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1.
J Biol Chem ; 295(43): 14618-14629, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-32817340

RESUMO

Motility in archaea is facilitated by a unique structure termed the archaellum. N-Glycosylation of the major structural proteins (archaellins) is important for their subsequent incorporation into the archaellum filament. The identity of some of these N-glycans has been determined, but archaea exhibit extensive variation in their glycans, meaning that further investigations can shed light not only on the specific details of archaellin structure and function, but also on archaeal glycobiology in general. Here we describe the structural characterization of the N-linked glycan modifications on the archaellins and S-layer protein of Methanothermococcus thermolithotrophicus, a methanogen that grows optimally at 65 °C. SDS-PAGE and MS analysis revealed that the sheared archaella are composed principally of two of the four predicted archaellins, FlaB1 and FlaB3, which are modified with a branched, heptameric glycan at all N-linked sequons except for the site closest to the N termini of both proteins. NMR analysis of the purified glycan determined the structure to be α-d-glycero-d-manno-Hep3OMe6OMe-(1-3)-[α-GalNAcA3OMe-(1-2)-]-ß-Man-(1-4)-[ß-GalA3OMe4OAc6CMe-(1-4)-α-GalA-(1-2)-]-α-GalAN-(1-3)-ß-GalNAc-Asn. A detailed investigation by hydrophilic interaction liquid ion chromatography-MS discovered the presence of several, less abundant glycan variants, related to but distinct from the main heptameric glycan. In addition, we confirmed that the S-layer protein is modified with the same heptameric glycan, suggesting a common N-glycosylation pathway. The M. thermolithotrophicus archaellin N-linked glycan is larger and more complex than those previously identified on the archaellins of related mesophilic methanogens, Methanococcus voltae and Methanococcus maripaludis This could indicate that the nature of the glycan modification may have a role to play in maintaining stability at elevated temperatures.


Assuntos
Proteínas Arqueais/química , Methanococcaceae/química , Polissacarídeos/análise , Sequência de Aminoácidos , Sequência de Carboidratos , Glicosilação , Espectrometria de Massas , Ressonância Magnética Nuclear Biomolecular
2.
Int J Syst Evol Microbiol ; 69(4): 1225-1230, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30843780

RESUMO

A novel hydrogenotrophic methanogen, strain HHBT, was isolated from a deep-sea hydrothermal vent chimney sample collected from Beebe Vent Field at the Mid-Cayman Spreading Center, Caribbean Sea. The cells were non-motile regular to irregular cocci possessing several flagella. The novel isolate grew at 60-80 °C, pH 5.0-7.4 and with 1-4 % of NaCl (w/v). The isolate utilized H2/CO2 as the only substrates for growth and methane production. The results of phylogenetic analyses of both 16S rRNA and mcrA gene sequences and comparative genome analysis indicated that HHBT represented a member of the order Methanococcales, and was closely related to the members of the genera Methanothermococcus and Methanotorris. The most closely related species were Methanothermococcus okinawensis IH1T and Methanotorris igneus Kol 5T in comparison of 16S rRNA gene sequences (each with 93 % identity), and Methanotorris formicicus Mc-S-70T in the case of deduced amino acid sequence similarity of mcrA genes (92 % similarity). The ANI and AAI values between HHBT and the members of the genera Methanothermococcus and Methanotorris were 69-72 % and 66-70 %, respectively. Although many of the morphological and physiological characteristics were quite similar between HHBT and the species of the genera Methanothermococcus and Methanotorris, they were distinguishable by the differences in susceptibility to antibiotics, formate utilization, growth temperature and NaCl ranges. On the basis of these phenotypic, phylogenetic and genomic properties, we propose that strain HHBT represents a novel species, of a novel genus, Methanofervidicoccus abyssi gen. nov., sp. nov. The type strain is HHBT (=JCM 32161T=DSM 105918T).


Assuntos
Fontes Hidrotermais/microbiologia , Methanococcaceae/classificação , Filogenia , Região do Caribe , DNA Arqueal/genética , Genes Arqueais , Methanococcaceae/isolamento & purificação , RNA Ribossômico 16S/genética , Água do Mar , Análise de Sequência de DNA , Temperatura
3.
FEBS Lett ; 593(5): 543-553, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30702149

RESUMO

3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyses the last step in mevalonate biosynthesis. HMGR is the target of statin inhibitors that regulate cholesterol concentration in human blood. Here, we report the properties and structures of HMGR from an archaeon Methanothermococcus thermolithotrophicus (mHMGR). The structures of the apoenzyme and the NADPH complex are highly similar to those of human HMGR. A notable exception is C-terminal helix (Lα10-11) that is straight in both mHMGR structures. This helix is kinked and closes the active site in the human enzyme ternary complex, pointing to a substrate-induced structural rearrangement of C-terminal in class-I HMGRs during the catalytic cycle.


