ARN/ultraestructura , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genéticaRESUMEN
Escherichia coli topoisomerase I (TopA), a regulator of global and local DNA supercoiling, is modified by Nε-Lysine acetylation. The NAD+-dependent protein deacetylase CobB can reverse both enzymatic and non-enzymatic lysine acetylation modification in E. coli. Here, we show that the absence of CobB in a ΔcobB mutant reduces intracellular TopA catalytic activity and increases negative DNA supercoiling. TopA expression level is elevated as topA transcription responds to the increased negative supercoiling. The slow growth phenotype of the ΔcobB mutant can be partially compensated by further increase of intracellular TopA level via overexpression of recombinant TopA. The relaxation activity of purified TopA is decreased by in vitro non-enzymatic acetyl phosphate mediated lysine acetylation, and the presence of purified CobB protects TopA from inactivation by such non-enzymatic acetylation. The specific activity of TopA expressed from His-tagged fusion construct in the chromosome is inversely proportional to the degree of in vivo lysine acetylation during growth transition and growth arrest. These findings demonstrate that E. coli TopA catalytic activity can be modulated by lysine acetylation-deacetylation, and prevention of TopA inactivation from excess lysine acetylation and consequent increase in negative DNA supercoiling is an important physiological function of the CobB protein deacetylase.
Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Sirtuinas/metabolismo , Acetilación , Biocatálisis , Cromosomas Bacterianos/metabolismo , ADN Bacteriano/metabolismo , ADN Superhelicoidal/metabolismo , Electroforesis en Gel de Agar , Escherichia coli/crecimiento & desarrollo , Lisina/metabolismo , Mutación/genética , Fenotipo , Unión Proteica , Proteómica , Proteínas Recombinantes/metabolismo , SolubilidadRESUMEN
Mycobacterium tuberculosis and the fast-growing species Mycobacterium abscessus are two important human pathogens causing persistent pulmonary infections that are difficult to cure and require long treatment times. The emergence of drug-resistant M. tuberculosis strains and the high level of intrinsic resistance of M. abscessus call for novel drug scaffolds that effectively target both pathogens. In this study, we evaluated the activity of bis(pyrrolide-imine) gold(III) macrocycles and chelates, originally designed as DNA intercalators capable of targeting human topoisomerase types I and II (Topo1 and Topo2), against M. abscessus and M. tuberculosis We identified a total of 5 noncytotoxic compounds active against both mycobacterial pathogens under replicating in vitro conditions. We chose one of these hits, compound 14, for detailed analysis due to its potent bactericidal mode of inhibition and scalable synthesis. The clinical relevance of this compound was demonstrated by its ability to inhibit a panel of diverse M. tuberculosis and M. abscessus clinical isolates. Prompted by previous data suggesting that compound 14 may target topoisomerase/gyrase enzymes, we demonstrated that it lacked cross-resistance with fluoroquinolones, which target the M. tuberculosis gyrase. In vitro enzyme assays confirmed the potent activity of compound 14 against bacterial topoisomerase 1A (Topo1) enzymes but not gyrase. Novel scaffolds like compound 14 with potent, selective bactericidal activity against M. tuberculosis and M. abscessus that act on validated but underexploited targets like Topo1 represent a promising starting point for the development of novel therapeutics for infections by pathogenic mycobacteria.
Asunto(s)
Oro/farmacología , Sustancias Intercalantes/farmacología , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Mycobacterium abscessus/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa II/farmacología , Tuberculosis Pulmonar/tratamiento farmacológico , Humanos , Compuestos Macrocíclicos/farmacología , Mycobacterium abscessus/aislamiento & purificación , Mycobacterium abscessus/metabolismo , Mycobacterium tuberculosis/aislamiento & purificación , Mycobacterium tuberculosis/metabolismoRESUMEN
Bacterial infections are serious health threats. Emerging drug resistance in bacteria further poses serious challenges to the treatment options involving traditional antibiotics. Antimicrobial polymers disrupt the physical cell membrane integrity of bacteria to address the drug resistance problems. Here, we introduce a conceptually new class of antimicrobial polymers containing positively charged guanylurea backbones for enhanced antimicrobial effects. The initial structure-activity relationship studies demonstrate that poly(guanylurea piperazine)s (PGU-Ps) exhibit excellent antimicrobial activity against different types of bacteria with high selectivity. The new design concept of using a positively charged guanylurea backbone will contribute to the development of future biocompatible, specific, and selective antimicrobial polymers.
