RESUMEN
GlgB (α-1,4-glucan branching enzyme) is the key enzyme involved in the biosynthesis of α-glucan, which plays a significant role in the virulence and pathogenesis of Mycobacterium tuberculosis. Because α-glucans are implicated in the survival of both replicating and non-replicating bacteria, there exists an exigent need for the identification and development of novel inhibitors for targeting enzymes, such as GlgB, involved in this pathway. We have used the existing structural information of M. tuberculosis GlgB for high throughput virtual screening and molecular docking. A diverse database of 330,000 molecules was used for identifying novel and efficacious therapeutic agents for targeting GlgB. We also used three-dimensional shape as well as two-dimensional similarity matrix methods to identify diverse molecular scaffolds that inhibit M. tuberculosis GlgB activity. Virtual hits were generated after structure and ligand-based screening followed by filters based on interaction with human GlgB and in silico pharmacokinetic parameters. These hits were experimentally evaluated and resulted in the discovery of a number of structurally diverse chemical scaffolds that target M. tuberculosis GlgB. Although a number of inhibitors demonstrated in vitro enzyme inhibition, two compounds in particular showed excellent inhibition of in vivo M. tuberculosis survival and its ability to get phagocytosed. This work shows that in silico docking and three-dimensional chemical similarity could be an important therapeutic approach for developing inhibitors to specifically target the M. tuberculosis GlgB enzyme.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/antagonistas & inhibidores , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Descubrimiento de Drogas , Mycobacterium tuberculosis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Enzima Ramificadora de 1,4-alfa-Glucano/química , Enzima Ramificadora de 1,4-alfa-Glucano/genética , Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Antituberculosos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Bases de Datos Farmacéuticas , Bases de Datos de Proteínas , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Glucanos/química , Glucanos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/crecimiento & desarrollo , Fagocitosis/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Homología Estructural de Proteína , Relación Estructura-Actividad , Interfaz Usuario-ComputadorRESUMEN
A wide-host-range bacteriophage (phage) PIS136 was isolated from PA136, a strain of Saccharomonospora belonging to the group actinomycetes. Here, we present the genome sequence of the PIS136 phage, which is 94,870 bp long and contains 132 putative coding sequences and one tRNA gene. An IS element-like region with two genes for putative transposases was identified in the genome. The presence of IS element-like sequences suggests that PIS136 is still under active evolution.
Asunto(s)
Actinomycetales/virología , Bacteriófagos/genética , Genoma Viral , Siphoviridae/genética , Bacteriófagos/clasificación , Bacteriófagos/aislamiento & purificación , Secuencia de Bases , Datos de Secuencia Molecular , Siphoviridae/clasificación , Siphoviridae/aislamiento & purificación , Microbiología del Suelo , Transposasas/genética , Proteínas Virales/genéticaRESUMEN
Patient safety, improved quality of care, and better patient satisfaction and functional outcomes are currently the topmost priorities in regional anaesthesia (RA) and all advancements in RA move in this direction. Ultrasonography-guided central neuraxial and peripheral nerve blocks, intracluster and intratruncal injections, fascial plane blocks, diaphragm-sparing blocks, use of continuous nerve block techniques, and continuous local anaesthetic wound infiltration catheters are now topics of popular clinical interest. The safety and efficacy of nerve blocks can be improved with the help of injection pressure monitoring and the incorporation of advanced technology in the ultrasound machine and needles. Novel procedure-specific and motor-sparing nerve blocks have come up. The anaesthesiologist of the current era, with a good understanding of the sonoanatomy of the target area and the microarchitecture of nerves, along with the backup of advanced technology, can be very successful in performing RA techniques. RA is rapidly evolving and revolutionising the practice of anaesthesia.