Assuntos
Hidroximetilglutaril-CoA Redutases/química , Methanococcaceae/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Hidroximetilglutaril-CoA Redutases/metabolismo , Cinética , NADP/metabolismo , Conformação Proteica , Especificidade por Substrato
4.
Nat Commun ; 9(1): 748, 2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29487311

RESUMO

The detection of silica-rich dust particles, as an indication for ongoing hydrothermal activity, and the presence of water and organic molecules in the plume of Enceladus, have made Saturn's icy moon a hot spot in the search for potential extraterrestrial life. Methanogenic archaea are among the organisms that could potentially thrive under the predicted conditions on Enceladus, considering that both molecular hydrogen (H2) and methane (CH4) have been detected in the plume. Here we show that a methanogenic archaeon, Methanothermococcus okinawensis, can produce CH4 under physicochemical conditions extrapolated for Enceladus. Up to 72% carbon dioxide to CH4 conversion is reached at 50 bar in the presence of potential inhibitors. Furthermore, kinetic and thermodynamic computations of low-temperature serpentinization indicate that there may be sufficient H2 gas production to serve as a substrate for CH4 production on Enceladus. We conclude that some of the CH4 detected in the plume of Enceladus might, in principle, be produced by methanogens.


Assuntos
Exobiologia , Meio Ambiente Extraterreno/química , Metano/biossíntese , Saturno , Atmosfera/química , Pressão Atmosférica , Hidrogênio/metabolismo , Methanobacteriaceae/crescimento & desenvolvimento , Methanobacteriaceae/metabolismo , Methanococcaceae/crescimento & desenvolvimento , Methanococcaceae/metabolismo , Mathanococcus/crescimento & desenvolvimento , Mathanococcus/metabolismo , Modelos Biológicos , Astronave
5.
Chembiochem ; 18(23): 2295-2297, 2017 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-28986941

RESUMO

Elaborate arrays of iron-sulfur clusters link active sites via a flavin that bifurcates electrons through two energetically independent paths. The structure of the heterodisulfide reductase provides insight into how methanogens conserve energy through coupling hydrogen oxidation to coordinated exergonic heterodisulfide and endergonic ferredoxin reduction in an overall thermodynamically favorable reaction.


Assuntos
Metano/metabolismo , Dióxido de Carbono/química , Transporte de Elétrons , Elétrons , Flavina-Adenina Dinucleotídeo/química , Hidrogênio/química , Hidrogenase/química , Hidrogenase/metabolismo , Metano/química , Methanococcaceae/enzimologia , Oxirredução , Oxirredutases/metabolismo
6.
Science ; 357(6352): 699-703, 2017 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-28818947

RESUMO

In methanogenic archaea, the carbon dioxide (CO2) fixation and methane-forming steps are linked through the heterodisulfide reductase (HdrABC)-[NiFe]-hydrogenase (MvhAGD) complex that uses flavin-based electron bifurcation to reduce ferredoxin and the heterodisulfide of coenzymes M and B. Here, we present the structure of the native heterododecameric HdrABC-MvhAGD complex at 2.15-angstrom resolution. HdrB contains two noncubane [4Fe-4S] clusters composed of fused [3Fe-4S]-[2Fe-2S] units sharing 1 iron (Fe) and 1 sulfur (S), which were coordinated at the CCG motifs. Soaking experiments showed that the heterodisulfide is clamped between the two noncubane [4Fe-4S] clusters and homolytically cleaved, forming coenzyme M and B bound to each iron. Coenzymes are consecutively released upon one-by-one electron transfer. The HdrABC-MvhAGD atomic model serves as a structural template for numerous HdrABC homologs involved in diverse microbial metabolic pathways.


