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1.
J Bacteriol ; 203(16): e0002521, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34096778

RESUMO

Serine kinase catalyzes the phosphorylation of free serine (Ser) to produce O-phosphoserine (Sep). An ADP-dependent Ser kinase in the hyperthermophilic archaeon Thermococcus kodakarensis (Tk-SerK) is involved in cysteine (Cys) biosynthesis and most likely Ser assimilation. An ATP-dependent Ser kinase in the mesophilic bacterium Staphylococcus aureus is involved in siderophore biosynthesis. Although proteins displaying various degrees of similarity with Tk-SerK are distributed in a wide range of organisms, it is unclear if they are actually Ser kinases. Here, we examined proteins from Desulfurococcales species in Crenarchaeota that display moderate similarity with Tk-SerK from Euryarchaeota (42 to 45% identical). Tk-serK homologs from Staphylothermus marinus (Smar_0555), Desulfurococcus amylolyticus (DKAM_0858), and Desulfurococcus mucosus (Desmu_0904) were expressed in Escherichia coli. All three partially purified recombinant proteins exhibited Ser kinase activity utilizing ATP rather than ADP as a phosphate donor. Purified Smar_0555 protein displayed activity for l-Ser but not other compounds, including d-Ser, l-threonine, and l-homoserine. The enzyme utilized ATP, UTP, GTP, CTP, and the inorganic polyphosphates triphosphate and tetraphosphate as phosphate donors. Kinetic analysis indicated that the Smar_0555 protein preferred nucleoside 5'-triphosphates over triphosphate as a phosphate donor. Transcript levels and Ser kinase activity in S. marinus cells grown with or without serine suggested that the Smar_0555 gene is constitutively expressed. The genes encoding Ser kinases examined here form an operon with genes most likely responsible for the conversion between Sep and 3-phosphoglycerate of central sugar metabolism, suggesting that the ATP-dependent Ser kinases from Desulfurococcales play a role in the assimilation of Ser. IMPORTANCE Homologs of the ADP-dependent Ser kinase from the archaeon Thermococcus kodakarensis (Tk-SerK) include representatives from all three domains of life. The results of this study show that even homologs from the archaeal order Desulfurococcales, which are the most structurally related to the ADP-dependent Ser kinases from the Thermococcales, are Ser kinases that utilize ATP, and in at least some cases inorganic polyphosphates, as the phosphate donor. The differences in properties between the Desulfurococcales and Thermococcales enzymes raise the possibility that Tk-SerK homologs constitute a group of kinases that phosphorylate free serine with a wide range of phosphate donors.


Assuntos
Proteínas Arqueais/metabolismo , Desulfurococcaceae/enzimologia , /metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Desulfurococcaceae/classificação , Desulfurococcaceae/genética , Temperatura Alta , Cinética , /genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
2.
Proc Natl Acad Sci U S A ; 118(25)2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34161262

RESUMO

The prokaryotic cell is traditionally seen as a "bag of enzymes," yet its organization is much more complex than in this simplified view. By now, various microcompartments encapsulating metabolic enzymes or pathways are known for Bacteria These microcompartments are usually small, encapsulating and concentrating only a few enzymes, thus protecting the cell from toxic intermediates or preventing unwanted side reactions. The hyperthermophilic, strictly anaerobic Crenarchaeon Ignicoccus hospitalis is an extraordinary organism possessing two membranes, an inner and an energized outer membrane. The outer membrane (termed here outer cytoplasmic membrane) harbors enzymes involved in proton gradient generation and ATP synthesis. These two membranes are separated by an intermembrane compartment, whose function is unknown. Major information processes like DNA replication, RNA synthesis, and protein biosynthesis are located inside the "cytoplasm" or central cytoplasmic compartment. Here, we show by immunogold labeling of ultrathin sections that enzymes involved in autotrophic CO2 assimilation are located in the intermembrane compartment that we name (now) a peripheric cytoplasmic compartment. This separation may protect DNA and RNA from reactive aldehydes arising in the I. hospitalis carbon metabolism. This compartmentalization of metabolic pathways and information processes is unprecedented in the prokaryotic world, representing a unique example of spatiofunctional compartmentalization in the second domain of life.