RESUMEN
A transformation system which is free of in vitro plant regeneration following Agrobacterium infection is established for the forage legume, Sunnhemp (Crotalaria juncea L.) where in the entire embryo axis of the germinating seed was used as the target tissue for transformation. After standardization of transformation conditions, the cotyledonary node of the embryo axis was infected with Agrobacterium host LBA 4404 harboring the recombinant vector pCAMBIA 2301. The bivalent 1D gene of the two major foot and mouth disease virus (FMDV) serotypes 'O' and 'A22' and the neomycin phosphotransferase (nptII) gene were used as the markers for optimization of the protocol. The embryo axes were pricked randomly on the cotyledonary node and co-cultivated with Agrobacterium. The germlings were then allowed to grow under standard growth room conditions in to mature fertile plants. 60 T0 plants were established from 3 separate experiments. Three hundred seeds from the 60 T0 plants were sown to raise the T1 generation of which 180 were analyzed for integration of bivalent FMDV gene 1D "O" and "A22" and the nptII gene. Eighteen out of these 180 plants amplified both the marker genes. Two independent transgenic lines 24 and 37, showed elevated levels of expression of 12 µg and 8 µg (per gm of fresh leaf) of the bivalent ID antigen "O" and "A22" . The results showed that the transformation efficiency was 3 %. To the best of our knowledge, this is the first successful attempt of Agrobacterium tumefaciens mediated transformation of Sunnhemp. The protocol can generate whole plant transformants with relative ease and should be compatible to all genotypes of Sunnhemp.
RESUMEN
The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an alpha-1,4-glucan branching enzyme (MtbGlgB, EC 2.4.1.18, Uniprot entry Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1-->4 bond and making a new 1-->6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-A resolution. MtbGlgBWT contains four domains: N1 beta-sandwich, N2 beta-sandwich, a central (beta/alpha)(8) domain that houses the catalytic site, and a C-terminal beta-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) MtbDelta108GlgB protein. The N1 beta-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 beta-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and MtbDelta108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1-->4 bond breakage) and isomerization (1-->6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and MtbDelta108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (ECDelta112GlgB).
Asunto(s)
Hidrolasas/química , Mycobacterium tuberculosis/enzimología , Conformación de Carbohidratos , Cristalografía por Rayos X , Cartilla de ADN , Glucógeno/química , Hidrolasas/genética , Hidrolasas/metabolismo , Cinética , Modelos Moleculares , Conformación Proteica , Eliminación de Secuencia , Especificidad por SustratoRESUMEN
Several synthetic dyes employed in textile and food industries are discharged into aquatic environment. These visible pollutants in water damage environment, as they are carcinogenic and toxic to humans. The use of cost effective and ecofriendly plant cellulose based adsorbents have been studied in batch experiments as an alternative and effective substitution of activated carbon for the removal of toxic dyes from waste water. Adsorbents prepared from sugarcane baggase, were successfully used to remove certain textile dye such as crystal violet from an aqueous solution. The present investigation potentiate the use of sugarcane baggase, pretreated with formaldehyde (referred as Raw Baggase) and sulphuric acid (referred as Chemically Activated Baggase), for the removal of crystal violet dye from simulated waste water. Experiments were carried out at neutral pH with various parameters like dye concentration, temperature, contact time and adsorbent dosage. Efficiency of raw baggase was found better than chemically activated baggase for adsorption of crystal violet dye. The data obtained perfectly fits in the Freundlich adsorption isotherm.
