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Victoria Guixe - One of the best experts on this subject based on the ideXlab platform.
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adp dependent phosphofructokinases from the archaeal order methanosarcinales display redundant glucokinase activity
Archives of Biochemistry and Biophysics, 2017Co-Authors: Ricardo A. Zamora, Victor Castrofernandez, Felipe Gonzalezordenes, Victoria GuixeAbstract:The genome of Methanosarcinales organisms presents both ADP-dependent glucokinase and phosphofructokinase genes. However, Methanococcoides burtonii has a truncate glucokinase gene with a large deletion at the C-terminal, where the catalytic GXGD motif is located. Characterization of its phosphofructokinase annotated protein shows that is a bifunctional enzyme able to supply the absence of the glucokinase activity. Moreover, kinetic analyses of the phosphofructokinase annotated enzyme from, Methanohalobium evestigatum demonstrated that this enzyme is also bifunctional. The high conservation of the active site residues of all the enzymes from the order Methanosarcinales suggest that they should be bifunctional, as was previously reported for the ADP-dependent kinases from Methanococcales, highlighting the redundancy of the glucokinase activity in this archaeal group. The presence of active glycolytic enzymes would be important when glycogen storage of these organisms needs to be degraded to be used as energy source. Kinetic and structural information allows us to establish a substrate specificity signature that identifies specific GK or PFK, and bifunctional enzymes in this family.
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bifunctional adp dependent phosphofructokinase glucokinase activity in the order Methanococcales biochemical characterization of the mesophilic enzyme from methanococcus maripaludis
FEBS Journal, 2014Co-Authors: Victor Castrofernandez, Felipe Bravomoraga, Alejandra Herreramorande, Victoria GuixeAbstract:In some archaea, the phosphorylation of glucose and fructose 6-phosphate (fructose 6P) is carried out by enzymes that are specific for either substrate and that use ADP as phosphoryl donor. In the hyperthermophilic archaeon Methanocaldococcus jannaschii, a bifunctional enzyme able to phosphorylate glucose and fructose 6P has been described. To determine whether the ability to phosphorylate both glucose and fructose 6P is a common feature for all enzymes of the order Methanococcales, we expressed, purified and characterized the unique homologous protein of the mesophilic archaea Methanococcus maripaludis. Assay of the enzyme activity with different sugars, metals and nucleotides allows us to conclude that the enzyme is able to phosphorylate both fructose 6P and glucose in the presence of ADP and a divalent metal cation. Kinetic characterization of the enzyme revealed complex regulation by the free Mg2+ concentration and AMP, with the latter appearing to be a key metabolite. To determine whether this enzyme could have a role in gluconeogenesis, we evaluated the reversibility of both reactions and found that glucokinase activity is reversible, whereas phosphofructokinase activity is not. To determine the important residues for glucose and fructose 6P binding, we modeled the bifunctional phosphofructokinase/glucokinase enzyme from M. maripaludis and its interactions with both sugar substrates using protein–ligand docking. Comparison of the active site of the phosphofructokinase/glucokinase enzyme from M. maripaludis with the structural models constructed for all the homology sequences present in the order Methanococcales shows that all of the ADP-dependent kinases from this order would be able to phosphorylate glucose and fructose 6P, which rules out the current annotation of these enzymes as specific phosphofructokinases. Database Model data are available in the Protein Model Data Base under accession numbers PM0079106, PM0079107, PM0079108, PM0079109, PM0079110, PM0079111, PM0079112, PM0079113, PM0079114, PM0079115 and PM0079116
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Conservation of cluster residues involved in domain opening/closure across the ADP-dependent sugar kinases family.
2013Co-Authors: Jaime Andrés Rivas-pardo, Alejandra Herrera-morande, Victor Castro-fernandez, Francisco J. Fernandez, Cristina M. Vega, Victoria GuixeAbstract:(A) Consensus phylogenetic tree for ADP-dependent sugar kinase family determined by Bayesian inference. Groups of enzymes from archaea are shown in color: GK from Thermococcales (blue), PFK from Thermococcales (pink), PFK from Methanococcales (purple) PFK from Methanosarcinales (dark green) and GK from Methanosarcinales (cyan). The group of enzymes from eukaryotic organisms that were used as an outgroup to establish the tree root is shown in gray. The posterior probability of some interesting groups is shown in its respective node. (B) Multiple sequence alignment of glucokinases from the Thermococcales group. Residues involved in clusters described in the texts are indicated by dots; cluster 1, red (Glu188-Thr446/Val447); cluster 2, yellow (Arg202-Tyr354); cluster 3, green (Arg117/Glu115-Gly386/Ser445) and brown (Lys382-Arg117). (C) Amino acids involved in clusters 1–3 are shown as spheres and colored as in B.