Assuntos
Compartimento Celular , Células Procarióticas/citologia , Células Procarióticas/metabolismo , Ciclo do Carbono , Dióxido de Carbono/metabolismo , DNA Arqueal/metabolismo , Desulfurococcaceae/citologia , Desulfurococcaceae/metabolismo , Desulfurococcaceae/ultraestrutura , Células Procarióticas/ultraestrutura , Frações Subcelulares/metabolismo
3.
Commun Biol ; 4(1): 132, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514861

RESUMO

The metallo-ß-lactamase fold is an ancient protein structure present in numerous enzyme families responsible for diverse biological processes. The crystal structure of the hyperthermostable crenarchaeal enzyme Igni18 from Ignicoccus hospitalis was solved at 2.3 Å and could resemble a possible first archetype of a multifunctional metallo-ß-lactamase. Ancestral enzymes at the evolutionary origin are believed to be promiscuous all-rounders. Consistently, Igni18´s activity can be cofactor-dependently directed from ß-lactamase to lactonase, lipase, phosphodiesterase, phosphotriesterase or phospholipase. Its core-domain is highly conserved within metallo-ß-lactamases from Bacteria, Archaea and Eukarya and gives insights into evolution and function of enzymes from this superfamily. Structural alignments with diverse metallo-ß-lactamase-fold-containing enzymes allowed the identification of Protein Variable Regions accounting for modulation of activity, specificity and oligomerization patterns. Docking of different substrates within the active sites revealed the basis for the crucial cofactor dependency of this enzyme superfamily.


Assuntos
Desulfurococcaceae/enzimologia , Evolução Molecular , beta-Lactamases/metabolismo , Cristalografia , Desulfurococcaceae/genética , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação Proteica , Dobramento de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , Temperatura , beta-Lactamases/química , beta-Lactamases/genética
4.
Arch Microbiol ; 203(4): 1299-1308, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33325001

RESUMO

Radiation of ionizing or non-ionizing nature has harmful effects on cellular components like DNA as radiation can compromise its proper integrity. To cope with damages caused by external stimuli including radiation, within living cells, several fast and efficient repair mechanisms have evolved. Previous studies addressing organismic radiation tolerance have shown that radiotolerance is a predominant property among extremophilic microorganisms including (hyper-) thermophilic archaea. The analysis of the ionizing radiation tolerance of the chemolithoautotrophic, obligate anaerobic, hyperthermophilic Crenarchaeon Ignicoccus hospitalis showed a D10-value of 4.7 kGy, fourfold exceeding the doses previously determined for other extremophilic archaea. The genome integrity of I. hospitalis after γ-ray exposure in relation to its survival was visualized by RAPD and qPCR. Furthermore, the discrimination between reproduction, and ongoing metabolic activity was possible for the first time indicating that a potential viable but non-culturable (VBNC) state may also account for I. hospitalis.


Assuntos
Replicação do DNA/efeitos da radiação , Desulfurococcaceae/efeitos da radiação , Desulfurococcaceae/genética , Desulfurococcaceae/crescimento & desenvolvimento , Desulfurococcaceae/metabolismo , Extremófilos , Genoma Arqueal/efeitos da radiação , Viabilidade Microbiana/efeitos da radiação , Doses de Radiação , Tolerância a Radiação , Radiação Ionizante
5.
J Struct Biol ; 211(3): 107572, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32652237

RESUMO

McrBC is a conserved modification-dependent restriction system that in Escherichia coli specifically targets foreign DNA containing methylated cytosines. Crystallographic data show that the N-terminal domain of Escherichia coli McrB binds substrates via a base flipping mechanism. This region is poorly conserved among the plethora of McrB homologs, suggesting that other species may use alternative binding strategies and/or recognize different targets. Here we present the crystal structure of the N-terminal domain from Stayphlothermus marinus McrB (Sm3-180) at 1.92 Å, which adopts a PUA-like EVE fold that is closely related to the YTH and ASCH RNA binding domains. Unlike most PUA-like domains, Sm3-180 binds DNA and can associate with different modified substrates. We find the canonical 'aromatic cage' binding pocket that confers specificity for methylated bases in other EVE/YTH domains is degenerate and occluded in Sm3-180, which may contribute to its promiscuity in target recognition. Further structural comparison between different PUA-like domains identifies motifs and conformational variations that correlate with the preference for binding either DNA or RNA. Together these data have important implications for PUA-like domain specificity and suggest a broader biological versatility for the McrBC family than previously described.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Desulfurococcaceae/química , Proteínas de Ligação a RNA/química , Proteínas Arqueais/genética , Sítios de Ligação , Cristalografia por Raios X , DNA Arqueal/química , DNA Arqueal/metabolismo , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Domínios Proteicos , Dobramento de Proteína , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo
6.
Nucleic Acids Res ; 48(12): 6906-6918, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32459340

