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1.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 536-544, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205016

RESUMO

The crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the hyperthermophilic archaeon Hyperthermus butylicus is presented at 1.8 Šresolution. Previous structures of archaeal Rubisco have been found to assemble into decamers, and this oligomerization was thought to be required for a highly thermally stable enzyme. In the current study, H. butylicus Rubisco is shown to exist as a dimer in solution, yet has a thermal denaturation midpoint of 114°C, suggesting that high thermal stability can be achieved without an increased oligomeric state. This increased thermal stability appears to be due to an increased number of electrostatic interactions within the monomeric subunit. As such, H. butylicus Rubisco presents a well characterized system in which to investigate the role of assembly and thermal stability in enzyme function.


Assuntos
Proteínas Arqueais/química , Modelos Moleculares , Pyrodictiaceae/enzimologia , Ribulose-Bifosfato Carboxilase/química , Cristalização , Cristalografia por Raios X/métodos , Estabilidade Enzimática , Estrutura Quaternária de Proteína , Eletricidade Estática
2.
Int J Syst Evol Microbiol ; 66(9): 3372-3376, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27260263

RESUMO

A hyperthermophilic, autotrophic iron and nitrate reducer, strain Su06T, was isolated from an active deep-sea hydrothermal vent chimney on the Endeavour Segment in the north-eastern Pacific Ocean. It was obligately anaerobic, hydrogenotrophic and reduced Fe(III) oxide to magnetite and NO3- to N2. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain was more than 97 % similar to other species of the genera Pyrodictium and Hyperthermus. Therefore, overall genome relatedness index analyses were performed to establish whether strain Su06T represents a novel species. For each analysis, strain Su06T was most similar to Pyrodictium occultum PL-19T. Relative to this strain, the average nucleotide identity score for strain Su06T was 72 %, the genome-to-genome direct comparison score was 13-19 % and the species identification score at the protein level was 89 %. For each analysis, strain Su06T was below the species delineation cutoff. Based on its whole genome sequence and its unique phenotypic characteristics, strain Su06T is suggested to represent a novel species of the genus Pyrodictium, for which the name Pyrodictium delaneyi is proposed. The type strain is Su06T (=DSM 28599T=ATCC BAA-2559T).


Assuntos
Compostos Férricos/metabolismo , Fontes Hidrotermais/microbiologia , Filogenia , Pyrodictiaceae/classificação , Composição de Bases , DNA Arqueal/genética , Óxido Ferroso-Férrico/metabolismo , Ferro/metabolismo , Nitratos/metabolismo , Oceano Pacífico , Pyrodictiaceae/genética , Pyrodictiaceae/isolamento & purificação , RNA Ribossômico 16S/genética , Água do Mar/microbiologia , Análise de Sequência de DNA
3.
Geobiology ; 12(3): 200-11, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24612368

RESUMO

Hyperthermophilic iron reducers are common in hydrothermal chimneys found along the Endeavour Segment in the northeastern Pacific Ocean based on culture-dependent estimates. However, information on the availability of Fe(III) (oxyhydr) oxides within these chimneys, the types of Fe(III) (oxyhydr) oxides utilized by the organisms, rates and environmental constraints of hyperthermophilic iron reduction, and mineral end products is needed to determine their biogeochemical significance and are addressed in this study. Thin-section petrography on the interior of a hydrothermal chimney from the Dante edifice at Endeavour showed a thin coat of Fe(III) (oxyhydr) oxide associated with amorphous silica on the exposed outer surfaces of pyrrhotite, sphalerite, and chalcopyrite in pore spaces, along with anhydrite precipitation in the pores that is indicative of seawater ingress. The iron sulfide minerals were likely oxidized to Fe(III) (oxyhydr) oxide with increasing pH and Eh due to cooling and seawater exposure, providing reactants for bioreduction. Culture-dependent estimates of hyperthermophilic iron reducer abundances in this sample were 1740 and 10 cells per gram (dry weight) of material from the outer surface and the marcasite-sphalerite-rich interior, respectively. Two hyperthermophilic iron reducers, Hyperthermus sp. Ro04 and Pyrodictium sp. Su06, were isolated from other active hydrothermal chimneys on the Endeavour Segment. Strain Ro04 is a neutrophilic (pH opt 7-8) heterotroph, while strain Su06 is a mildly acidophilic (pH opt 5), hydrogenotrophic autotroph, both with optimal growth temperatures of 90-92 °C. Mössbauer spectroscopy of the iron oxides before and after growth demonstrated that both organisms form nanophase (<12 nm) magnetite [Fe3 O4 ] from laboratory-synthesized ferrihydrite [Fe10 O14 (OH)2 ] with no detectable mineral intermediates. They produced up to 40 mm Fe(2+) in a growth-dependent manner, while all abiotic and biotic controls produced <3 mm Fe(2+) . Hyperthermophilic iron reducers may have a growth advantage over other hyperthermophiles in hydrothermal systems that are mildly acidic where mineral weathering at increased temperatures occurs.