Guixé Leguía, Victoria Cristina - One of the best experts on this subject based on the ideXlab platform.
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ADP-dependent phosphofructokinases from the archaeal order Methanosarcinales display redundant glucokinase activity
'Elsevier BV', 2017Co-Authors: Zamora, Ricardo A., González Órdenes Felipe, Castro Fernández Víctor, Guixé Leguía, Victoria CristinaAbstract:© 2017 Elsevier Inc. The genome of Methanosarcinales organisms presents both ADP-dependent glucokinase and phosphofructokinase genes. However, Methanococcoides burtonii has a truncate glucokinase gene with a large deletion at the C-terminal, where the catalytic GXGD motif is located. Characterization of its phosphofructokinase annotated protein shows that is a bifunctional enzyme able to supply the absence of the glucokinase activity. Moreover, kinetic analyses of the phosphofructokinase annotated enzyme from, Methanohalobium evestigatum demonstrated that this enzyme is also bifunctional. The high conservation of the active site residues of all the enzymes from the order Methanosarcinales suggest that they should be bifunctional, as was previously reported for the ADP-dependent kinases from Methanococcales, highlighting the redundancy of the glucokinase activity in this archaeal group. The presence of active glycolytic enzymes would be important when glycogen storage of these organism
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Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases
'American Society for Biochemistry & Molecular Biology (ASBMB)', 2017Co-Authors: Castro Fernández Víctor, González Órdenes Felipe, Herrera Morandé Alejandra, Zamorano Sarria Ricardo, Merino Felipe, Padilla Salinas Felipe, Pereira, Humberto M., Brandao Neto José, Garratt, Richard C., Guixé Leguía, Victoria CristinaAbstract:One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales, Methanosarcinales, and Methanococcales, as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales.Fondo Nacional de Desarrollo Cientifico y Tecnologico from Chile FONDECYT 1150460 316033
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Bifunctional ADP-dependent phosphofructokinase/ glucokinase activity in the order Methanococcales – biochemical characterization of the mesophilic enzyme from Methanococcus maripaludis
FEBS, 2014Co-Authors: Castro Fernández Víctor, Bravo Moraga Felipe, Herrera Morandé Alejandra, Guixé Leguía, Victoria CristinaAbstract:Artículo de publicación ISIIn some archaea, the phosphorylation of glucose and fructose 6-phosphate (fructose 6P) is carried out by enzymes that are specific for either substrate and that use ADP as phosphoryl donor. In the hyperthermophilic archaeon Methanocaldococcus jannaschii, a bifunctional enzyme able to phosphorylate glucose and fructose 6P has been described. To determine whether the ability to phosphorylate both glucose and fructose 6P is a common feature for all enzymes of the order Methanococcales, we expressed, purified and characterized the unique homologous protein of the mesophilic archaea Methanococcus maripaludis. Assay of the enzyme activity with different sugars, metals and nucleotides allows us to conclude that the enzyme is able to phosphorylate both fructose 6P and glucose in the presence of ADP and a divalent metal cation. Kinetic characterization of the enzyme revealed complex regulation by the free Mg2+ concentration and AMP, with the latter appearing to be a key metabolite. To determine whether this enzyme could have a role in gluconeogenesis, we evaluated the reversibility of both reactions and found that glucokinase activity is reversible, whereas phosphofructokinase activity is not. To determine the important residues for glucose and fructose 6P binding, we modeled the bifunctional phosphofructokinase/ glucokinase enzyme from M. maripaludis and its interactions with both sugar substrates using protein–ligand docking. Comparison of the active site of the phosphofructokinase/glucokinase enzyme from M. maripaludis with the structural models constructed for all the homology sequences present in the order Methanococcales shows that all of the ADPdependent kinases from this order would be able to phosphorylate glucose and fructose 6P, which rules out the current annotation of these enzymes as specific phosphofructokinases.This work was supported by Fondo Nacional de Desarrollo Cientifico y Tecnologico (Fondecyt, Chile) Grant 1110137 and CONICYT scholarship 24121448
Herrera Morandé, Alejandra Margarita - One of the best experts on this subject based on the ideXlab platform.