RESUMO

The universal L-shaped tertiary structure of tRNAs is maintained with the help of nucleotide modifications within the D- and T-loops, and these modifications are most extensive within hyperthermophilic species. The obligate-commensal Nanoarchaeum equitans and its phylogenetically-distinct host Ignicoccus hospitalis grow physically coupled under identical hyperthermic conditions. We report here two fundamentally different routes by which these archaea modify the key conserved nucleotide U54 within their tRNA T-loops. In N. equitans, this nucleotide is methylated by the S-adenosylmethionine-dependent enzyme NEQ053 to form m5U54, and a recombinant version of this enzyme maintains specificity for U54 in Escherichia coli. In N. equitans, m5U54 is subsequently thiolated to form m5s2U54. In contrast, I. hospitalis isomerizes U54 to pseudouridine prior to methylating its N1-position and thiolating the O4-position of the nucleobase to form the previously uncharacterized nucleotide m1s4Ψ. The methyl and thiol groups in m1s4Ψ and m5s2U are presented within the T-loop in a spatially identical manner that stabilizes the 3'-endo-anti conformation of nucleotide-54, facilitating stacking onto adjacent nucleotides and reverse-Hoogsteen pairing with nucleotide m1A58. Thus, two distinct structurally-equivalent solutions have evolved independently and convergently to maintain the tertiary fold of tRNAs under extreme hyperthermic conditions.


Assuntos
Desulfurococcaceae/genética , Nanoarchaeota/genética , Conformação de Ácido Nucleico , RNA de Transferência/ultraestrutura , Archaea/genética , Archaea/ultraestrutura , Escherichia coli/genética , Metilação , Filogenia , RNA de Transferência/genética , tRNA Metiltransferases/genética , tRNA Metiltransferases/ultraestrutura
7.
Microsc Res Tech ; 83(6): 691-705, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32057162

RESUMO

For nearly 50 years immunogold labeling on ultrathin sections has been successfully used for protein localization in laboratories worldwide. In theory and in practice, this method has undergone continual improvement over time. In this study, we carefully analyzed circulating protocols for postembedding labeling to find out if they are still valid under modern laboratory conditions, and in addition, we tested unconventional protocols. For this, we investigated immunolabeling of Epon-embedded cells, immunolabeling of cells treated with osmium, and the binding behavior of differently sized gold particles. Here we show that (in contrast to widespread belief) immunolabeling of Epon-embedded cells and of cells treated with osmium tetroxide is actually working. Furthermore, we established a "speed protocol" for immunolabeling by reducing antibody incubation times. Finally, we present our results on three-dimensional immunogold labeling.


Assuntos
Compostos de Epóxi/química , Técnicas Histológicas , Imuno-Histoquímica/métodos , Microscopia Imunoeletrônica/métodos , Tetróxido de Ósmio/química , Anticorpos/química , Desulfurococcaceae/ultraestrutura , Microalgas/ultraestrutura , Microtomia/métodos
8.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 4): 307-311, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30950832

RESUMO

The hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN4/I possesses at least 35 putative genes encoding enzymes that belong to the α/ß-hydrolase superfamily. One of those genes, the metallo-hydrolase-encoding igni18, was cloned and heterologously expressed in Pichia pastoris. The enzyme produced was purified in its catalytically active form. The recombinant enzyme was successfully crystallized and the crystal diffracted to a resolution of 2.3 Å. The crystal belonged to space group R32, with unit-cell parameters a = b = 67.42, c = 253.77 Å, α = ß = 90.0, γ = 120.0°. It is suggested that it contains one monomer of Igni18 within the asymmetric unit.