Assuntos
Compostos Férricos/metabolismo , Óxido Ferroso-Férrico/metabolismo , Fontes Hidrotermais/microbiologia , Pyrodictiaceae/metabolismo , Fontes Hidrotermais/química , Ferro/química , Oxirredução , Oceano Pacífico , Fotomicrografia , Pyrodictiaceae/crescimento & desenvolvimento , Espectroscopia de Mossbauer , Sulfetos/metabolismo
4.
Extremophiles ; 17(3): 515-22, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23546841

RESUMO

The nucleotide cofactor specificity of the DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus (Hbu) was studied to investigate the evolutionary relationship of DNA ligases. The Hbu DNA ligase gene was expressed under control of the T7lac promoter of pTARG in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was purified using the IMPACT™-CN system (intein-mediated purification with an affinity chitin-binding tag) and cation-ion (Arg-tag) chromatography. The optimal temperature for Hbu DNA ligase activity was 75 °C, and the optimal pH was 8.0 in Tris-HCl. The activity was highly dependent on MgCl2 or MnCl2 with maximal activity above 5 mM MgCl2 and 2 mM MnCl2. Notably, Hbu DNA ligase can use ADP and GTP in addition to ATP. The broad nucleotide cofactor specificity of Hbu DNA ligase might exemplify an undifferentiated ancestral stage in the evolution of DNA ligases. This study provides new evidence for possible evolutionary relationships among DNA ligases.


Assuntos
Proteínas Arqueais/metabolismo , Coenzimas/metabolismo , DNA Ligases/metabolismo , Evolução Molecular , Pyrodictiaceae/enzimologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/genética , DNA Ligases/genética , Guanosina Trifosfato/metabolismo , Cinética , Filogenia
5.
J Am Chem Soc ; 134(48): 19731-8, 2012 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-23145461

RESUMO

The electronic structure and geometry of redox-active metal cofactors in proteins are tuned by the pattern of hydrogen bonding with the backbone peptide matrix. In this study we developed a method for selective amino acid labeling of a hyperthermophilic archaeal metalloprotein with engineered Escherichia coli auxotroph strains, and we applied this to resolve the hydrogen bond interactions with the reduced Rieske-type [2Fe-2S] cluster by two-dimensional pulsed electron spin resonance technique. Because deep electron spin-echo envelope modulation of two histidine (14)N(δ) ligands of the cluster decreased non-coordinating (15)N signal intensities via the cross-suppression effect, an inverse labeling strategy was employed in which (14)N amino acid-labeled archaeal Rieske-type ferredoxin samples were examined in an (15)N-protein background. This has directly identified Lys45 N(α) as providing the major pathway for the transfer of unpaired electron spin density from the reduced cluster by a "through-bond" mechanism. All other backbone peptide nitrogens interact more weakly with the reduced cluster. The extension of this approach will allow visualizing the three-dimensional landscape of preferred pathways for the transfer of unpaired spin density from a paramagnetic metal center onto the protein frame, and will discriminate specific interactions by a "through-bond" mechanism from interactions which are "through-space" in various metalloproteins.