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Estudios funcionales y estructurales en quinasas dependientes de ADP relacionadas con el metabolismo de glucosa en Archaea
'Edicions de la Universitat de Barcelona', 2015Co-Authors: Herrera Morandé, Alejandra MargaritaAbstract:En algunos organismos Archaeas, la vía glicolítica presenta modificaciones entre las que destaca las enzimas quinasas que fosforilan glucosa y fructosa 6-fosfato utilizando ADP en vez de ATP como dador de grupo fosforilo. Estas enzimas pertenecen a la superfamilia riboquinasa, puntualmente a la familia de quinasas dependientes de ADP, y están conformadas por un dominio menor y un dominio mayor el cual presenta un plegamiento tipo riboquinasa, común a todos los miembros esta superfamilia. La mayoría de estas quinasas han sido descritas en arqueas hipertermófilas como Thermococcus litoral (TlGK), metanogénicas bifuncionales, con actividad glucoquinasa (GK) y fosfofructoquinasa (PFK), de Methanococcus jannaschii o Methanococcus maripaludis, aunque también se han encontrado en organismo eucariontes como humano y ratones. En esta tesis se estudió la enzima glucoquinasa de TlGK como modelo de una quinasa hipertermófila dependiente de ADP pata conocer los determinantes estructurales en el mecanismo cinético y la especificidad por ADP o ATP mediante cristalografía de rayos –X y estudios cinética enzimática. Los resultados confirmaron que la TlGK experimenta cambios en su conformación como consecuencia de la unión de sus ligandos demostrado en las estructuras de la proteína, donde se distinguió que la proteína en su forma apo (2.0 Å) presenta una mayor distancia entre sus dominios (conformación abierta), mientras que en la estructura en complejo ternario (2.5 Å) esta distancia se reduce mostrando una estructura cerrada. Estos resultados se relacionan con el mecanismo cinético ordenado en secuencia, donde la unión de MgADP, induce el primer cambio conformacional en la enzima, seguido por la unión de glucosa provocando el cierre de los dominios de la enzima. Dichos cambios conformacionales son estabilizados por una serie de interacciones que se coordinan entre los dominios menor y mayor. La especificidad por el nucleótido fue evaluada generando una versión permutante de la TlGK (perGK), que imita la topología de la región β-meandro de las quinasas dependientes de ATP, que constituye casi por completo el sitio de unión del nucleótido. Los resultados indican que la enzima perGK utiliza ADP, en vez de a ATP como dador de grupo fosforilo, revelando que la permutación no es suficiente para el cambio en la especificidad del nucleótido. Estudios cinéticos señalan que el mecanismo de perGK es ordenado en secuencia, no obstante, el orden de unión de los sustratos varió, respecto a lo observado para la enzima TlGK. Estos cambios fueron apoyados con las estructuras cristalinas de la proteína perGK en su forma apo (2.14 Å), en complejo con glucosa (1.95 Å) y el complejo ternario (2.44 Å), generadas por cristalografía de rayos-X, donde se infiere que la enzima perGK sufre un cambio en su conformación luego que se une glucosa, no así con la unión de MgADP, en claro contraste con TlGK. Por tanto, este cambio en la topología, posiblemente, haya sido uno de los cambios estructurales que experimentaron estas enzimas, de la superfamilia de riboquinasas, a lo largo de su historia evolutiva para transformarse a quinasas dependientes de ADP. La enzima dependiente de ADP, MjPfk/GK, que posee las dos actividades PFK y GK en la misma cadena polipeptidica, es la única enzima caracterizada de este tipo del orden de las Methanococcales, por lo que se desconoce si este carácter bifuncional de la enzima es un rasgo particular de MjPfk/GK, o si es una característica común de los miembros de este orden. En este estudio se determinó que el mecanismo cinético de la enzima MjPfk/GK es secuencial para ambas reacciones, y solo tiene la capacidad de fosforilar glucosa y fructosa-6-fosfato, siendo muy específica de la vía glicolítica. Además, se confirmó que la enzima fosfofructoquinasa del mesófilo M. maripaludis presenta actividad GK además de actividad PFK sugiriendo que este carácter bifuncional sea un rasgo compartido por otras enzimas homologas del orden Methanococcales. Finalmente, se dilucidó que la actividad GK en ambas enzimas bifuncionales se ve afectada por las concentración libre de Mg2+ y AMP, lo que nos lleva a pensar que estos metabolitos pueden ser claves para promover la vía glicolítica actuando como un sensor de la disponibilidad de energía celular aportada por ADP o ATP.In some Archaea, the glucose degradation proceeds through a modified version of glycolytic pathway where phosphorylation of glucose and fructose 6-phosphate is performed by kinases that use ADP instead ADP as phosphoryl donor. These enzymes belong to ribokinase