Assuntos
Clonagem Molecular , Desulfurococcaceae/enzimologia , Expressão Gênica , Hidrolases/química , Hidrolases/isolamento & purificação , Metais/química , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X , Hidrolases/genética
9.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 2): 89-97, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30713159

RESUMO

The influenza neuraminidase (NA) is a homotetramer with head, stalk, transmembrane and cytoplasmic regions. The structure of the NA head with a stalk has never been determined. The NA head from an N9 subtype influenza A virus, A/tern/Australia/G70C/1975 (H1N9), was expressed with an artificial stalk derived from the tetrabrachion (TB) tetramerization domain from Staphylothermus marinus. The NA was successfully crystallized both with and without the TB stalk, and the structures were determined to 2.6 and 2.3 Šresolution, respectively. Comparisons of the two NAs with the native N9 NA structure from egg-grown virus showed that the artificial TB stalk maintained the native NA head structure, supporting previous biological observations.


Assuntos
Proteínas de Bactérias/química , Virus da Influenza A Subtipo H5N1/enzimologia , Neuraminidase/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Cristalização , Cristalografia por Raios X , Desulfurococcaceae/metabolismo , Humanos , Influenza Humana/virologia , Modelos Moleculares , Neuraminidase/metabolismo , Conformação Proteica , Domínios Proteicos
10.
J Bacteriol ; 201(7)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30642991

RESUMO

In this study, the ATP synthase of Ignicoccus hospitalis was purified, characterized, and structurally compared to the respective enzymes of the other Ignicoccus species, to shed light on energy conservation in this unique group of archaea. The crenarchaeal genus Ignicoccus comprises three described species, i.e., I. hospitalis and Ignicoccus islandicus from hot marine sediments near Iceland and Ignicoccus pacificus from a hydrothermal vent system in the Pacific Ocean. This genus is unique among all archaea due to the unusual cell envelope, consisting of two membranes that enclose a large intermembrane compartment (IMC). I. hospitalis is the best studied member of this genus, mainly because it is the only known host for the potentially parasitic archaeon Nanoarchaeum equitans I. hospitalis grows chemolithoautotrophically, and its sole energy-yielding reaction is the reduction of elemental sulfur with molecular hydrogen, forming large amounts of hydrogen sulfide. This reaction generates an electrochemical gradient, which is used by the ATP synthase, located in the outer cellular membrane, to generate ATP inside the IMC. The genome of I. hospitalis encodes nine subunits of an A-type ATP synthase, which we could identify in the purified complex. Although the maximal in vitro activity of the I. hospitalis enzyme was measured around pH 6, the optimal stability of the A1AO complex seemed to be at pH 9. Interestingly, the soluble A1 subcomplexes of the different Ignicoccus species exhibited significant differences in their apparent molecular masses in native electrophoresis, although their behaviors in gel filtration and chromatography-mass spectrometry were very similar.IMPORTANCE The Crenarchaeota represent one of the major phyla within the Archaea domain. This study describes the successful purification of a crenarchaeal ATP synthase. To date, all information about A-type ATP synthases is from euryarchaeal enzymes. The fact that it has not been possible to purify this enzyme complex from a member of the Crenarchaeota until now points to significant differences in stability, possibly caused by structural alterations. Furthermore, the study subject I. hospitalis has a particular importance among crenarchaeotes, since it is the only known host of N. equitans The energy metabolism in this system is still poorly understood, and our results can help elucidate the unique relationship between these two microbes.