Assuntos
Proteínas Arqueais/química , Proteínas Ferro-Enxofre/química , Methanococcaceae/enzimologia , Oxirredutases/química , Motivos de Aminoácidos , Proteínas Arqueais/ultraestrutura , Coenzimas/química , Coenzimas/ultraestrutura , Cristalografia por Raios X , Transporte de Elétrons , Ferredoxinas/química , Ferro/química , Proteínas Ferro-Enxofre/ultraestrutura , Redes e Vias Metabólicas , Oxirredução , Oxirredutases/ultraestrutura , Domínios Proteicos , Enxofre/química
7.
Science ; 354(6308): 114-117, 2016 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-27846502

RESUMO

Biological methane formation starts with a challenging adenosine triphosphate (ATP)-independent carbon dioxide (CO2) fixation process. We explored this enzymatic process by solving the x-ray crystal structure of formyl-methanofuran dehydrogenase, determined here as Fwd(ABCDFG)2 and Fwd(ABCDFG)4 complexes, from Methanothermobacter wolfeii The latter 800-kilodalton apparatus consists of four peripheral catalytic sections and an electron-supplying core with 46 electronically coupled [4Fe-4S] clusters. Catalysis is separately performed by subunits FwdBD (FwdB and FwdD), which are related to tungsten-containing formate dehydrogenase, and subunit FwdA, a binuclear metal center carrying amidohydrolase. CO2 is first reduced to formate in FwdBD, which then diffuses through a 43-angstrom-long tunnel to FwdA, where it condenses with methanofuran to formyl-methanofuran. The arrangement of [4Fe-4S] clusters functions as an electron relay but potentially also couples the four tungstopterin active sites over 206 angstroms.


Assuntos
Aldeído Oxirredutases/química , Proteínas Arqueais/química , Dióxido de Carbono/química , Proteínas Ferro-Enxofre/química , Metano/síntese química , Methanococcaceae/enzimologia , Amidoidrolases/química , Biocatálise , Cristalografia por Raios X , Estradiol/análogos & derivados , Oxirredução , Conformação Proteica em Folha beta , Tungstênio/química
8.
Appl Biochem Biotechnol ; 176(4): 1012-28, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25894951

RESUMO

Anaerobic incubations using crude oil and brine from a North Sea reservoir were conducted to gain increased understanding of indigenous microbial community development, metabolite production, and the effects on the oil-brine system after addition of a complex carbon source, molasses, with or without nitrate to boost microbial growth. Growth of the indigenous microbes was stimulated by addition of molasses. Pyrosequencing showed that specifically Anaerobaculum, Petrotoga, and Methanothermococcus were enriched. Addition of nitrate favored the growth of Petrotoga over Anaerobaculum. The microbial growth caused changes in the crude oil-brine system: formation of oil emulsions, and reduction of interfacial tension (IFT). Reduction in IFT was associated with microbes being present at the oil-brine interphase. These findings suggest that stimulation of indigenous microbial growth by addition of molasses has potential as microbial enhanced oil recovery (MEOR) strategy in North Sea oil reservoirs.


Assuntos
Methanococcaceae/metabolismo , Campos de Petróleo e Gás/microbiologia , Petróleo/provisão & distribuição , Águas Salinas/química , Thermotoga maritima/metabolismo , Dinamarca , Methanococcaceae/efeitos dos fármacos , Methanococcaceae/crescimento & desenvolvimento , Consórcios Microbianos/efeitos dos fármacos , Consórcios Microbianos/fisiologia , Melaço/análise , Nitratos/farmacologia , Mar do Norte , Indústria de Petróleo e Gás/métodos , Tensão Superficial , Tensoativos/farmacologia , Thermotoga maritima/efeitos dos fármacos , Thermotoga maritima/crescimento & desenvolvimento
9.
FEBS Lett ; 587(18): 3083-8, 2013 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-23954289

RESUMO

SecYEG functions as a membrane channel for protein export. SecY constitutes the protein-conducting pore, which is enwrapped by SecE in a V-shaped manner. In its minimal form SecE consists of a single transmembrane segment that is connected to a surface-exposed amphipathic α-helix via a flexible hinge. These two domains are the major sites of interaction between SecE and SecY. Specific cleavage of SecE at the hinge region, which destroys the interaction between the two SecE domains, reduced translocation. When SecE and SecY were disulfide bonded at the two sites of interaction, protein translocation was not affected. This suggests that the SecY and SecE interactions are static, while the hinge region provides flexibility to allow the SecY pore to open.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Motivos de Aminoácidos , Proteínas Arqueais/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Dissulfetos/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Methanococcaceae/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Transporte Proteico , Canais de Translocação SEC , Proteínas SecA
10.
Biochemistry ; 52(17): 2949-54, 2013 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-23560717