superfamily due to share ribokinase folding type. Also these enzymes are found in Archaea from Thermococcales and Methanococcales orders. In these studies we reported that the glucokinase of T.litoralis (TlGK) undergoes changes in its conformation due to the binding of its ligands shown in the structures of the protein in the absence and presence of their ligands. These structural changes are relate to sequentially ordered kinetic mechanism, where MgADP binding induces the firth conformational change, followed by binding of glucose, causing the approach of the domains of the enzyme. These conformational changes are stabilized by a series of interactions that are coordinated between the smallest and largest domains. Also, it was revealed that the permutation in the β- meander regions of TlGK, which is almost entirely the nucleotide binding site, is not sufficient to change the specificity of the nucleotide. The kinetic studies indicated that the kinetic mechanism perGK is ordered in sequence, however, the order of binding of the substrates varied with respect to that observed for the enzyme TlGK. These changes were supported by the crystal structures of the perGK protein in their apo form, with glucose and the ternary complex, generated by crystallography X-rays. Therefore, this change in topology possibly has been one of the structural changes experienced by these enzymes, riboquinasas superfamily, along their evolutionary history to transform ADP-dependent kinases. On the other hand, we study on the ADP-dependent MjPfk/GK kinase, which owns both phosphofructokinase and glucokinase activity in the same polypeptide chain, it was determined that the enzyme has a sequential mechanism for both reactions and that this enzyme is specific for glucose and fructose 6P, being highly specific in the glycolytic pathway. Furthermore, it was confirmed that the enzyme phosphofructokinase mesophyll M. maripaludis GK also has activity PFK activity suggesting that this bifunctional character is shared by other homologous enzymes enforcement Methanococcales order. Finally, it was elucidated that the GK activity in both bifunctional enzyme is affected by the free concentration of Mg2 + and AMP, which leads us to believe that these metabolites may be key to promoting glycolytic pathway acting as a sensor of energy availability cell provided by ADP or ATP
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Estudios funcionales y estructurales en quinasas dependientes de ADP relacionadas con el metabolismo de glucosa en Archaea
'Edicions de la Universitat de Barcelona', 2015Co-Authors: Herrera Morandé, Alejandra MargaritaAbstract:[spa] En algunos organismos Archaeas, la vía glicolítica presenta modificaciones entre las que destaca las enzimas quinasas que fosforilan glucosa y fructosa 6-fosfato utilizando ADP en vez de ATP como dador de grupo fosforilo. Estas enzimas pertenecen a la superfamilia riboquinasa, puntualmente a la familia de quinasas dependientes de ADP, y están conformadas por un dominio menor y un dominio mayor el cual presenta un plegamiento tipo riboquinasa, común a todos los miembros esta superfamilia. La mayoría de estas quinasas han sido descritas en arqueas hipertermófilas como Thermococcus litoral (TlGK), metanogénicas bifuncionales, con actividad glucoquinasa (GK) y fosfofructoquinasa (PFK), de Methanococcus jannaschii o Methanococcus maripaludis, aunque también se han encontrado en organismo eucariontes como humano y ratones. En esta tesis se estudió la enzima glucoquinasa de TlGK como modelo de una quinasa hipertermófila dependiente de ADP pata conocer los determinantes estructurales en el mecanismo cinético y la especificidad por ADP o ATP mediante cristalografía de rayos –X y estudios cinética enzimática. Los resultados confirmaron que la TlGK experimenta cambios en su conformación como consecuencia de la unión de sus ligandos demostrado en las estructuras de la proteína, donde se distinguió que la proteína en su forma apo (2.0 Å) presenta una mayor distancia entre sus dominios (conformación abierta), mientras que en la estructura en complejo ternario (2.5 Å) esta distancia se reduce mostrando una estructura cerrada. Estos resultados se relacionan con el mecanismo cinético ordenado en secuencia, donde la unión de MgADP, induce el primer cambio conformacional en la enzima, seguido por la unión de glucosa provocando el cierre de los dominios de la enzima. Dichos cambios conformacionales son estabilizados por una serie de interacciones que se coordinan entre los dominios menor y mayor. La especificidad por el nucleótido fue evaluada generando una versión permutante de la TlGK (perGK), que imita la topología de la región β-meandro de las quinasas dependientes de ATP, que constituye casi por completo el sitio de unión del nucleótido. Los resultados indican que la