Assuntos
Complexos de ATP Sintetase/isolamento & purificação , Complexos de ATP Sintetase/metabolismo , Desulfurococcaceae/enzimologia , Complexos de ATP Sintetase/química , Desulfurococcaceae/isolamento & purificação , Estabilidade Enzimática , Sedimentos Geológicos , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo
11.
Syst Appl Microbiol ; 42(1): 94-106, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30195930

RESUMO

Three thermophilic Nanoarchaeota-Crenarchaeota symbiotic systems have been described. We obtained another stable anaerobic enrichment culture at 80°C, pH 6.0 from a New Zealand hot spring. The nanoarchaeote (Ncl-1) and its host (NZ3T) were isolated in co-culture and their genomes assembled. The small (∼200nm) flagellated cocci were often attached to larger cocci. Based on 16S rRNA gene similarity (88.4%) and average amino acid identity (52%), Ncl-1 is closely related to Candidatus Nanopusillus acidilobi. Their genomes both encode for archaeal flagella and partial glycolysis and gluconeogenesis pathways, but lack ATP synthase genes. Like Nanoarchaeum equitans, Ncl-1 has a CRISPR-Cas system. Ncl-1 also relies on its crenarchaeotal host for most of its biosynthetic needs. The host NZ3T was isolated and grows on proteinaceous substrates but not on sugars, alcohols, or fatty acids. NZ3T requires thiosulfate and grows best at 82°C, pH 6.0. NZ3T is most closely related to the Desulfurococcaceae, Ignisphaera aggregans (∼92% 16S rRNA gene sequence similarity, 45% AAI). Based on phylogenetic, physiological and genomic data, Ncl-1 and NZ3T represent novel genera in the Nanoarchaeota and the Desulfurococcaceae, respectively, with the proposed names Candidatus Nanoclepta minutus and Zestosphaera tikiterensis gen. nov., sp. nov., type strain NZ3T (=DSMZ 107634T=OCM 1213T).


Assuntos
Desulfurococcaceae/classificação , Fontes Termais/microbiologia , Filogenia , Simbiose , Desulfurococcaceae/genética , Genoma Arqueal , Nova Zelândia , RNA Ribossômico 16S/genética
12.
Biochemistry ; 57(36): 5271-5281, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-29939726

RESUMO

Superoxide reductases (SORs) are enzymes that detoxify the superoxide anion through its reduction to hydrogen peroxide and exist in both prokaryotes and eukaryotes. The substrate is transformed at an iron catalytic center, pentacoordinated in the ferrous state by four histidines and one cysteine. SORs have a highly conserved motif, (E)(K)HxP-, in which the glutamate is associated with a redox-driven structural change, completing the octahedral coordination of the iron in the ferric state, whereas the lysine may be responsible for stabilization and donation of a proton to catalytic intermediates. We aimed to understand at the structural level the role of these two residues, by determining the X-ray structures of the SORs from the hyperthermophilic archaea Ignicoccus hospitalis and Nanoarchaeum equitans that lack the quasi-conserved lysine and glutamate, respectively, but have catalytic rate constants similar to those of the canonical enzymes, as we previously demonstrated. Furthermore, we have determined the crystal structure of the E23A mutant of I. hospitalis SOR, which mimics several enzymes that lack both residues. The structures revealed distinct structural arrangements of the catalytic center that simulate several catalytic cycle intermediates, namely, the reduced and the oxidized forms, and the glutamate-free and deprotonated ferric forms. Moreover, the structure of the I. hospitalis SOR provides evidence for the presence of an alternative lysine close to the iron center in the reduced state that may be a functional substitute for the "canonical" lysine.


Assuntos
Proteínas Arqueais/química , Desulfurococcaceae/enzimologia , Nanoarchaeota/enzimologia , Oxirredutases/química , Superóxidos/química , Sequência de Aminoácidos , Proteínas Arqueais/metabolismo , Catálise , Crioprotetores , Cristalização , Cristalografia por Raios X , Oxirredução , Oxirredutases/metabolismo , Conformação Proteica , Homologia de Sequência , Superóxidos/metabolismo
13.
Folia Microbiol (Praha) ; 63(6): 713-723, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29797222

RESUMO

Desulfurococcus amylolyticus DSM 16532 is an anaerobic and hyperthermophilic crenarchaeon known to grow on a variety of different carbon sources, including monosaccharides and polysaccharides. Furthermore, D. amylolyticus is one of the few archaea that are known to be able to grow on cellulose. Here, we present the metabolic reconstruction of D. amylolyticus' central carbon metabolism. Based on the published genome, the metabolic reconstruction was completed by integrating complementary information available from the KEGG, BRENDA, UniProt, NCBI, and PFAM databases, as well as from available literature. The genomic analysis of D. amylolyticus revealed genes for both the classical and the archaeal version of the Embden-Meyerhof pathway. The metabolic reconstruction highlighted gaps in carbon dioxide-fixation pathways. No complete carbon dioxide-fixation pathway such as the reductive citrate cycle or the dicarboxylate-4-hydroxybutyrate cycle could be identified. However, the metabolic reconstruction indicated that D. amylolyticus harbors all genes necessary for glucose metabolization. Closed batch experimental verification of glucose utilization by D. amylolyticus was performed in chemically defined medium. The findings from in silico analyses and from growth experiments are discussed with respect to physiological features of hyperthermophilic organisms.