Asunto(s)
Antiinfecciosos Locales/aislamiento & purificación , Celulosa/química , Violeta de Genciana/aislamiento & purificación , Eliminación de Residuos Líquidos/métodos , Contaminantes Químicos del Agua/aislamiento & purificación , Adsorción , Residuos Industriales , Saccharum , Temperatura , Eliminación de Residuos Líquidos/economíaRESUMEN
BACKGROUND: Mycobacterium tuberculosis, an intracellular pathogen encounters redox stress throughout its life inside the host. In order to protect itself from the redox onslaughts of host immune system, M. tuberculosis appears to have developed accessory thioredoxin-like proteins which are represented by ORFs encoding WhiB-like proteins. We have earlier reported that WhiB1/Rv3219 is a thioredoxin like protein of M. tuberculosis and functions as a protein disulfide reductase. Generally thioredoxins have many substrate proteins. The current study aims to identify the substrate protein(s) of M. tuberculosis WhiB1. RESULTS: Using yeast two-hybrid screen, we identified alpha (1,4)-glucan branching enzyme (GlgB) of M. tuberculosis as a interaction partner of WhiB1. In vitro GST pull down assay confirmed the direct physical interaction between GlgB and WhiB1. Both mass spectrometry data of tryptic digests and in vitro labeling of cysteine residues with 4-acetamido-4' maleimidyl-stilbene-2, 2'-disulfonic acid showed that in GlgB, C95 and C658 are free but C193 and C617 form an intra-molecular disulfide bond. WhiB1 has a C37XXC40 motif thus a C40S mutation renders C37 to exist as a free thiol to form a hetero-disulfide bond with the cysteine residue of substrate protein. A disulfide mediated binary complex formation between GlgB and WhiB1C40S was shown by both in-solution protein-protein interaction and thioredoxin affinity chromatography. Finally, transfer of reducing equivalent from WhiB1 to GlgB disulfide was confirmed by 4-acetamido-4' maleimidyl-stilbene-2, 2'-disulfonic acid trapping by the reduced disulfide of GlgB. Two different thioredoxins, TrxB/Rv1471 and TrxC/Rv3914 of M. tuberculosis could not perform this reaction suggesting that the reduction of GlgB by WhiB1 is specific. CONCLUSION: We conclude that M. tuberculosis GlgB has one intra-molecular disulfide bond which is formed between C193 and C617. WhiB1, a thioredoxin like protein interacts with GlgB and transfers its electrons to the disulfide thus reduces the intra-molecular disulfide bond of GlgB. For the first time, we report that GlgB is one of the in vivo substrate of M. tuberculosis WhiB1.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/enzimología , Tiorredoxinas/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucano/antagonistas & inhibidores , Enzima Ramificadora de 1,4-alfa-Glucano/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Cromatografía de Afinidad , Disulfuros/química , Espectrometría de Masas , Datos de Secuencia Molecular , Mycobacterium tuberculosis/metabolismo , Oxidación-Reducción , Dominios y Motivos de Interacción de Proteínas , Estilbenos/síntesis química , Estilbenos/química , Ácidos Sulfónicos/síntesis química , Ácidos Sulfónicos/química , Tiorredoxinas/química , Técnicas del Sistema de Dos HíbridosRESUMEN
Redox stress is one of the major challenges faced by Mycobacterium tuberculosis during early infection and latency. The mechanism of sensing and adaptation to altered redox conditions is poorly understood. whiB family of Mtb is emerging as an important class of stress responsive genes. WhiB3/Rv3416 has been shown to be important for pathogenesis in animal model and was recently shown to co-ordinate a Fe-S cluster. Here, we report a simple, rapid and efficient matrix-assisted refolding method and important redox properties of WhiB3. Similar to other WhiB proteins, WhiB3 also has four conserved cysteine residues, where two of them are present in a CXXC motif. The Fe-S cluster of WhiB3 remained bound in the presence of strong protein denaturant. Upon cluster removal due to oxidation, the four cysteine residues which are ligands of Fe-S cluster, formed two intra-molecular disulfide bridges where one of them is possibly between the cysteines of CXXC motif, an important feature of several thiol-disulfide oxido-reductases. Far-UV CD spectroscopy revealed the presence of both alpha-helices and beta-strands in apo WhiB3. The secondary structural elements of apo WhiB3 were found resistant for thermal denaturation. The results demonstrated that apo WhiB3 functions as a protein disulfide reductase similar to thioredoxins. The importance of WhiB3 in redox sensing and its possible role in mycobacterial physiology has been discussed.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Cromatografía en Gel/métodos , Dicroismo Circular , Cartilla de ADN , Espectrometría de Masas , Oxidación-Reducción , Pliegue de Proteína , Espectrometría de FluorescenciaRESUMEN
