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1.
PLoS Biol ; 17(1): e3000122, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30657780

RESUMEN

PolD is an archaeal replicative DNA polymerase (DNAP) made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2). Recently, we reported the individual crystal structures of the DP1 and DP2 catalytic cores, thereby revealing that PolD is an atypical DNAP that has all functional properties of a replicative DNAP but with the catalytic core of an RNA polymerase (RNAP). We now report the DNA-bound cryo-electron microscopy (cryo-EM) structure of the heterodimeric DP1-DP2 PolD complex from Pyrococcus abyssi, revealing a unique DNA-binding site. Comparison of PolD and RNAPs extends their structural similarities and brings to light the minimal catalytic core shared by all cellular transcriptases. Finally, elucidating the structure of the PolD DP1-DP2 interface, which is conserved in all eukaryotic replicative DNAPs, clarifies their evolutionary relationships with PolD and sheds light on the domain acquisition and exchange mechanism that occurred during the evolution of the eukaryotic replisome.


Asunto(s)
Proteínas de Unión al ADN/ultraestructura , Factor de Transcripción DP1/ultraestructura , Factores de Transcripción/ultraestructura , Secuencia de Aminoácidos/genética , Sitios de Unión/genética , Dominio Catalítico , Microscopía por Crioelectrón/métodos , ADN/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/ultraestructura , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/ultraestructura , Dominios Proteicos/genética , Subunidades de Proteína/metabolismo , Pyrococcus abyssi/metabolismo , Pyrococcus abyssi/ultraestructura , Factor de Transcripción DP1/metabolismo , Factores de Transcripción/metabolismo
2.
Nucleic Acids Res ; 47(12): 6386-6395, 2019 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-31114911

RESUMEN

Casposons are a group of bacterial and archaeal DNA transposons encoding a specific integrase, termed casposase, which is homologous to the Cas1 enzyme responsible for the integration of new spacers into CRISPR loci. Here, we characterized the sequence motifs recognized by the casposase from a thermophilic archaeon Aciduliprofundum boonei. We identified a stretch of residues, located in the leader region upstream of the actual integration site, whose deletion or mutagenesis impaired the concerted integration reaction. However, deletions of two-thirds of the target site were fully functional. Various single-stranded 6-FAM-labelled oligonucleotides derived from casposon terminal inverted repeats were as efficiently incorporated as duplexes into the target site. This result suggests that, as in the case of spacer insertion by the CRISPR Cas1-Cas2 integrase, casposon integration involves splaying of the casposon termini, with single-stranded ends being the actual substrates. The sequence critical for incorporation was limited to the five terminal residues derived from the 3' end of the casposon. Furthermore, we characterize the casposase from Nitrosopumilus koreensis, a marine member of the phylum Thaumarchaeota, and show that it shares similar properties with the A. boonei enzyme, despite belonging to a different family. These findings further reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the CRISPR-Cas systems.


Asunto(s)
Euryarchaeota/enzimología , Integrasas/metabolismo , Archaea/enzimología , Elementos Transponibles de ADN , ADN de Archaea/química , Euryarchaeota/genética , Motivos de Nucleótidos , Oligonucleótidos
3.
EMBO J ; 34(8): 1126-42, 2015 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-25762590

RESUMEN

Eukaryotic DNA polymerase mu of the PolX family can promote the association of the two 3'-protruding ends of a DNA double-strand break (DSB) being repaired (DNA synapsis) even in the absence of the core non-homologous end-joining (NHEJ) machinery. Here, we show that terminal deoxynucleotidyltransferase (TdT), a closely related PolX involved in V(D)J recombination, has the same property. We solved its crystal structure with an annealed DNA synapsis containing one micro-homology (MH) base pair and one nascent base pair. This structure reveals how the N-terminal domain and Loop 1 of Tdt cooperate for bridging the two DNA ends, providing a templating base in trans and limiting the MH search region to only two base pairs. A network of ordered water molecules is proposed to assist the incorporation of any nucleotide independently of the in trans templating base. These data are consistent with a recent model that explains the statistics of sequences synthesized in vivo by Tdt based solely on this dinucleotide step. Site-directed mutagenesis and functional tests suggest that this structural model is also valid for Pol mu during NHEJ.


Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , ADN Polimerasa Dirigida por ADN/química , Células Eucariotas/metabolismo , Animales , Secuencia de Bases , Cristalografía por Rayos X , ADN Nucleotidilexotransferasa/química , ADN Nucleotidilexotransferasa/fisiología , ADN Polimerasa Dirigida por ADN/fisiología , Ratones , Modelos Moleculares , Conformación Proteica , Recombinación V(D)J
4.
Nucleic Acids Res ; 44(21): 10367-10376, 2016 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-27655632

RESUMEN

Casposons are a recently discovered group of large DNA transposons present in diverse bacterial and archaeal genomes. For integration into the host chromosome, casposons employ an endonuclease that is homologous to the Cas1 protein involved in protospacer integration by the CRISPR-Cas adaptive immune system. Here we describe the site-preference of integration by the Cas1 integrase (casposase) encoded by the casposon of the archaeon Aciduliprofundum boonei Oligonucleotide duplexes derived from the terminal inverted repeats (TIR) of the A. boonei casposon as well as mini-casposons flanked by the TIR inserted preferentially at a site reconstituting the original A. boonei target site. As in the A. boonei genome, the insertion was accompanied by a 15-bp direct target site duplication (TSD). The minimal functional target consisted of the 15-bp TSD segment and the adjacent 18-bp sequence which comprises the 3' end of the tRNA-Pro gene corresponding to the TΨC loop. The functional casposase target site bears clear resemblance to the leader sequence-repeat junction which is the target for protospacer integration catalyzed by the Cas1-Cas2 adaptation module of CRISPR-Cas. These findings reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the prokaryotic adaptive immunity systems.


Asunto(s)
Sitios de Unión , Sistemas CRISPR-Cas , Elementos Transponibles de ADN/genética , Endodesoxirribonucleasas/metabolismo , Orden Génico , Resistencia a la Kanamicina/genética , Mutagénesis Insercional , Conformación de Ácido Nucleico , Plásmidos/genética , Secuencias Repetidas Terminales
5.
Nucleic Acids Res ; 42(6): 3707-19, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24445805

RESUMEN

We report the characterization of a DNA primase/polymerase protein (PolpTN2) encoded by the pTN2 plasmid from Thermococcus nautilus. Sequence analysis revealed that this protein corresponds to a fusion between an N-terminal domain homologous to the small catalytic subunit PriS of heterodimeric archaeal and eukaryotic primases (AEP) and a C-terminal domain related to their large regulatory subunit PriL. This unique domain configuration is not found in other virus- and plasmid-encoded primases in which PriS-like domains are typically fused to different types of helicases. PolpTN2 exhibited primase, polymerase and nucleotidyl transferase activities and specifically incorporates dNTPs, to the exclusion of rNTPs. PolpTN2 could efficiently prime DNA synthesis by the T. nautilus PolB DNA polymerase, suggesting that it is used in vivo as a primase for pTN2 plasmid replication. The N-terminal PriS-like domain of PolpTN2 exhibited all activities of the full-length enzyme but was much less efficient in priming cellular DNA polymerases. Surprisingly, the N-terminal domain possesses reverse transcriptase activity. We speculate that this activity could reflect an ancestral function of AEP proteins in the transition from the RNA to the DNA world.