Assuntos
Desulfurococcaceae/metabolismo , Glucose/metabolismo , Biomassa , Dióxido de Carbono/metabolismo , Desulfurococcaceae/genética , Fermentação , Genoma Bacteriano , Gluconeogênese , Glicólise , Redes e Vias Metabólicas
14.
Enzyme Microb Technol ; 114: 15-21, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29685348

RESUMO

Desulfurococcus amylolyticus is an anaerobic and hyperthermophilic crenarchaeon that can use various carbohydrates as energy sources. We found a gene encoding a glycoside hydrolase family 57 amylolytic enzymes (DApu) in a putative carbohydrate utilization gene cluster in the genome of D. amylolyticus. This gene has an open reading frame of 1,878 bp and consists of 626 amino acids with a molecular mass of 71 kDa. Recombinant DApu (rDApu) completely hydrolyzed pullulan to maltotriose by attacking α-1,6-glycosidic linkages, and was able to produce glucose and maltose from soluble starch and amylopectin. Although rDApu showed no activity toward α-cyclodextrin (CD) and ß-CD, maltooctaose (G8) was detected from reaction with γ-CD. The highest activity of rDApu was measured at pH 5.0 and 95 °C. The half-life of rDApu was 12.7 h at 95 °C and 27 min at 98 °C. Interestingly, rDApu was able to transfer a maltose unit to 6-O-α-maltosyl-ß-CD via transglycosylation. Structure analysis using MALDI-TOF/TOF MS and nuclear magnetic resonance revealed that the new transglycosylated products were 61, 64-di-O-maltosyl-ß-CD and 61, 63, 65-tri-O-maltosyl-ß-CD.


Assuntos
Proteínas Arqueais/química , Ciclodextrinas/metabolismo , Desulfurococcaceae/enzimologia , Glicosídeo Hidrolases/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Clonagem Molecular , Ciclodextrinas/química , Desulfurococcaceae/química , Desulfurococcaceae/genética , Estabilidade Enzimática , Glucanos/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Glicosilação , Maltose/análogos & derivados , Maltose/química , Maltose/metabolismo , Peso Molecular , Fases de Leitura Aberta , Especificidade por Substrato , Trissacarídeos/metabolismo
15.
RNA Biol ; 15(4-5): 614-622, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28901837

RESUMO

tRNAHis guanylyltransferase (Thg1) has unique reverse (3'-5') polymerase activity occurring in all three domains of life. Most eukaryotic Thg1 homologs are essential genes involved in tRNAHis maturation. These enzymes normally catalyze a single 5' guanylation of tRNAHis lacking the essential G-1 identity element required for aminoacylation. Recent studies suggest that archaeal type Thg1, which includes most archaeal and bacterial Thg1 enzymes is phylogenetically distant from eukaryotic Thg1. Thg1 is evolutionarily related to canonical 5'-3' forward polymerases but catalyzes reverse 3'-5'polymerization. Similar to its forward polymerase counterparts, Thg1 encodes the conserved catalytic palm domain and fingers domain. Here we investigate the minimal requirements for reverse polymerization. We show that the naturally occurring minimal Thg1 enzyme from Ignicoccus hospitalis (IhThg1), which lacks parts of the conserved fingers domain, is catalytically active. And adds all four natural nucleotides to RNA substrates, we further show that the entire fingers domain of Methanosarcina acetivorans Thg1 and Pyrobaculum aerophilum Thg1 (PaThg1) is dispensable for enzymatic activity. In addition, we identified residues in yeast Thg1 that play a part in preventing extended polymerization. Mutation of these residues with alanine resulted in extended reverse polymerization. PaThg1 was found to catalyze extended, template dependent tRNA repair, adding up to 13 nucleotides to a truncated tRNAHis substrate. Sequencing results suggest that PaThg1 fully restored the near correct sequence of the D- and acceptor stem, but also produced incompletely and incorrectly repaired tRNA products. This research forms the basis for future engineering efforts towards a high fidelity, template dependent reverse polymerase.