Asunto(s)
Antibacterianos/síntesis química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Guanidinas/síntesis química , Guanidinas/farmacología , Polímeros/síntesis química , Polímeros/farmacología , Urea/análogos & derivados , Antibacterianos/química , Infecciones Bacterianas/tratamiento farmacológico , Guanidinas/química , Humanos , Pruebas de Sensibilidad Microbiana , Polímeros/química , Relación Estructura-Actividad , Urea/síntesis química , Urea/química , Urea/farmacologíaRESUMEN
DNA Topoisomerases are essential to resolve topological problems during DNA metabolism in all species. However, the prevalence and function of RNA topoisomerases remain uncertain. Here, we show that RNA topoisomerase activity is prevalent in Type IA topoisomerases from bacteria, archaea, and eukarya. Moreover, this activity always requires the conserved Type IA core domains and the same catalytic residue used in DNA topoisomerase reaction; however, it does not absolutely require the non-conserved carboxyl-terminal domain (CTD), which is necessary for relaxation reactions of supercoiled DNA. The RNA topoisomerase activity of human Top3ß differs from that of Escherichia coli topoisomerase I in that the former but not the latter requires the CTD, indicating that topoisomerases have developed distinct mechanisms during evolution to catalyze RNA topoisomerase reactions. Notably, Top3ß proteins from several animals associate with polyribosomes, which are units of mRNA translation, whereas the Top3 homologs from E. coli and yeast lack the association. The Top3ß-polyribosome association requires TDRD3, which directly interacts with Top3ß and is present in animals but not bacteria or yeast. We propose that RNA topoisomerases arose in the early RNA world, and that they are retained through all domains of DNA-based life, where they mediate mRNA translation as part of polyribosomes in animals.
Asunto(s)
ADN-Topoisomerasas de Tipo I/genética , Evolución Molecular , Polirribosomas/genética , Proteínas/genética , Secuencia de Aminoácidos/genética , Dominio Catalítico/genética , ADN Superhelicoidal/genética , Escherichia coli/enzimología , Escherichia coli/genética , Humanos , ARN/genética , ARN Mensajero/genética , Homología de Secuencia de AminoácidoRESUMEN
The relaxation activity of E. coli topoisomerase I is required for regulation of global and local DNA supercoiling. The in vivo topoisomerase I enzyme activity is sensitive to lysine acetylationâ»deacetylation and can affect DNA supercoiling and growth as a result. Nonenzymatic lysine acetylation by acetyl phosphate has been shown to reduce the relaxation activity of E. coli topoisomerase I. In this work, the biochemical consequence of topoisomerase I modification by acetyl phosphate with enzymatic assays was studied. Results showed that noncovalent binding to DNA and DNA cleavage by the enzyme were reduced as a result of the acetylation, with greater effect on DNA cleavage. Four lysine acetylation sites were identified using bottom-up proteomics: Lys13, Lys45, Lys346, and Lys488. The Lys13 residue modified by acetyl phosphate has not been reported previously as a lysine acetylation site for E. coli topoisomerase I. We discuss the potential biochemical consequence of lysine acetylation at this strictly conserved lysine and other lysine residues on the enzyme based on available genetic and structural information.