Asunto(s)
Proteínas Arqueales/metabolismo , ADN Primasa/metabolismo , Thermococcus/enzimología , Secuencia de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/genética , ADN/biosíntesis , ADN Primasa/química , ADN Primasa/genética , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , ADN Polimerasa Dirigida por ADN/metabolismo , Datos de Secuencia Molecular , Plásmidos/genética , Estructura Terciaria de Proteína , ARN/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Thermococcus/genética
6.
Extremophiles ; 19(1): 69-76, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25420601

RESUMEN

A combination of three enzymes from the hyperthermophilic archaeon Thermococcus nautili, DNA primase PolpTN2, DNA polymerase PolB, and pTN2 DNA helicase, was found to synthesize up to 300-400 ng/µl dsDNA from deoxynucleotide triphosphates in less than 30 min in the absence of added template DNA and oligonucleotide primer. The reaction did not occur below 64 °C. No synthesis was observed if PolpTN2 or PolB were left out; helicase was not essential but accelerated the reaction. The DNA synthesized consisted of highly reiterated palindromic sequences reaching up to more that 10 kb. Sequence analysis of three independent reaction products synthesized at different temperatures showed that the palindromes shared a common pentanucleotide core, suggesting that random nucleic acid fragments were not responsible for priming the reaction. When enzymes were added sequentially, preincubation with primase plus helicase followed by PolB led to a shorter delay before the onset of the reaction as compared to preincubation with PolB plus helicase followed by primase. This suggests that the primase generates seeds that are subsequently amplified and elongated in synergy with PolB by a mechanism involving hairpin formation and slippage synthesis.


Asunto(s)
ADN Helicasas/genética , ADN Polimerasa II/genética , ADN Primasa/genética , ADN/biosíntesis , Thermococcus/enzimología , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , Electroforesis en Gel de Agar , Nucleasa Microcócica/química , Datos de Secuencia Molecular , Ácidos Nucleicos/química , Temperatura
7.
Extremophiles ; 18(4): 779-87, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24889120

RESUMEN

We expressed, purified, and characterized the helicase encoded by ORF1 of the Thermococcus nautili pTN2 plasmid (Soler et al. Nucl Acids Res 38, 5088-5104, 2010). The enzyme, which belongs to the SF1 family of helicases, possesses NTPase activity, with a strong preference for ATP and GTP as compared to CTP and TTP; dATP was also a substrate. Triphosphatase activity was strongly stimulated by single-stranded DNA and, to a lesser extent, by double-stranded DNA. Unwinding of duplexes comprising a fluorescent oligonucleotide was monitored by fluorescence polarization spectroscopy and by polyacrylamide gel electrophoresis. As observed for enzymes of the same family, pTN2 helicase displays a strong preference for duplexes comprising a 3' single-stranded extension and proceeds from the 3' to the 5' end of the loading strand. Under the conditions of the in vitro assay, pTN2 helicase did not appear to be recycled, but stayed bound to single-stranded DNA, which explains why high concentrations of enzyme are required to unwind long stretches of duplex DNA. The helicase enhances the synthesis of double-stranded DNA by pTN2 primase and by T. nautili PolB polymerase primed by pTN2 primase but it did not enhance synthesis by Taq DNA polymerase.


Asunto(s)
Proteínas Arqueales/metabolismo , ADN Helicasas/metabolismo , Thermococcus/enzimología , Proteínas Arqueales/genética , ADN Helicasas/genética , Replicación del ADN
8.
J Pineal Res ; 54(1): 46-57, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22775292

RESUMEN

Melatonin is a synchronizer of many physiological processes. Abnormal melatonin signaling is associated with human disorders related to sleep, metabolism, and neurodevelopment. Here, we present the X-ray crystal structure of human N-acetyl serotonin methyltransferase (ASMT), the last enzyme of the melatonin biosynthesis pathway. The polypeptide chain of ASMT consists of a C-terminal domain, which is typical of other SAM-dependent O-methyltransferases, and an N-terminal domain, which intertwines several helices with another monomer to form the physiologically active dimer. Using radioenzymology, we analyzed 20 nonsynonymous variants identified through the 1000 genomes project and in patients with neuropsychiatric disorders. We found that the majority of these mutations reduced or abolished ASMT activity including one relatively frequent polymorphism in the Han Chinese population (N17K, rs17149149). Overall, we estimate that the allelic frequency of ASMT deleterious mutations ranges from 0.66% in Europe to 2.97% in Asia. Mapping of the variants on to the 3-dimensional structure clarifies why some are harmful and provides a structural basis for understanding melatonin deficiency in humans.