Assuntos
Desulfurococcaceae/enzimologia , Methanosarcina/enzimologia , Nucleotidiltransferases/metabolismo , Pyrobaculum/enzimologia , RNA de Transferência de Histidina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Sequência Conservada , Desulfurococcaceae/genética , Expressão Gênica , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Methanosarcina/genética , Modelos Moleculares , Mutação , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Polimerização , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas/métodos , Pyrobaculum/genética , RNA de Transferência de Histidina/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
16.
Int J Biol Macromol ; 107(Pt A): 413-417, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28887188

RESUMO

The most extremely thermostable maltogenic amylase (SMMA) from archaeon Staphylothermus marinus has many potential applications in food processing. To ensure safety of microbial origin, a recombinant plasmid containing the enzymic gene and a constitutive promoter AmyR2 was constructed, and then transformed into a GRAS microorganism Bacillus subtilis. The purified SMMA from the liquid cultures of Bacillus has a specific activity of 66.96U/mg, two times more than that from Escherichia coli. SMMA was further employed to catalyze the genistion glycosylation using γ-CD as both glucosyl donors and solubilizer. Glycosylated genistins with one to four additional α-glucosyls and a molar percentage of 69.87% in genistin reaction mixture were identified and quantified by HPLC-UV-MS. The glycosylated genistins at 0.2-1.2mM showed an enhanced DPPH free radical scavenging capacity. To our knowledge, this is the first report on the Bacillus expression of archaeal maltogenic amylase.


Assuntos
Desulfurococcaceae/enzimologia , Manipulação de Alimentos , Glicosídeo Hidrolases/genética , Bacillus subtilis/genética , Estabilidade Enzimática/genética , Escherichia coli/genética , Glicosídeo Hidrolases/biossíntese , Glicosídeo Hidrolases/química , Glicosilação , Isoflavonas/química , Transformação Genética
17.
J Comput Aided Mol Des ; 31(12): 1063-1072, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29177929

RESUMO

I-DmoI, from the hyperthermophilic archaeon Desulfurococcus mobilis, belongs to the LAGLIDADG homing endonuclease protein family. Its members are highly specific enzymes capable of recognizing long DNA target sequences, thus providing potential tools for genome manipulation. Working towards this particular application, many efforts have been made to generate mesophilic variants of I-DmoI that function at lower temperatures than the wild-type. Here, we report a structural and computational analysis of two I-DmoI mesophilic mutants. Despite very limited structural variations between the crystal structures of these variants and the wild-type, a different dynamical behaviour near the cleavage sites is observed. In particular, both the dynamics of the water molecules and the protein perturbation effect on the cleavage site correlate well with the changes observed in the experimental enzymatic activity.


Assuntos
Desoxirribonucleases de Sítio Específico do Tipo I , Modelos Moleculares , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Simulação por Computador , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo I/química , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Desulfurococcaceae/enzimologia , Mutação , Ligação Proteica , Conformação Proteica
18.
Proteins ; 85(12): 2209-2216, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28905430

RESUMO

Elemental sulfur exists primarily as an S80 ring and serves as terminal electron acceptor for a variety of sulfur-fermenting bacteria. Hyperthermophilic archaea from black smoker vents are an exciting research tool to advance our knowledge of sulfur respiration under extreme conditions. Here, we use a hybrid method approach to demonstrate that the proteinaceous cavities of the S-layer nanotube of the hyperthermophilic archaeon Staphylothermus marinus act as a storage reservoir for cyclo-octasulfur S8. Fully atomistic molecular dynamics (MD) simulations were performed and the method of multiconfigurational thermodynamic integration was employed to compute the absolute free energy for transferring a ring of elemental sulfur S8 from an aqueous bath into the largest hydrophobic cavity of a fragment of archaeal tetrabrachion. Comparisons with earlier MD studies of the free energy of hydration as a function of water occupancy in the same cavity of archaeal tetrabrachion show that the sulfur ring is energetically favored over water.