RESUMO

UreG proteins are small GTP binding (G) proteins that catalyze the hydrolysis of GTP necessary for the maturation of urease, a virulence factor in bacterial pathogenesis. UreG proteins are the first documented cases of intrinsically disordered enzymes. The comprehension of the dynamics of folding-unfolding events occurring in this protein could shed light on the enzymatic mechanism of UreG. Here, we used the recently developed replica exchange with solute tempering (REST2) computational methodology to explore the conformational space of UreG from Helicobacter pylori (HpUreG) and to identify its structural fluctuations. The same simulation and analysis protocol has been applied to HypB from Methanocaldococcus jannaschii (MjHypB), which is closely related to UreG in both sequence and function, even though it is not intrinsically disordered. A comparison of the two systems reveals that both HpUreG and MjHypB feature a substantial rigidity of the protein regions involved in catalysis, justifying its residual catalytic activity. On the other hand, HpUreG tends to unfold more than MjHypB in portions involved in protein-protein interactions with metallochaperones necessary for the formation of multiprotein complexes known to be involved in urease activation.


Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Methanococcaceae/química , Modelos Moleculares , Proteínas de Ligação a Fosfato , Conformação Proteica
11.
Artigo em Inglês | MEDLINE | ID: mdl-23295494

RESUMO

MJ0927 is a member of the Nif3 family and is widely distributed across living organisms. Although several crystal structures of Nif3 proteins have been reported, structural information on archaeal Nif3 is still limited. To understand the structural differences between bacterial and archaeal Nif3 proteins, MJ0927 from Methanocaldococcus jannaschii was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.47 Šand belonged to the orthorhombic space group C222, with unit-cell parameters a = 81.21, b = 172.94, c = 147.42 Å. Determination of this structure may provide insights into the function of MJ0927.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/isolamento & purificação , Methanococcaceae/química , Proteínas Arqueais/genética , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Conformação Proteica
12.
Proc Natl Acad Sci U S A ; 109(52): 21325-9, 2012 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-23236183

RESUMO

Unusual tRNA genes, found in some algae, have their mature terminal 3' portion in front of their 5' portion in the genome. The transcripts from such genes must be cleaved by a pre-tRNA endonuclease to form a functional tRNA. We present a mechanism for the generation of "corrected" tRNAs from such a "permuted" pre-tRNA configuration. We used two avatar (av) or model pre-tRNAs and two splicing endonucleases with distinct mechanisms of recognition of the pre-tRNA. The splicing results are compatible with an evolutionary route in which permuted genes result from a duplication event followed by DNA rearrangement. The model pre-tRNAs permit description of the features that a transcript, derived from a rearranged duplicated gene, must have to give rise to functional tRNA. The two tRNA endonucleases are a eukaryal enzyme that normally acts in a mature domain-dependent mode and an archaeal enzyme that acts in a mature domain-independent mode. Both av pre-tRNAs are able to fold into two conformations: 1 and 2. We find that only conformation 2 can yield a corrected functional tRNA. This result is consistent with contemporary algae representing snapshots of different evolutionary stages, with duplicated genes preceding recombinatorial events generating a permutated gene. In a scenario elucidated by the use of the av pre-tRNAs, algal permuted tRNA genes could have further lost one of two mature domains, eliminating steric problems for the algal tRNA endonuclease, which remains a typical eukaryal enzyme capable of correcting the permuted transcript to a functional tRNA.


Assuntos
Endorribonucleases/metabolismo , Genes/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Sequência de Bases , Methanococcaceae/enzimologia , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Precursores de RNA/química , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/química , Schizosaccharomyces/enzimologia
13.
Biochemistry ; 51(12): 2378-89, 2012 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-22401293

RESUMO

Hydrogenotrophic methanogens possessing the hydrogen-dependent dehydrogenase Hmd also encode paralogs of this protein whose function is poorly understood. Here we present biochemical evidence that the two inactive Hmd paralogs of Methanocaldococcus jannaschii, HmdII and HmdIII, form binary and ternary complexes with several components of the protein translation apparatus. HmdII and HmdIII, but not the active dehydrogenase Hmd, bind with micromolar binding affinities to a number of tRNAs and form ternary complexes with tRNA(Pro) and prolyl-tRNA synthetase (ProRS). Fluorescence spectroscopy experiments also suggest that binding of HmdII and ProRS involves distinct binding determinants on the tRNA. These biochemical data suggest the possibility of a regulatory link between energy production and protein translation pathways that may allow a rapid cellular response to altered environmental conditions.