Asunto(s)
Acetilserotonina O-Metiltransferasa/química , Acetilserotonina O-Metiltransferasa/genética , Acetilserotonina O-Metiltransferasa/metabolismo , Secuencia de Aminoácidos , Pueblo Asiatico/genética , Cristalografía por Rayos X , Frecuencia de los Genes , Humanos , Melatonina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Polimorfismo Genético , Alineación de Secuencia
9.
Infect Immun ; 80(8): 2963-70, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22753372

RESUMEN

Recent studies suggest shared pathogenic pathways during malaria and allergy. Indeed, IgE, histamine, and the parasite-derived Plasmodium falciparum histamine-releasing factor translationally controlled tumor protein (PfTCTP) can be found at high levels in serum from patients experiencing malaria, but their relationship with basophil activation remains unknown. We recruited P. falciparum-infected patients in Senegal with mild malaria (MM; n = 19) or severe malaria (SM; n = 9) symptoms and healthy controls (HC; n = 38). Levels of serum IgE, PfTCTP, and IgG antibodies against PfTCTP were determined by enzyme-linked immunosorbent assays (ELISA). Basophil reactivities to IgE-dependent and -independent stimulations were measured ex vivo using fresh blood by looking at the expression level of the basophil activation marker CD203c with flow cytometry. Unstimulated basophils from MM had significantly lower levels of CD203c expression compared to those from HC and SM. After normalization on this baseline level, basophils from SM showed an enhanced reactivity to calcimycin (A23187) and hemozoin. Although SM reached higher median levels of activation after anti-IgE stimulation, great interindividual differences did not allow the results to reach statistical significance. When primed with recombinant TCTP before anti-IgE, qualitative differences in terms of a better ability to control excessive activation could be described for SM. IgE levels were very high in malaria patients, but concentrations in MM and SM were similar and were not associated with basophil responses, which demonstrates that the presence of IgE alone cannot explain the various basophil reactivities. Indeed, PfTCTP could be detected in 32% of patients, with higher concentrations for SM. These PfTCTP-positive patients displayed significantly higher basophil reactivities to any stimulus. Moreover, the absence of anti-PfTCTP IgG was associated with higher responses in SM but not MM. Our results show an association between basophil reactivity and malaria severity and suggest a pathogenic role for plasmodial PfTCTP in the induction of this allergy-like mechanism.


Asunto(s)
Basófilos/fisiología , Malaria Falciparum/parasitología , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Adulto , Anticuerpos Antiprotozoarios/sangre , Femenino , Regulación de la Expresión Génica/inmunología , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Malaria Falciparum/sangre , Malaria Falciparum/epidemiología , Masculino , Plasmodium falciparum/genética , Plasmodium falciparum/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Senegal/epidemiología , Proteína Tumoral Controlada Traslacionalmente 1
10.
Protein Expr Purif ; 75(1): 114-8, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20688166

RESUMEN

N-acetyl serotonin methyl transferase (ASMT) is the last enzyme in the melatonin synthesis pathway. Evidence linking autism-related disorders with disorders of melatonin metabolism, and, more specifically, with mutations of the gene encoding ASMT, prompted us to investigate the properties and localization of this enzyme. As a first step, we undertook to overproduce the protein in a recombinant host. Early attempts to produce ASMT in recombinant Escherichia coli yielded only insoluble and heavily degraded material. However, recombinant ASMT (rASMT) could be produced in soluble, active form and purified in milligram amounts when the gene was cloned and expressed in Leishmania tarentolae.