Assuntos
Ferredoxinas/química , Ferro/química , Enxofre/química , Sítios de Ligação , Escherichia coli/genética , Ligação de Hidrogênio , Marcação por Isótopo , Modelos Moleculares , Oxirredução , Pyrodictiaceae/química , Especificidade por Substrato , Sulfolobus solfataricus/química
6.
Microbiology ; 156(Pt 1): 256-69, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19850614

RESUMO

Two new autotrophic carbon fixation cycles have been recently described in Crenarchaeota. The 3-hydroxypropionate/4-hydroxybutyrate cycle using acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the carboxylating enzyme has been identified for (micro)aerobic members of the Sulfolobales. The dicarboxylate/4-hydroxybutyrate cycle using oxygen-sensitive pyruvate synthase and phosphoenolpyruvate carboxylase as carboxylating enzymes has been found in members of the anaerobic Desulfurococcales and Thermoproteales. However, Sulfolobales include anaerobic and Desulfurococcales aerobic autotrophic representatives, raising the question of which of the two cycles they use. We studied the mechanisms of autotrophic CO(2) fixation in the strictly anaerobic Stygiolobus azoricus (Sulfolobales) and in the facultatively aerobic Pyrolobus fumarii (Desulfurococcales). The activities of all enzymes of the 3-hydroxypropionate/4-hydroxybutyrate cycle were found in the anaerobic S. azoricus. In contrast, the aerobic or denitrifying P. fumarii possesses all enzyme activities of the dicarboxylate/4-hydroxybutyrate cycle. We conclude that autotrophic Crenarchaeota use one of the two cycles, and that their distribution correlates with the 16S rRNA-based phylogeny of this group, rather than with the aerobic or anaerobic lifestyle.


Assuntos
Processos Autotróficos , Dióxido de Carbono/metabolismo , Pyrodictiaceae/enzimologia , Sulfolobaceae/enzimologia , Ácidos Dicarboxílicos/metabolismo , Hidroxibutiratos/metabolismo , Ácido Láctico/análogos & derivados , Ácido Láctico/metabolismo , Dados de Sequência Molecular , Filogenia , Pyrodictiaceae/genética , RNA Arqueal/genética , RNA Ribossômico 16S/genética , Sulfolobaceae/genética
7.
FEBS Lett ; 583(21): 3519-24, 2009 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-19822146

RESUMO

Polyamines are ubiquitously present in all organisms. In addition to the common polyamines, thermophilic archaea synthesize long-chain polyamines. In the present study polyamine synthases from Hyperthermus butylicus and Pyrobaculum aerophilum were cloned and their substrate specificity was analyzed. The polyamine synthase HbSpeE II from H. butylicus synthesized long-chain polyamines with high activity using the same mechanism that is used by a wide range of organisms to synthesize common polyamines, in which the aminopropyl residue derives from decarboxylated S-adenosylmethionine. This is the first polyamine synthase described that synthesizes a polyamine longer than a tetramine with high activity.


Assuntos
Ligases/metabolismo , Poliaminas/química , Poliaminas/metabolismo , Pyrobaculum/enzimologia , Pyrodictiaceae/enzimologia , Clonagem Molecular , Ligases/biossíntese , Ligases/genética , Filogenia , Poliaminas/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Especificidade por Substrato
8.
BMC Genomics ; 10: 145, 2009 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-19341479

RESUMO

BACKGROUND: Staphylothermus marinus is an anaerobic, sulfur-reducing peptide fermenter of the archaeal phylum Crenarchaeota. It is the third heterotrophic, obligate sulfur reducing crenarchaeote to be sequenced and provides an opportunity for comparative analysis of the three genomes. RESULTS: The 1.57 Mbp genome of the hyperthermophilic crenarchaeote Staphylothermus marinus has been completely sequenced. The main energy generating pathways likely involve 2-oxoacid:ferredoxin oxidoreductases and ADP-forming acetyl-CoA synthases. S. marinus possesses several enzymes not present in other crenarchaeotes including a sodium ion-translocating decarboxylase likely to be involved in amino acid degradation. S. marinus lacks sulfur-reducing enzymes present in the other two sulfur-reducing crenarchaeotes that have been sequenced -- Thermofilum pendens and Hyperthermus butylicus. Instead it has three operons similar to the mbh and mbx operons of Pyrococcus furiosus, which may play a role in sulfur reduction and/or hydrogen production. The two marine organisms, S. marinus and H. butylicus, possess more sodium-dependent transporters than T. pendens and use symporters for potassium uptake while T. pendens uses an ATP-dependent potassium transporter. T. pendens has adapted to a nutrient-rich environment while H. butylicus is adapted to a nutrient-poor environment, and S. marinus lies between these two extremes. CONCLUSION: The three heterotrophic sulfur-reducing crenarchaeotes have adapted to their habitats, terrestrial vs. marine, via their transporter content, and they have also adapted to environments with differing levels of nutrients. Despite the fact that they all use sulfur as an electron acceptor, they are likely to have different pathways for sulfur reduction.