Assuntos
Proteínas Arqueais/biossíntese , Hidrogênio/metabolismo , Methanococcaceae/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Termodinâmica
14.
FEBS Lett ; 586(1): 60-3, 2012 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-22166682

RESUMO

In methanogenic archaea, Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep-tRNA(Cys) to Cys-tRNA(Cys). The mechanism of tRNA-dependent cysteine formation remains unclear due to the lack of functional studies. In this work, we mutated 19 conserved residues in Methanocaldococcus jannaschii SepCysS, and employed an in vivo system to determine the activity of the resulting variants. Our results show that three active-site cysteines (Cys39, Cys42 and Cys247) are essential for SepCysS activity. In addition, combined with structural modeling, our mutational and functional analyses also reveal multiple residues that are important for the binding of PLP, Sep and tRNA. Our work thus represents the first systematic functional analysis of conserved residues in archaeal SepCysSs, providing insights into the catalytic and substrate binding mechanisms of this poorly characterized enzyme.


Assuntos
Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Methanococcaceae/enzimologia , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/química , Sítios de Ligação , Domínio Catalítico , Sequência Conservada , Cisteína/química , Cisteína/metabolismo , Análise Mutacional de DNA , Modelos Moleculares , Conformação Proteica , Fosfato de Piridoxal/metabolismo , Aminoacil-RNA de Transferência/metabolismo
15.
Biomol NMR Assign ; 6(2): 193-6, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22203461

RESUMO

Sequence specific resonance assignments have been obtained for (1)H, (13)C and (15)N nuclei of the 21 kDa (188 residues long) glutamine amido transferase subunit of guanosine monophosphate synthetase from Methanocaldococcus jannaschii. From an analysis of (1)H and (13)C(α), (13)C(ß) secondary chemical shifts, (3) JH(N)H(α) scalar coupling constants and sequential, short and medium range (1)H-(1)H NOEs, it was deduced that the glutamine amido transferase subunit has eleven strands and five helices as the major secondary structural elements in its tertiary structure.


Assuntos
Aciltransferases/química , Guanosina Monofosfato/metabolismo , Ligases/química , Methanococcaceae/enzimologia , Ressonância Magnética Nuclear Biomolecular , Subunidades Proteicas/química , Prótons , Isótopos de Carbono , Isótopos de Nitrogênio , Estrutura Secundária de Proteína
16.
Arch Microbiol ; 194(2): 141-5, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22002406

RESUMO

The protein derived from the Methanocaldococcus jannaschii MJ0458 gene is annotated as a δ-1-pyrroline 5-carboxylate synthetase and is predicted to be related to aspartokinase and uridylate kinase. Analysis of the predicted protein sequence indicated that it is a unique kinase with few similarities to either uridylate or adenylate kinase. Here, we report that the MJ0458 gene product is a second type of archaeal adenylate kinase, AdkB. This enzyme is different from the established archaeal-specific adenylate kinase in both sequence and predicted tertiary structure.


Assuntos
Adenilato Quinase/metabolismo , Methanococcaceae/enzimologia , Núcleosídeo-Fosfato Quinase/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Adenilato Quinase/química , Archaea/enzimologia , Methanococcaceae/classificação , Núcleosídeo-Fosfato Quinase/química , Fosforilação , Filogenia , Especificidade por Substrato
17.
Biochemistry ; 50(23): 5301-13, 2011 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-21542645

RESUMO

In archaea and bacteria, the late steps in adenosylcobalamin (AdoCbl) biosynthesis are collectively known as the nucleotide loop assembly (NLA) pathway. In the archaeal and bacterial NLA pathways, two different guanylyltransferases catalyze the activation of the corrinoid. Structural and functional studies of the bifunctional bacterial guanylyltransferase that catalyze both ATP-dependent corrinoid phosphorylation and GTP-dependent guanylylation are available, but similar studies of the monofunctional archaeal enzyme that catalyzes only GTP-dependent guanylylation are not. Herein, the three-dimensional crystal structure of the guanylyltransferase (CobY) enzyme from the archaeon Methanocaldococcus jannaschii (MjCobY) in complex with GTP is reported. The model identifies the location of the active site. An extensive mutational analysis was performed, and the functionality of the variant proteins was assessed in vivo and in vitro. Substitutions of residues Gly8, Gly153, or Asn177 resulted in ≥94% loss of catalytic activity; thus, variant proteins failed to support AdoCbl synthesis in vivo. Results from isothermal titration calorimetry experiments showed that MjCobY(G153D) had 10-fold higher affinity for GTP than MjCobY(WT) but failed to bind the corrinoid substrate. Results from Western blot analyses suggested that the above-mentioned substitutions render the protein unstable and prone to degradation; possible explanations for the observed instability of the variants are discussed within the framework of the three-dimensional crystal structure of MjCobY(G153D) in complex with GTP. The fold of MjCobY is strikingly similar to that of the N-terminal domain of Mycobacterium tuberculosis GlmU (MtbGlmU), a bifunctional acetyltransferase/uridyltransferase that catalyzes the formation of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc).