Asunto(s)
Acetilserotonina O-Metiltransferasa/genética , Acetilserotonina O-Metiltransferasa/metabolismo , Expresión Génica , Leishmania/genética , Acetilserotonina O-Metiltransferasa/aislamiento & purificación , Clonación Molecular , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Solubilidad
11.
J Bacteriol ; 192(4): 1075-87, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20008066

RESUMEN

Proteins that bind sigma factors typically attenuate the function of the sigma factor by restricting its access to the RNA polymerase (RNAP) core enzyme. An exception to this general rule is the Crl protein that binds the stationary-phase sigma factor sigma(S) (RpoS) and enhances its affinity for the RNAP core enzyme, thereby increasing expression of sigma(S)-dependent genes. Analyses of sequenced bacterial genomes revealed that crl is less widespread and less conserved at the sequence level than rpoS. Seventeen residues are conserved in all members of the Crl family. Site-directed mutagenesis of the crl gene from Salmonella enterica serovar Typhimurium and complementation of a Deltacrl mutant of Salmonella indicated that substitution of the conserved residues Y22, F53, W56, and W82 decreased Crl activity. This conclusion was further confirmed by promoter binding and abortive transcription assays. We also used a bacterial two-hybrid system (BACTH) to show that the four substitutions in Crl abolish Crl-sigma(S) interaction and that residues 1 to 71 in sigma(S) are dispensable for Crl binding. In Escherichia coli, it has been reported that Crl also interacts with the ferric uptake regulator Fur and that Fur represses crl transcription. However, the Salmonella Crl and Fur proteins did not interact in the BACTH system. In addition, a fur mutation did not have any significant effect on the expression level of Crl in Salmonella. These results suggest that the relationship between Crl and Fur is different in Salmonella and E. coli.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Mapeo de Interacción de Proteínas , Salmonella typhimurium/fisiología , Factor sigma/genética , Factor sigma/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Sitios de Unión , ADN Bacteriano/metabolismo , Eliminación de Gen , Prueba de Complementación Genética , Peróxido de Hidrógeno/toxicidad , Viabilidad Microbiana , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Represoras/metabolismo , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Alineación de Secuencia , Técnicas del Sistema de Dos Híbridos
12.
Curr Opin Microbiol ; 38: 36-43, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-28472712

RESUMEN

A casposon, a member of a distinct superfamily of archaeal and bacterial self-synthesizing transposons that employ a recombinase (casposase) homologous to the Cas1 endonuclease, appears to have given rise to the adaptation module of CRISPR-Cas systems as well as the CRISPR repeats themselves. Comparison of the mechanistic features of the reactions catalyzed by the casposase and the Cas1-Cas2 heterohexamer, the CRISPR integrase, reveals close similarity but also important differences that explain the requirement of Cas2 for integration of short DNA fragments, the CRISPR spacers.


Asunto(s)
Adaptación Biológica , Archaea/genética , Bacterias/genética , Sistemas CRISPR-Cas , Secuencias Repetitivas Esparcidas
13.
J Mol Biol ; 316(5): 1061-9, 2002 Mar 08.
Artículo en Inglés | MEDLINE | ID: mdl-11884144

RESUMEN

The crystal structure of Clostridium thermocellum endoglucanase CelA in complex with cellopentaose has been determined at 0.94 A resolution. The oligosaccharide occupies six D-glucosyl-binding subsites, three on either side of the scissile glycosidic linkage. The substrate and product of the reaction occupy different positions at the reducing end of the cleft, where an extended array of hydrogen-bonding interactions with water molecules fosters the departure of the leaving group. Severe torsional strain upon the bound substrate forces a distorted boat(2,5) B conformation for the glucosyl residue bound at subsite -1, which facilitates the formation of an oxocarbenium ion intermediate and might favor the breakage of the sugar ring concomitant with catalysis.