Assuntos
Desulfurococcaceae/genética , Genoma Arqueal , Pyrodictiaceae/genética , Enxofre/metabolismo , Thermofilaceae/genética , Sequência de Aminoácidos , Carboxiliases/metabolismo , Desulfurococcaceae/classificação , Desulfurococcaceae/metabolismo , Transporte de Elétrons , Genômica , Metilmalonil-CoA Descarboxilase/metabolismo , Dados de Sequência Molecular , Filogenia , Pyrodictiaceae/metabolismo , Thermofilaceae/metabolismo , Transposases/genética
9.
Extremophiles ; 12(3): 383-9, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18286223

RESUMO

The archaeon Pyrolobus fumarii, one of the most extreme members of hyperthermophiles known thus far, is able to grow at temperatures up to 113 degrees C. Over a decade after the description of this organism our knowledge about the structures and strategies underlying its remarkable thermal resistance remains incipient. The accumulation of a restricted number of charged organic solutes is a common response to heat stress in hyperthermophilic organisms and accordingly their role in thermoprotection has been often postulated. In this work, the organic solute pool of P. fumarii was characterized using 1H, 13C, and 31P NMR. Di-myo-inositol phosphate was the major solute (0.21 micromol/mg protein), reinforcing the correlation between the occurrence of this solute and hyperthermophily; in addition, UDP-sugars (total concentration 0.11 micromol/mg protein) were present. The structures of the two major UDP-sugars were identified as UDP-alpha-GlcNAc3NAc and UDP-alpha-GlcNAc3NAc-(4<--1)-beta-GlcpNAc3NAc. Interestingly, the latter compound appears to be derived from the first one by addition of a 2,3-N-acetylglucoronic acid unit, suggesting that these UDP-sugars are intermediates of an N-linked glycosylation pathway. To our knowledge the UDP-disaccharide has not been reported elsewhere. The physiological roles of these organic solutes are discussed.


Assuntos
Temperatura Alta , Fosfatos de Inositol/metabolismo , Pyrodictiaceae/metabolismo , Açúcares de Uridina Difosfato/metabolismo , Adaptação Fisiológica , Dissacarídeos/metabolismo , Glicosilação , Espectroscopia de Ressonância Magnética
11.
Archaea ; 2(2): 127-35, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17350933

RESUMO

Hyperthermus butylicus, a hyperthermophilic neutrophile and anaerobe, is a member of the archaeal kingdom Crenarchaeota. Its genome consists of a single circular chromosome of 1,667,163 bp with a 53.7% G+C content. A total of 1672 genes were annotated, of which 1602 are protein-coding, and up to a third are specific to H. butylicus. In contrast to some other crenarchaeal genomes, a high level of GUG and UUG start codons are predicted. Two cdc6 genes are present, but neither could be linked unambiguously to an origin of replication. Many of the predicted metabolic gene products are associated with the fermentation of peptide mixtures including several peptidases with diverse specificities, and there are many encoded transporters. Most of the sulfur-reducing enzymes, hydrogenases and electron-transfer proteins were identified which are associated with energy production by reducing sulfur to H(2)S. Two large clusters of regularly interspaced repeats (CRISPRs) are present, one of which is associated with a crenarchaeal-type cas gene superoperon; none of the spacer sequences yielded good sequence matches with known archaeal chromosomal elements. The genome carries no detectable transposable or integrated elements, no inteins, and introns are exclusive to tRNA genes. This suggests that the genome structure is quite stable, possibly reflecting a constant, and relatively uncompetitive, natural environment.


Assuntos
Genoma Arqueal , Pyrodictiaceae/genética , Carbono/metabolismo , Reparo do DNA , Replicação do DNA , Fermentação , Genes Arqueais , Temperatura Alta , Dados de Sequência Molecular , Oxirredução , Peptídeos/metabolismo , Filogenia , Biossíntese de Proteínas , Pyrodictiaceae/classificação , Pyrodictiaceae/crescimento & desenvolvimento , Pyrodictiaceae/metabolismo , Análise de Sequência de DNA , Enxofre/metabolismo , Transcrição Genética
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