Assuntos
Desulfurococcaceae/química , Nanotubos/química , Enxofre/química , Água/química , Motivos de Aminoácidos , Proteínas Arqueais , Cristalografia por Raios X , Desulfurococcaceae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Interações Hidrofóbicas e Hidrofílicas , Fontes Hidrotermais , Simulação de Dinâmica Molecular , Nanotubos/ultraestrutura , Plasmídeos/química , Plasmídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Enxofre/metabolismo , Termodinâmica , Água/metabolismo
19.
Biochim Biophys Acta Gen Subj ; 1861(9): 2218-2227, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28591626

RESUMO

BACKGROUND: Studies of interspecies interactions are inherently difficult due to the complex mechanisms which enable these relationships. A model system for studying interspecies interactions is the marine hyperthermophiles Ignicoccus hospitalis and Nanoarchaeum equitans. Recent independently-conducted 'omics' analyses have generated insights into the molecular factors modulating this association. However, significant questions remain about the nature of the interactions between these archaea. METHODS: We jointly analyzed multiple levels of omics datasets obtained from published, independent transcriptomics, proteomics, and metabolomics analyses. DAVID identified functionally-related groups enriched when I. hospitalis is grown alone or in co-culture with N. equitans. Enriched molecular pathways were subsequently visualized using interaction maps generated using STRING. RESULTS: Key findings of our multi-level omics analysis indicated that I. hospitalis provides precursors to N. equitans for energy metabolism. Analysis indicated an overall reduction in diversity of metabolic precursors in the I. hospitalis-N. equitans co-culture, which has been connected to the differential use of ribosomal subunits and was previously unnoticed. We also identified differences in precursors linked to amino acid metabolism, NADH metabolism, and carbon fixation, providing new insights into the metabolic adaptions of I. hospitalis enabling the growth of N. equitans. CONCLUSIONS: This multi-omics analysis builds upon previously identified cellular patterns while offering new insights into mechanisms that enable the I. hospitalis-N. equitans association. GENERAL SIGNIFICANCE: Our study applies statistical and visualization techniques to a mixed-source omics dataset to yield a more global insight into a complex system, that was not readily discernable from separate omics studies.


Assuntos
Desulfurococcaceae/metabolismo , Nanoarchaeota/metabolismo , Aminoácidos/metabolismo , Metabolismo Energético , Metabolômica , NAD/metabolismo , Proteômica , Proteínas Ribossômicas/metabolismo , Transcriptoma
20.
Nucleic Acids Res ; 45(4): 2007-2015, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28204608

RESUMO

In all free-living organisms a late-stage checkpoint in the biogenesis of the small ribosomal subunit involves rRNA modification by an RsmA/Dim1 methyltransferase. The hyperthermophilic archaeon Nanoarchaeum equitans, whose existence is confined to the surface of a second archaeon, Ignicoccus hospitalis, lacks an RsmA/Dim1 homolog. We demonstrate here that the I. hospitalis host possesses the homolog Igni_1059, which dimethylates the N6-positions of two invariant adenosines within helix 45 of 16S rRNA in a manner identical to other RsmA/Dim1 enzymes. However, Igni_1059 is not transferred from I. hospitalis to N. equitans across their fused cell membrane structures and the corresponding nucleotides in N. equitans 16S rRNA remain unmethylated. An alternative mechanism for ribosomal subunit maturation in N. equitans is suggested by sRNA interactions that span the redundant RsmA/Dim1 site to introduce 2΄-O-ribose methylations within helices 44 and 45 of the rRNA.


Assuntos
Adenosina/metabolismo , Metiltransferases/metabolismo , Nanoarchaeota/genética , RNA Ribossômico 16S/metabolismo , Desulfurococcaceae/enzimologia , Desulfurococcaceae/genética , Escherichia coli/genética , Metilação , Metiltransferases/genética , Nanoarchaeota/enzimologia , RNA Ribossômico 16S/química , Subunidades Ribossômicas Menores de Arqueas/metabolismo
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