Asunto(s)
Celulasa/química , Celulasa/metabolismo , Clostridium/enzimología , Oligosacáridos/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Enlace de Hidrógeno , Ligandos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Agua/metabolismo
14.
J Mol Biol ; 425(22): 4334-52, 2013 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-23856622

RESUMEN

Terminal deoxynucleotidyltransferase (Tdt) is a non-templated eukaryotic DNA polymerase of the polX family that is responsible for the random addition of nucleotides at the V(D)J junctions of immunoglobulins and T-cell receptors. Here we describe a series of high-resolution X-ray structures that mimic the pre-catalytic state, the post-catalytic state and a competent state that can be transformed into the two other ones in crystallo via the addition of dAMPcPP and Zn(2+), respectively. We examined the effect of Mn(2+), Co(2+) and Zn(2+) because they all have a marked influence on the kinetics of the reaction. We demonstrate a dynamic role of divalent transition metal ions bound to site A: (i) Zn(2+) (or Co(2+)) in Metal A site changes coordination from octahedral to tetrahedral after the chemical step, which explains the known higher affinity of Tdt for the primer strand when these ions are present, and (ii) metal A has to leave to allow the translocation of the primer strand and to clear the active site, a typical feature for a ratchet-like mechanism. Except for Zn(2+), the sugar puckering of the primer strand 3' terminus changes from C2'-endo to C3'-endo during catalysis. In addition, our data are compatible with a scheme where metal A is the last component that binds to the active site to complete its productive assembly, as already inferred in human pol beta. The new structures have potential implications for modeling pol mu, a closely related polX implicated in the repair of DNA double-strand breaks, in a complex with a DNA synapsis.


Asunto(s)
ADN Nucleotidilexotransferasa/química , Catálisis , Dominio Catalítico , Cobalto/química , Cobalto/metabolismo , Cristalografía por Rayos X , ADN Nucleotidilexotransferasa/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Humanos , Iones/química , Iones/metabolismo , Ligandos , Metales/química , Metales/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Especificidad por Sustrato , Zinc/química , Zinc/metabolismo
15.
Protein Expr Purif ; 55(1): 9-16, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17574862

RESUMEN

The S24F mutant of the chemokine RANTES was found to be partly acetylated when produced in recombinant Escherichia coli BL21(DE3)(pDIA17)(CCL5-S24F-pET-26b). Mass spectrometry and Edman sequencing of peptides generated by lys-C endopeptidase indicated that Lys-26, Lys-34, Lys-46, and Lys-57 were susceptible to acetylation. The extent of acetylation of the RANTES S24F polypeptide increased with temperature and with the time during which the culture was incubated after adding the inducer isopropyl-beta-D-thiogalactoside (IPTG). These findings suggest that induction at low temperature and for a short period of time should be preferred when spurious acetylation is a problem for the production of genuine recombinant polypeptides. Acetylation of the polypeptide was not affected by deleting acs, yfiQ, or speG, which encode acetyl-CoA synthetase, acetyl-CoA synthetase acetylase, and spermidine acetyl transferase, respectively, nor by the presence or absence of the pDIA17 plasmid, which harbours the cat gene encoding chloramphenicol acetyl transferase. By contrast, spontaneous acetylation of RANTES could be demonstrated by incubating either the purified polypeptide or inclusion bodies derived from an induced culture in the presence of acetyl-CoA.


Asunto(s)
Quimiocina CCL5/metabolismo , Frío , Escherichia coli/genética , Proteínas Recombinantes/metabolismo , Acetilcoenzima A/química , Acetilación , Acetiltransferasas/genética , Secuencia de Aminoácidos , Quimiocina CCL5/química , Quimiocina CCL5/genética , Quimiocinas/química , Quimiocinas/genética , Quimiocinas/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/genética , Eliminación de Gen , Humanos , Lisina/metabolismo , Datos de Secuencia Molecular , Mutación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Análisis de Secuencia de Proteína , Temperatura , Factores de Tiempo
16.
Microbiology (Reading) ; 144 ( Pt 1): 211-217, 1998 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-9467913