Assuntos
Proteínas Arqueais/química , Guanosina Trifosfato/metabolismo , Methanococcaceae/enzimologia , Nucleotidiltransferases/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Cobamidas/química , Cobamidas/metabolismo , Dimerização , Guanosina Trifosfato/química , Modelos Moleculares , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Conformação Proteica , Uridina Difosfato N-Acetilglicosamina/metabolismo
18.
Methods Enzymol ; 494: 111-8, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21402212

RESUMO

Hydrogen (H(2)) is a primary electron donor for methanogenesis and its availability can have profound effects on gene expression and the physiology of energy conservation. The rigorous evaluation of the effects of hydrogen conditions requires the comparison of cultures that are grown under hydrogen limitation and hydrogen excess. The growth of methanogens under defined hydrogen conditions is complicated by the dynamics of hydrogen dissolution and its utilization by the cells. In batch culture, gassing and agitation conditions must be carefully calibrated, and even then variations in growth rate and cell density are hard to avoid. Using chemostats, continuous cultures can be achieved whose nutritional states are known, while growth rate and cell density are invariant. Cultures whose growth is limited by hydrogen can be compared to cultures whose growth is limited by some other nutrient and are therefore under hydrogen excess.


Assuntos
Archaea/crescimento & desenvolvimento , Archaea/metabolismo , Hidrogênio/metabolismo , Metano/metabolismo , Methanobacteriaceae/crescimento & desenvolvimento , Methanobacteriaceae/metabolismo , Methanococcaceae/crescimento & desenvolvimento , Methanococcaceae/metabolismo
19.
Artigo em Inglês | MEDLINE | ID: mdl-21206036

RESUMO

Type 2 isopentenyl diphosphate isomerase (IDI-2) is a flavoprotein. Recently, flavin has been proposed to play a role as a general acid-base catalyst with no redox role during the enzyme reaction. To clarify the detailed enzyme reaction mechanism of IDI-2 and the unusual role of flavin, structural analysis of IDI-2 from Methanocaldococcus jannaschii (MjIDI) was performed. Recombinant MjIDI was crystallized at 293 K using calcium acetate as a precipitant. The diffraction of the crystal extended to 2.08 Šresolution at 100 K. The crystal belonged to the tetragonal space group I422, with unit-cell parameters a=126.46, c=120.03 Å. The presence of one monomer per asymmetric unit gives a crystal volume per protein weight (VM) of 3.0 Å3 Da(-1) and a solvent constant of 59.0% by volume.


Assuntos
Proteínas de Bactérias/química , Isomerases de Ligação Dupla Carbono-Carbono/química , Methanococcaceae/enzimologia , Proteínas de Bactérias/genética , Isomerases de Ligação Dupla Carbono-Carbono/genética , Cristalização , Cristalografia por Raios X , Hemiterpenos , Dados de Sequência Molecular
20.
Artigo em Inglês | MEDLINE | ID: mdl-21206044

RESUMO

Nitrogenases are protein complexes that are only found in Azotobacter and are required for biological nitrogen fixation. They are made up of a nitrogenase, which is a NifD2/NifK2 heterotetramer, and a nitrogenase reductase, which is a homodimer of NifH. Many homologues of nitrogenase have been found in various non-nitrogen-fixing prokaryotes; in particular, they are found in all known methanogens. This indicates that these homologues may play a role in methane production. Here, the cloning of NifH2, a homologue of the NifH nitrogenase component, from Methanocaldococcus jannaschii (MjNifH2) and its expression in Escherichia coli with a polyhistidine tag, purification and crystallization are described. MjNifH2 crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 2.85 Å. The crystals belonged to space group P2, with unit-cell parameters a=64.01, b=94.38, c=98.08 Å, α=γ=90, ß=98.85°.


Assuntos
Proteínas de Bactérias/química , Methanococcaceae/enzimologia , Nitrogenase/química , Proteínas de Bactérias/genética , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Dados de Sequência Molecular , Nitrogenase/genética , Conformação Proteica
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