enzima perGK utiliza ADP, en vez de a ATP como dador de grupo fosforilo, revelando que la permutación no es suficiente para el cambio en la especificidad del nucleótido. Estudios cinéticos señalan que el mecanismo de perGK es ordenado en secuencia, no obstante, el orden de unión de los sustratos varió, respecto a lo observado para la enzima TlGK. Estos cambios fueron apoyados con las estructuras cristalinas de la proteína perGK en su forma apo (2.14 Å), en complejo con glucosa (1.95 Å) y el complejo ternario (2.44 Å), generadas por cristalografía de rayos-X, donde se infiere que la enzima perGK sufre un cambio en su conformación luego que se une glucosa, no así con la unión de MgADP, en claro contraste con TlGK. Por tanto, este cambio en la topología, posiblemente, haya sido uno de los cambios estructurales que experimentaron estas enzimas, de la superfamilia de riboquinasas, a lo largo de su historia evolutiva para transformarse a quinasas dependientes de ADP. La enzima dependiente de ADP, MjPfk/GK, que posee las dos actividades PFK y GK en la misma cadena polipeptidica, es la única enzima caracterizada de este tipo del orden de las Methanococcales, por lo que se desconoce si este carácter bifuncional de la enzima es un rasgo particular de MjPfk/GK, o si es una característica común de los miembros de este orden. En este estudio se determinó que el mecanismo cinético de la enzima MjPfk/GK es secuencial para ambas reacciones, y solo tiene la capacidad de fosforilar glucosa y fructosa-6-fosfato, siendo muy específica de la vía glicolítica. Además, se confirmó que la enzima fosfofructoquinasa del mesófilo M. maripaludis presenta actividad GK además de actividad PFK sugiriendo que este carácter bifuncional sea un rasgo compartido por otras enzimas homologas del orden Methanococcales. Finalmente, se dilucidó que la actividad GK en ambas enzimas bifuncionales se ve afectada por las concentración libre de Mg2+ y AMP, lo que nos lleva a pensar que estos metabolitos pueden ser claves para promover la vía glicolítica actuando como un sensor de la disponibilidad de energía celular aportada por ADP o ATP.[eng] In some Archaea, the glucose degradation proceeds through a modified version of glycolytic pathway where phosphorylation of glucose and fructose 6-phosphate is performed by kinases that use ADP instead ADP as phosphoryl donor. These enzymes belong to ribokinase superfamily due to share ribokinase folding type. Also these enzymes are found in Archaea from Thermococcales and Methanococcales orders. In these studies we reported that the glucokinase of T.litoralis (TlGK) undergoes changes in its conformation due to the binding of its ligands shown in the structures of the protein in the absence and presence of their ligands. These structural changes are relate to sequentially ordered kinetic mechanism, where MgADP binding induces the firth conformational change, followed by binding of glucose, causing the approach of the domains of the enzyme. These conformational changes are stabilized by a series of interactions that are coordinated between the smallest and largest domains. Also, it was revealed that the permutation in the β- meander regions of TlGK, which is almost entirely the nucleotide binding site, is not sufficient to change the specificity of the nucleotide. The kinetic studies indicated that the kinetic mechanism perGK is ordered in sequence, however, the order of binding of the substrates varied with respect to that observed for the enzyme TlGK. These changes were supported by the crystal structures of the perGK protein in their apo form, with glucose and the ternary complex, generated by crystallography X-rays. Therefore, this change in topology possibly has been one of the structural changes experienced by these enzymes, riboquinasas superfamily, along their evolutionary history to transform ADP-dependent kinases. On the other hand, we study on the ADP-dependent MjPfk/GK kinase, which owns both phosphofructokinase and glucokinase activity in the same polypeptide chain, it was determined that the enzyme has a sequential mechanism for both reactions and that this enzyme is specific for glucose and fructose 6P, being highly specific in the glycolytic pathway. Furthermore, it was confirmed that the enzyme phosphofructokinase mesophyll M. maripaludis GK also has activity PFK activity suggesting that this bifunctional character is shared by other homologous enzymes enforcement Methanococcales order. Finally, it was elucidated that the GK activity in both bifunctional enzyme is affected by the free concentration of Mg2 + and AMP, which leads us to believe that these metabolites may be key to promoting glycolytic pathway acting as a sensor of energy availability cell provided by ADP or ATP
Reinhard Wirth - One of the best experts on this subject based on the ideXlab platform.