RESUMEN

The protomer forming the S-layer of Clostridium thermocellum was identified as a 140 kDa protein which was non-covalently bound to the cell wall. Cloning and sequencing of the corresponding gene revealed an open reading frame of 3108 nucleotides encoding a polypeptide of 1036 amino acids, termed SlpA. The amino acid composition of SlpA matches the composition of a previously described exocellular glycoprotein. SlpA shared extensive similarity with the S-layer protein of Bacillus sphaericus and with the outer wall protein of Bacillus brevis. In addition, the amino-terminal region of SlpA contained a segment presenting similarities with segments termed SLH (S-layer homologous), which are found in several bacterial exoproteins. A polypeptide of 209 residues comprising this segment was shown to bind to cell walls extracted from C. thermocellum cells.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas , Clostridium/metabolismo , Secuencia de Aminoácidos , Bacillus/genética , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Sitios de Unión , Pared Celular/metabolismo , Clostridium/genética , Clostridium/ultraestructura , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Alineación de Secuencia , Homología de Secuencia de Aminoácido
17.
Biochemistry ; 41(7): 2115-9, 2002 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-11841201

RESUMEN

To locate the region involved in binding dockerin domains, 15 mutations were introduced across the surface of the seventh cohesin domain of the scaffolding protein CipA, which holds together the cellulosome of Clostridium thermocellum. Mutated residues were located on both faces of the nine-stranded beta-sandwich forming the cohesin domain and on the loops connecting beta-strands 4 and 5, 6 and 7, and 8 and 9. The loop region was previously proposed, on the basis of sequence comparisons, to form a contiguous "recognition strip". Individual mutants of four residues, D39, Y74, E86, and G89, formed no complexes detectable by nondenaturing gel electrophoresis after incubation with CelD664, a shortened form of endoglucanase CelD lacking the residues linking the catalytic domain with the dockerin domain. The four sensitive residues encompass a hydrophobic region on the 5-6-3-8 face of the molecule, which overlaps partially with the recognition strip and with a hydrophobic zone involved in the formation of cohesin-cohesin dimers. Isothermal titration calorimetry showed that single cohesin mutations affecting the binding of CelD664 had significant effects on the enthalpy or entropy of binding of wild-type CelD but much lesser effects on the association constant, owing to enthalpy-entropy compensation. However, the affinity for wild-type CelD of the triple mutant affecting D39, Y74, and E86 was reduced by 2 orders of magnitude, due to negative cooperativity between mutations affecting D39 + Y74 on one hand and E86 on the other hand.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clostridium/enzimología , Clostridium/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Sitios de Unión/genética , Proteínas de Ciclo Celular , Celulasa/metabolismo , Proteínas Cromosómicas no Histona , Proteínas Fúngicas , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Mapeo Peptídico/métodos , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Cohesinas
18.
Biochemistry ; 41(7): 2106-14, 2002 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-11841200

RESUMEN

Mutagenized dockerin domains of endoglucanase CelD (type I) and of the cellulosome-integrating protein CipA (type II) were constructed by swapping residues 10 and 11 of the first or the second duplicated segment between the two polypeptides. These residues have been proposed to determine the specificity of cohesin-dockerin interactions. The dockerin domain of CelD still bound to the seventh cohesin domain of CipA (CohCip7), provided that mutagenesis occurred in one segment only. Binding was no longer detected by nondenaturing gel electrophoresis when both segments were mutagenized. The dockerin domain of CipA bound to the cohesin domain of SdbA as long as the second segment was intact. None of the mutated dockerins displayed detectable binding to the noncognate cohesin domain. Isothermal titration calorimetry showed that binding of the CelD dockerin to CohCip7 occurred with a high affinity [K(a) = (2.6 +/- 0.5) x 10(9) M(-1)] and a 1:1 stoichiometry. The reaction was weakly exothermic (DeltaHdegrees = -2.22 +/- 0.2 kcal x mol(-1)) and largely entropy driven (TDeltaSdegrees = 10.70 +/- 0.5 kcal x mol(-1)). The heat capacity change on complexation was negative (DeltaC(p) = -305 +/- 15 cal x mol(-1) x K(-1)). These values show that cohesin-dockerin binding is mainly hydrophobic. Mutations in the first or the second dockerin segment reduced or enhanced, respectively, the hydrophobic character of the interaction. Due to partial enthalpy-entropy compensation, these mutations induced only small changes in binding affinity. However, the binding affinity was strongly decreased when both segments were mutated, indicating strong negative cooperativity between the two mutated sites.