Tuberculosis continues to be a deadly infectious disease, mainly due to the existence of persistent bacterial populations that survive drug treatment and obstruct complete eradication of infection. The enzymes GlgE and GlgB, which are involved in the glycan pathway, have recently been identified as promising drug targets for combating persistent bacillus strains. In the glycan pathway, enzymes GlgE, GlgA, and Tre-xyz produce linear α-glucans, which are then converted to essential branched α-glucan by GlgB. This α-glucan is a vital cell-wall and storage polysaccharide, critical for Mtb virulence and persistence. We highlight recent insights into the significance of both GlgE and GlgB in the glycan pathway and also discuss drug strategies for tuberculosis such as polypharmcological targeting of GlgB and GlgE. Small molecule-based modulation of GlgB and GlgE to boost the design and development of novel and improved drugs for more selective and efficient targeting of tuberculosis are also discussed.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/antagonistas & inhibidores , Proteínas Bacterianas/antagonistas & inhibidores , Glucógeno/metabolismo , Mycobacterium tuberculosis/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucano/clasificación , Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Antituberculosos/química , Antituberculosos/metabolismo , Antituberculosos/uso terapéutico , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/metabolismo , Sitios de Unión , Humanos , Simulación de Dinámica Molecular , Mycobacterium tuberculosis/enzimología , Estructura Terciaria de Proteína , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Tuberculosis/patologíaRESUMEN
whiB-like genes have been found in all actinomycetes sequenced so far. The amino-acid sequences of WhiB proteins of Mycobacterium tuberculosis H37Rv are highly conserved and participate in several cellular functions. Unlike other WhiB proteins of M. tuberculosis that have properties of protein disulfide reductases, WhiB2 showed properties like a chaperone as it suppressed the aggregation of several model substrates (e.g. citrate synthase, rhodanese and luciferase). Suppression of aggregation of the model substrates did not require ATP. Four cysteine residues of WhiB2 form two intramolecular disulfide bonds; however, chaperone function was unaffected by the redox state of the cysteines. WhiB2 also restored the activity of chemically denatured citrate synthase and did not require either ATP or a co-chaperone for refolding. The results indicate that WhiB2, which has been shown to be associated with cell division in mycobacteria and streptomyces, has evolved independently of other WhiBs, although it retains basic properties of this group of proteins. This is the first report to show that a WhiB protein has chaperone-like function; therefore, this report will have major implications in attempts to understand the role of WhiB proteins in mycobacteria, particularly in cell division.
Asunto(s)
Proteínas Bacterianas/fisiología , Chaperonas Moleculares/fisiología , Mycobacterium tuberculosis/fisiología , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , División Celular/genética , División Celular/fisiología , Precipitación Química , Citrato (si)-Sintasa/química , Citrato (si)-Sintasa/metabolismo , Cisteína/química , ADN Bacteriano/genética , Genes Bacterianos , Insulina/química , Insulina/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutagénesis Sitio-Dirigida , Mycobacterium tuberculosis/genética , Oxidación-Reducción , Desnaturalización Proteica , Serina/químicaRESUMEN
The alternate sigma factor sigH of Mycobacterium tuberculosis is expressed under stress and acts as a major regulator of several genes, including some other sigma factors and redox systems. While it is auto-regulated by its own promoter at the transcriptional level, its regulation at the post-translational level is through its cognate protein, an anti-sigma factor, RshA. Hither before RshA was believed to be a zinc-associated anti-sigma factor (ZAS) and the binding of RshA to SigH is redox dependent. Here, we show that RshA coordinates a [2Fe-2S] cluster using cysteines as ligands and native RshA has more affinity to [2Fe-2S] cluster than to zinc. Furthermore, we used amide hydrogen deuterium exchange mass spectrometry (HDX-MS), followed by site-directed mutagenesis in SigH and RshA, to elucidate the interaction mechanism of RshA and SigH and the potential role of metal ion clustering in SigH regulation. Three regions in SigH, comprising of residues 1-25, 58-69, 90-111, 115-132 and 157-196 and residues 35-57 of RshA show decreased deuterium exchange and reflect decreased solvent accessibility upon complexation with SigH. Of the three RshA mutants, created based on the HDX results, the RsHA E37A mutant shows stronger interaction with SigH, relative to WT RshA, while the H49A mutant abolishes interactions and the C(53)XXC(56)AXXA mutant has no effect on complexation with SigH. The D22A, D160A and E162 SigH mutants show significantly decreased binding to RshA and the E168A mutant completely abolished interactions with RshA, indicating that the SigH-RshA interaction is mediated by salt bridges. In addition, SigH-RshA interaction does not require clustering of metal ions. Based on our results, we propose a molecular model of the SigH-RshA interaction.