Asunto(s)
ADN-Topoisomerasas de Tipo I/química , ADN-Topoisomerasas de Tipo I/metabolismo , Escherichia coli/enzimología , Lisina/metabolismo , Acetilación , ADN/metabolismo , División del ADN , Organofosfatos/metabolismo , Unión Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
Escherichia coli topoisomerase I has an essential function in preventing hypernegative supercoiling of DNA. A full length structure of E. coli topoisomerase I reported here shows how the C-terminal domains bind single-stranded DNA (ssDNA) to recognize the accumulation of negative supercoils in duplex DNA. These C-terminal domains of E. coli topoisomerase I are known to interact with RNA polymerase, and two flexible linkers within the C-terminal domains may assist in the movement of the ssDNA for the rapid removal of transcription driven negative supercoils. The structure has also unveiled for the first time how the 4-Cys zinc ribbon domain and zinc ribbon-like domain bind ssDNA with primarily π-stacking interactions. This novel structure, in combination with new biochemical data, provides important insights into the mechanism of genome regulation by type IA topoisomerases that is essential for life, as well as the structures of homologous type IA TOP3α and TOP3ß from higher eukaryotes that also have multiple 4-Cys zinc ribbon domains required for their physiological functions.
Asunto(s)
ADN-Topoisomerasas de Tipo I/química , ADN de Cadena Simple/química , ADN Superhelicoidal/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , ADN-Topoisomerasas de Tipo I/metabolismo , ADN de Cadena Simple/metabolismo , ADN Superhelicoidal/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Bacterial topoisomerase functions are required for regulation of DNA supercoiling and overcoming the DNA topological barriers that are encountered during many vital cellular processes. DNA gyrase and topoisomerase IV of the type IIA bacterial topoisomerase family are important clinical targets for antibacterial therapy. Topoisomerase I, belonging to the type IA topoisomerase family, has recently been validated as a potential antitubercular target. The topoisomerase I activity has been shown to be essential for bacterial viability and infection in a murine model of tuberculosis. Mixture-based combinatorial libraries were screened in this study to identify novel bacterial topoisomerase I inhibitors. Using positional-scanning deconvolution, selective small-molecule inhibitors of bacterial topoisomerase I were identified starting from a polyamine scaffold. Antibacterial assays demonstrated that four of these small-molecule inhibitors of bacterial topoisomerase I are bactericidal against Mycobacterium smegmatis and Mycobacterium tuberculosis The MICs for growth inhibition of M. smegmatis increased with overexpression of recombinant M. tuberculosis topoisomerase I, consistent with inhibition of intracellular topoisomerase I activity being involved in the antimycobacterial mode of action.
Asunto(s)
Antituberculosos/farmacología , ADN-Topoisomerasas de Tipo I/metabolismo , Inhibidores de Topoisomerasa I/farmacología , Antibacterianos/farmacología , Girasa de ADN/genética , Girasa de ADN/metabolismo , Topoisomerasa de ADN IV/genética , Topoisomerasa de ADN IV/metabolismo , Pruebas de Sensibilidad Microbiana , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismoRESUMEN
A novel complex, [Cu(acetylethTSC)Cl]Clâ¢0.25C2H5OH 1 (where acetylethTSC = (E)-N-ethyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide), was shown to have anti-proliferative activity against various colon and aggressive breast cancer cell lines. In vitro studies showed that complex 1 acted as a poison inhibitor of human topoisomerase IIα, which may account for the observed anti-cancer effects.
RESUMEN
Topoisomerase functions are required in all organisms for many vital cellular processes, including transcription elongation. The C terminus domains (CTD) of Escherichia coli topoisomerase I interact directly with RNA polymerase to remove transcription-driven negative supercoiling behind the RNA polymerase complex. This interaction prevents inhibition of transcription elongation from hypernegative supercoiling and R-loop accumulation. The physiological function of bacterial topoisomerase I in transcription is especially important for a rapid network response to an antibiotic challenge. In this study, Escherichia coli with a topA66 single nucleotide deletion mutation, which results in a frameshift in the TopA CTD, was shown to exhibit increased sensitivity to trimethoprim and quinolone antimicrobials. The topoisomerase I-RNA polymerase interaction and the SOS response to the antimicrobial agents were found to be significantly reduced by this topA66 mutation. Consequently, the mutation frequency measured by rifampin selection following SOS induction was diminished in the topA66 mutant. The increased antibiotic sensitivity for the topA66 mutant can be reversed by the expression of recombinant E. coli topoisomerase I but not by the expression of recombinant Mycobacterium tuberculosis topoisomerase I that has a nonhomologous CTD even though the recombinant M. tuberculosis topoisomerase I can restore most of the plasmid DNA linking number deficiency caused by the topA66 mutation. Direct interactions of E. coli topoisomerase I as part of transcription complexes are likely to be required for the rapid network response to an antibiotic challenge. Inhibitors of bacterial topoisomerase I functions and interactions may sensitize pathogens to antibiotic treatment and limit the mutagenic response.