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Methanocaldococcus villosus sp. nov., a heavily flagellated archaeon that adheres to surfaces and forms cell-cell contacts.
International Journal of Systematic and Evolutionary Microbiology, 2011Co-Authors: Annett Bellack, Harald Huber, Reinhard Rachel, Gerhard Wanner, Reinhard WirthAbstract:A novel chemolithoautotrophic, hyperthermophilic methanogen was isolated from a submarine hydrothermal system at the Kolbeinsey Ridge, north of Iceland. Based on its 16S rRNA gene sequence, the strain belongs to the order Methanococcales within the genus Methanocaldococcus, with approximately 95 % sequence similarity to Methanocaldococcus jannaschii as its closest relative. Cells of the novel organism stained Gram-negative and appeared as regular to irregular cocci possessing more than 50 polar flagella. These cell appendages mediated not only motility but also adherence to abiotic surfaces and the formation of cell–cell contacts. The new isolate grew at 55–90 °C, with optimum growth at 80 °C. The optimum NaCl concentration for growth was 2.5 % (w/v), and the optimal pH was 6.5. The cells gained their energy exclusively by reduction of CO2 with H2. Selenate, tungstate and yeast extract stimulated growth significantly. The genome size was determined to be in the range 1.8–2.0 kb, and the G+C content of the genomic DNA was 30 mol%. Despite being physiologically nearly identical to the other members of the genus Methanocaldococcus, analysis of whole-cell proteins revealed significant differences. Based on the results from phylogenetic, morphological and protein analyses, we conclude that the novel strain represents a novel species of the genus Methanocaldococcus, for which the name Methanocaldococcus villosus sp. nov. is proposed (type strain KIN24-T80T = DSM 22612T = JCM 16315T).
Zvi Kelman - One of the best experts on this subject based on the ideXlab platform.
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The CMG (CDC45/RecJ, MCM, GINS) complex is a conserved component of the DNA replication system in all archaea and eukaryotes
Biology Direct, 2012Co-Authors: Kira S Makarova, Zvi KelmanAbstract:Background In eukaryotes, the CMG (CDC45, MCM, GINS) complex containing the replicative helicase MCM is a key player in DNA replication. Archaeal homologs of the eukaryotic MCM and GINS proteins have been identified but until recently no homolog of the CDC45 protein was known. Two recent developments, namely the discovery of archaeal G INS- a ssociated n uclease (GAN) that belongs to the RecJ family of the DHH hydrolase superfamily and the demonstration of homology between the DHH domains of CDC45 and RecJ, show that at least some Archaea possess a full complement of homologs of the CMG complex subunits. Here we present the results of in-depth phylogenomic analysis of RecJ homologs in archaea. Results We confirm and extend the recent hypothesis that CDC45 is the eukaryotic ortholog of the bacterial and archaeal RecJ family nucleases. At least one RecJ homolog was identified in all sequenced archaeal genomes, with the single exception of Caldivirga maquilingensis . These proteins include previously unnoticed remote RecJ homologs with inactivated DHH domain in Thermoproteales . Combined with phylogenetic tree reconstruction of diverse eukaryotic, archaeal and bacterial DHH subfamilies, this analysis yields a complex scenario of RecJ family evolution in Archaea which includes independent inactivation of the nuclease domain in Crenarchaeota and Halobacteria, and loss of this domain in Methanococcales. Conclusions The archaeal complex of a CDC45/RecJ homolog, MCM and GINS is homologous and most likely functionally analogous to the eukaryotic CMG complex, and appears to be a key component of the DNA replication machinery in all Archaea. It is inferred that the last common archaeo-eukaryotic ancestor encoded a CMG complex that contained an active nuclease of the RecJ family. The inactivated RecJ homologs in several archaeal lineages most likely are dedicated structural components of replication complexes. Reviewers This article was reviewed by Prof. Patrick Forterre, Dr. Stephen John Aves (nominated by Dr. Purificacion Lopez-Garcia) and Prof. Martijn Huynen. For the full reviews, see the Reviewers' Comments section.