Asunto(s)
Proteínas Bacterianas/genética , ADN Bacteriano/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Mycobacterium tuberculosis/genética , Factor sigma/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutagénesis Sitio-Dirigida , Mycobacterium tuberculosis/metabolismo , Factor sigma/metabolismo , Transcripción GenéticaRESUMEN
The whiB-like genes (1-7) of Mycobacterium tuberculosis are involved in cell division, nutrient starvation, pathogenesis, antibiotic resistance and stress sensing. Although the biochemical properties of WhiB1, WhiB3 and WhiB4 are known, there is no information about the other proteins. Here, we elucidate in detail the biochemical and biophysical properties of WhiB2, WhiB5, WhiB6 and WhiB7 of M. tuberculosis and present a comprehensive comparative study on the molecular properties of all WhiB proteins. UV-Vis spectroscopy has suggested the presence of a redox-sensitive [2Fe-2S] cluster in each of the WhiB proteins, which remains stably bound to the proteins in the presence of 8 m urea. The [2Fe-2S] cluster of each protein was oxidation labile but the rate of cluster loss decreased under reducing environments. The [2Fe-2S] cluster of each WhiB protein responded differently to the oxidative effect of air and oxidized glutathione. In all cases, disassembly of the [2Fe-2S] cluster was coupled with the oxidation of cysteine-thiols and the formation of two intramolecular disulfide bonds. Both CD and fluorescence spectroscopy revealed that WhiB proteins are structurally divergent members of the same family. Similar to WhiB1, WhiB3 and WhiB4, apo WhiB5, WhiB6 and WhiB7 also reduced the disulfide of insulin, a model substrate. However, the reduction efficiency varied significantly. Surprisingly, WhiB2 did not reduce the insulin disulfide, even though its basic properties were similar to those of others. The structural and functional divergence among WhiB proteins indicated that each WhiB protein is a distinguished member of the same family and together they may represent a novel redox system for M. tuberculosis.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteínas Bacterianas/genética , División Celular , Secuencia Conservada , Variación Genética , Proteínas Hierro-Azufre/metabolismo , Cinética , Mycobacterium tuberculosis/citología , Mycobacterium tuberculosis/patogenicidad , Proteína Disulfuro Reductasa (Glutatión)/metabolismo , Espectrofotometría , Factores de Transcripción/genéticaRESUMEN
The genome sequence of Mycobacterium tuberculosis H37Rv revealed the presence of seven whiB-like open reading frames. In spite of several genetic studies on whiB genes, the biochemical properties of WhiB proteins are poorly understood. All WhiB-like proteins have four conserved cysteine residues, out of which two are present in a CXXC motif. We report for the first time the detailed biochemical and biophysical properties of M. tuberculosis WhiB4/Rv3681c and demonstrate the functional relevance of four conserved cysteines and the CXXC motif. UV-visible absorption spectra of freshly purified mWhiB4 showed the presence of a [2Fe-2S] cluster, whereas the electron paramagnetic resonance (EPR) spectra of reconstituted protein showed the presence of a [4Fe-4S] cluster. The iron-sulphur cluster was redox sensitive but stably co-ordinated to the protein even in the presence of high concentration of chaotropic agents. Despite primary sequence divergence from thioredoxin family proteins, the apo mWhiB4 has properties similar to thioredoxins and functions as a protein disulphide reductase, whereas holo mWhiB4 is enzymatically inactive. Apart from the cysteine thiol of CXXC motif the distantly placed thiol pair also contributes equally to the enzymatic activity of mWhiB4. A functional model of mWhiB4 in redox signaling during oxidative stress in M. tuberculosis has been presented.