Asunto(s)
Antibacterianos/farmacología , ADN-Topoisomerasas de Tipo I/genética , Proteínas de Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Mutación del Sistema de Lectura , Regulación Bacteriana de la Expresión Génica , ADN-Topoisomerasas de Tipo I/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Prueba de Complementación Genética , Pruebas de Sensibilidad Microbiana , Tasa de Mutación , Mycobacterium tuberculosis/química , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Quinolonas/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rifampin/farmacología , Respuesta SOS en Genética/efectos de los fármacos , Elongación de la Transcripción Genética , Trimetoprim/farmacologíaRESUMEN
To date, the bacterial DNA topoisomerases are one of the major target biomolecules for the discovery of new antibacterial drugs. DNA topoisomerase regulates the topological state of DNA, which is very important for replication, transcription and recombination. The relaxation of negatively supercoiled DNA is catalyzed by bacterial DNA topoisomerase I (topoI) and this reaction requires Mg(2+). In this report, we first quantitatively studied the intermolecular interactions between Escherichia coli topoisomerase I (EctopoI) and pBAD/Thio supercoiled plasmid DNA using surface plasmon resonance (SPR) technique. The equilibrium dissociation constant (Kd) for EctopoI-pBAD/Thio interactions was determined to be about 8 nM. We then studied the effect of Mg(2+) on the catalysis of EctopoI-pBAD/Thio reaction. A slightly higher equilibrium dissociation constant (~15 nM) was obtained for Mg(2+) coordinated EctopoI (Mg(2+)EctopoI)-pBAD/Thio interactions. In addition, we observed a larger dissociation rate constant (kd) for Mg(2+)EctopoI-pBAD/Thio interactions (~0.043 s(-1)), compared to EctopoI-pBAD/Thio interactions (~0.017 s(-1)). These results suggest that enzyme turnover during plasmid DNA relaxation is enhanced due to the presence of Mg(2+) and furthers the understanding of importance of the Mg(2+) ion for bacterial topoisomerase I catalytic activity.
Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , ADN Superhelicoidal/metabolismo , Escherichia coli/enzimología , Plásmidos/metabolismo , Magnesio/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Bacterial and archaeal topoisomerase I display selectivity for a cytosine base 4 nt upstream from the DNA cleavage site. Recently, the solved crystal structure of Escherichia coli topoisomerase I covalently linked to a single-stranded oligonucleotide revealed that R169 and R173 interact with the cytosine base at the -4 position via hydrogen bonds while the phenol ring of Y177 wedges between the bases at the -4 and the -5 position. Substituting R169 to alanine changed the selectivity of the enzyme for the base at the -4 position from a cytosine to an adenine. The R173A mutant displayed similar sequence selectivity as the wild-type enzyme, but weaker cleavage and relaxation activity. Mutation of Y177 to serine or alanine rendered the enzyme inactive. Although mutation of each of these residues led to different outcomes, R169, R173 and Y177 work together to interact with a cytosine base at the -4 position to facilitate DNA cleavage. These strictly conserved residues might act after initial substrate binding as a Molecular Ruler to form a protein-DNA complex with the scissile phosphate positioned at the active site for optimal DNA cleavage by the tyrosine hydroxyl nucleophile to facilitate DNA cleavage in the reaction pathway.