Asunto(s)
Proteínas Bacterianas/metabolismo , Celulasa/metabolismo , Clostridium/enzimología , Proteínas de la Membrana/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas Nucleares/metabolismo , Secuencias Repetitivas de Aminoácido , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión/genética , Proteínas de Ciclo Celular , Celulasa/química , Celulasa/genética , Proteínas Cromosómicas no Histona , Clostridium/genética , Cisteína/genética , Proteínas Fúngicas , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Secuencias Repetitivas de Aminoácido/genética , Termodinámica , Cohesinas
19.
Biosci Biotechnol Biochem ; 68(4): 924-6, 2004 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15118324

RESUMEN

The interaction between the type-II dockerin domain of the scaffoldin protein CipA and the type-II cohesin domain of the outer layer protein SdbA is the fundamental mechanism for anchoring the cellulosome to the cell surface of Clostridium thermocellum. We constructed and purified a dockerin polypeptide and a cohesin polypeptide, and determined affinity constants of the interaction between them by the surface plasmon resonance method. The dissociation constant (K(D)) value was 1.8 x 10(-9) M, which is a little larger than that for the combination of a type-I dockerin and a type-I cohesin.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Celulosomas/química , Celulosomas/metabolismo , Clostridium/química , Proteínas Nucleares/química , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , Clostridium/metabolismo , Proteínas Fúngicas , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Resonancia por Plasmón de Superficie , Cohesinas
20.
J Biol Chem ; 279(11): 9867-74, 2004 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-14688277

RESUMEN

The cellulosome components are assembled into the cellulosome complex by the interaction between one of the repeated cohesin domains of a scaffolding protein and the dockerin domain of an enzyme component. We prepared five recombinant cohesin polypeptides of the Clostridium thermocellum scaffolding protein CipA, two dockerin polypeptides of C. thermocellum Xyn11A and Xyn10C, four cohesin polypeptides of Clostridium josui CipA, and two dockerin polypeptides of C. josui Aga27A and Cel8A, and qualitatively and quantitatively examined the cohesin-dockerin interactions within C. thermocellum and C. josui, respectively, and the species specificity of the cohesin-dockerin interactions between these two bacteria. Surface plasmon resonance (SPR) analysis indicated that there was a certain selectivity, with a maximal 34-fold difference in the K(D) values, in the cohesin-dockerin interactions within a combination of C. josui, although this was not detected by qualitative analysis. Affinity blotting analysis suggested that there was at least one exception to the species specificity in the cohesin-dockerin interactions, although species specificity was generally conserved among the cohesin and dockerin polypeptides from C. thermocellum and C. josui, i.e. the dockerin polypeptides of C. thermocellum Xyn11A exceptionally bound to the cohesin polypeptides from C. josui CipA. SPR analysis confirmed this exceptional binding. We discuss the relationship between the species specificity of the cohesin-dockerin binding and the conserved amino acid residues in the dockerin domains.


Asunto(s)
Proteínas Bacterianas/química , Celulasa/química , Clostridium/metabolismo , Proteínas de la Membrana/química , Proteínas Nucleares/química , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Proteínas de Ciclo Celular , Celulasa/metabolismo , Proteínas Cromosómicas no Histona , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Proteínas Fúngicas , Cinética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Péptidos/química , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Especificidad por Sustrato , Resonancia por Plasmón de Superficie , Factores de Tiempo , Cohesinas
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