Asunto(s)
Mycobacterium tuberculosis/enzimología , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Espectroscopía de Resonancia por Spin del Electrón , Hierro/análisis , Luz , Modelos Biológicos , Datos de Secuencia Molecular , Oxidación-Reducción , Estrés Oxidativo , Oxidorreductasas/aislamiento & purificación , Alineación de Secuencia , Análisis Espectral , Azufre/análisis , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Rayos UltravioletaRESUMEN
Glycogen branching enzyme (GlgB, EC 2.4.1.18) catalyzes the third step of glycogen biosynthesis by the cleavage of an alpha-(1,4)-glucosidic linkage and subsequent transfer of cleaved oligosaccharide to form a new alpha-(1,6)-branch. A single glgB gene Rv1326c is present in Mycobacterium tuberculosis. The predicted amino acid sequence of GlgB of M. tuberculosis has all the conserved regions of alpha-amylase family proteins. The overall amino acid identity to other GlgBs ranges from 48.5 to 99%. The glgB gene of M. tuberculosis was cloned and expressed in Escherichia coli. The recombinant protein was purified to homogeneity using metal affinity and ion exchange chromatography. The recombinant protein is a monomer as evidenced by gel filtration chromatography, is active as an enzyme, and uses amylose as the substrate. Enzyme activity was optimal at pH 7.0, 30 degrees C and divalent cations such as Zn2+ and Cu2+ inhibited activity. CD spectroscopy, proteolytic cleavage and mass spectroscopy analyses revealed that cysteine residues of GlgB form structural disulfide bond(s), which allow the protein to exist in two different redox-dependent conformational states. These conformations have different surface hydrophobicities as evidenced by ANS-fluorescence of oxidized and reduced GlgB. Although the conformational change did not affect the branching enzyme activity, the change in surface hydrophobicity could influence the interaction or dissociation of different cellular proteins with GlgB in response to different physiological states.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/biosíntesis , Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Mycobacterium tuberculosis/enzimología , Secuencia de Aminoácidos , Cromatografía en Gel , Dicroismo Circular , Clonación Molecular , Datos de Secuencia Molecular , Oxidación-Reducción , Filogenia , Conformación Proteica , Alineación de SecuenciaRESUMEN
WhiB family of protein is emerging as one of the most fascinating group and is implicated in stress response as well as pathogenesis via their involvement in diverse cellular processes. Surprisingly, available in vivo data indicate an organism specific physiological role for each of these proteins. The WhiB proteins have four conserved cysteine residues where two of them are present in a C-X-X-C motif. In thioredoxins and similar proteins, this motif works as an active site and confers thiol-disulfide oxidoreductase activity to the protein. The recombinant WhiB1/Rv3219 was purified in a single step from Escherichia coli using Ni(2+)-NTA affinity chromatography and was found to exist as a homodimer. Mass spectrometry of WhiB1 shows that the four cysteine residues form two intramolecular disulfide bonds. Using intrinsic tryptophan fluorescence as a measure of redox state, the redox potential of WhiB1 was calculated as -236+/-2mV, which corresponds to the redox potential of many cytoplasmic thioredoxin-like proteins. WhiB1 catalyzed the reduction of insulin disulfide thus clearly demonstrating that it functions as a protein disulfide reductase. Present study for the first time suggests that WhiB1 may be a part of the redox network of Mycobacterium tuberculosis through its involvement in thiol-disulfide exchange with other cellular proteins.
Asunto(s)
Mycobacterium tuberculosis/enzimología , Proteína Disulfuro Reductasa (Glutatión)/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Cisteína/química , Expresión Génica , Datos de Secuencia Molecular , Mycobacterium tuberculosis/química , Homología de Secuencia de Aminoácido , Tiorredoxinas/químicaRESUMEN
DNA transposition is central to the propagation of temperate phage Mu. A long-standing problem in Mu biology has been the mechanism by which the linear genome of an infecting phage, which is linked at both ends to DNA acquired from a previous host, integrates into the new host chromosome. If Mu were to use its well-established cointegrate mechanism for integration (single-strand nicks at Mu ends, joined to a staggered double-strand break in the target), the flanking host sequences would remain linked to Mu; target-primed replication of the linear integrant would subsequently break the chromosome. The absence of evidence for chromosome breaks has led to speculation that infecting Mu might use a cut-and-paste mechanism, whereby Mu DNA is cut away from the flanking sequences prior to integration. In this study we have followed the fate of the flanking DNA during the time course of Mu infection. We have found that these sequences are still attached to Mu upon integration and that they disappear soon after. The data rule out a cut-and-paste mechanism and suggest that infecting Mu integrates to generate simple insertions by a variation of its established cointegrate mechanism in which, instead of a "nick, join, and replicate" pathway, it follows a "nick, join, and process" pathway. The results show similarities with human immunodeficiency virus integration and provide a unifying mechanism for development of Mu along either the lysogenic or lytic pathway.