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51.
Martin Vaupel Julia A. Vorholt R. K. Thauer 《Extremophiles : life under extreme conditions》1998,2(1):15-22
N
5,N
10-Methenyltetrahydromethanopterin cyclohydrolase (Mch) is an enzyme involved in methanogenesis from CO2 and H2 which represents the energy metabolism of Methanopyrus kandleri, a methanogenic Archaeon growing at a temperature optimum of 98°C. The gene mch from M. kandleri was cloned, sequenced, and expressed in Escherichia coli. The overproduced enzyme could be purified in yields above 90% in one step by chromatography on phenyl Sepharose in 80% ammonium
sulfate. From 3.5 g cells (250 mg protein), approximately 18 mg cyclohydrolase was obtained. The purified enzyme showed essentially
the same catalytic properties as the enzyme purified from M. kandleri cells. The primary structure and properties of the cyclohydrolase are compared with those of the enzyme from Methanococcus jannaschii (growth temperature optimum 85°C), from Methanobacterium thermoautotrophicum (65°C), and from Methanosarcina barkeri (37°C). Of the four enzymes, that from M. kandleri has the lowest isoelectric point (3.8) and the lowest hydrophobicity of amino acid composition. Besides, it has the highest
relative content of glutamate, leucine, and valine and the lowest relative content of isoleucine, serine, and lysine. Some
of these properties are unusual for enzymes from hyperthermophilic organisms. They may reflect the observation that the cyclohydrolase
from M. kandleri is not only adapted to hyperthermophilic conditions but also to the high intracellular concentrations of lyotrophic salts
prevailing in this organism.
Received: July 14, 1997 / Accepted: August 28, 1997 相似文献
52.
Gentisate 1,2-dioxygenase from the extreme halophile Haloferax sp. D1227 (Hf. D1227) was purified using a three-step procedure. The enzyme was found to be a homotetramer of 42 000 ± 1000 Da subunits,
with a native molecular weight of 174 000 ± 6000 Da. The optimal salt concentration, temperature, and pH for enzyme activity
were 2 M KCl or NaCl, 45°C, and pH 7.2, respectively. The gene encoding Hf. D1227 gentisate 1,2-dioxygenase was cloned, sequenced, and expressed in Haloferax volcanii. The deduced amino acid sequence exhibited a 9.2% excess acidic over basic amino acids typical of halophilic enzymes. Four
novel histidine clusters and a possible extradiol dioxygenase fingerprint region were identified.
Received: November 19, 1997 / Accepted: May 12, 1998 相似文献
53.
54.
Methanomicrococcus blatticola is an obligately anaerobic methanogen that derives the energy for growth exclusively from the reduction of methylated compounds to methane with molecular hydrogen as energy source. Competition for methanol (concentration below 10 microM) and H(2) (concentration below 500 Pa), as well as oxidative stress due to the presence of oxygen are likely to occur in the peripheral region of the cockroach hindgut, the species' normal habitat. We investigated the ecophysiological properties of M. blatticola to explain how it can successfully compete for its methanogenic substrates. The organism showed affinities for methanol (K(m)=5 microM; threshold<1 microM) and hydrogen (K(m)=200 Pa; threshold <0.7 Pa) that are superior to other methylotrophic methanogens (Methanosphaera stadtmanae, Methanosarcina barkeri) investigated here. Thermodynamic considerations indicated that 'methanol respiration', i.e. the use of methanol as the terminal electron acceptor, represents an attractive mode of energy generation, especially at low hydrogen concentrations. Methanomicrococcus blatticola exploits the opportunities by specific growth rates (>0.2 h(-1)) and specific growth yields (up to 7 g of dry cells per mole of methane formed) that are particularly high within the realm of mesophilic methanogens. Upon oxygen exposure, part of the metabolic activity may be diverted into oxygen removal, thus establishing appropriate anaerobic conditions for survival and growth. 相似文献
55.
Permafrost environments in the Arctic are characterized by extreme environmental conditions that demand a specific resistance from microorganisms to enable them to survive. In order to understand the carbon dynamics in the climate-sensitive Arctic permafrost environments, the activity and diversity of methanogenic communities were studied in three different permafrost soils of the Siberian Laptev Sea coast. The effect of temperature and the availability of methanogenic substrates on CH4 production was analysed. In addition, the diversity of methanogens was analysed by PCR with specific methanogenic primers and by denaturing gradient gel electrophoresis (DGGE) followed by sequencing of DGGE bands reamplified from the gel. Our results demonstrated methanogenesis with a distinct vertical profile in each investigated permafrost soil. The soils on Samoylov Island showed at least two optima of CH4 production activity, which indicated a shift in the methanogenic community from mesophilic to psychrotolerant methanogens with increasing soil depth. Furthermore, it was shown that CH4 production in permafrost soils is substrate-limited, although these soils are characterized by the accumulation of organic matter. Sequence analyses revealed a distinct diversity of methanogenic archaea affiliated to Methanomicrobiaceae, Methanosarcinaceae and Methanosaetaceae. However, a relationship between the activity and diversity of methanogens in permafrost soils could not be shown. 相似文献
56.
Le Breton M Henneke G Norais C Flament D Myllykallio H Querellou J Raffin JP 《Journal of molecular biology》2007,374(5):1172-1185
We report on the characterization of the DNA primase complex of the hyperthermophilic archaeon Pyrococcus abyssi (Pab). The Pab DNA primase complex is composed of the proteins Pabp41 and Pabp46, which show sequence similarities to the p49 and p58 subunits, respectively, of the eukaryotic polymerase α–primase complex. Both subunits were expressed, purified, and characterized. The Pabp41 subunit alone had no RNA synthesis activity but could synthesize long (up to 3 kb) DNA strands. Addition of the Pabp46 subunit increased the rate of DNA synthesis but decreased the length of the DNA fragments synthesized and conferred RNA synthesis capability. Moreover, in our experimental conditions, Pab DNA primase had comparable affinities for ribonucleotides and deoxyribonucleotides, and its activity was dependent on the presence of Mg2+ and Mn2+. Interestingly, Pab DNA primase also displayed DNA polymerase, gap-filling, and strand-displacement activities. Genetic analyses undertaken in Haloferax volcanii suggested that the eukaryotic-type heterodimeric primase is essential for survival in archaeal cells. Our results are in favor of a multifunctional archaeal primase involved in priming and repair. 相似文献
57.
Iron-uptake is well studied in a plethora of pro- and eukaryotic organisms with the exception of Archaea, which thrive mainly
in extreme environments. In this study, the mechanism of iron transport in the extremely halophilic Euryarchaeon Halobacterium salinarum strain JW 5 was analyzed. Under low-iron growth conditions no siderophores were detectable in culture supernatants. However,
various xenosiderophores support growth of H. salinarum. In [55Fe]–[14C] double-label experiments, H. salinarum displays uptake of iron but not of the chelator citrate. Uptake of iron was inhibited by cyanide and at higher concentrations
by Ga. Furthermore, a KM for iron uptake in cells of 2.36 μM and a Vmax of approximately 67 pmol Fe/min/mg protein was determined. [55Fe]-uptake kinetics were measured in the absence and presence of Ga. Uptake of iron was inhibited merely at very high Ga concentrations.
The results indicate an energy dependent iron uptake process in H. salinarum and suggest reduction of the metal at the membrane level. 相似文献
58.
Edgcomb VP Molyneaux SJ Böer S Wirsen CO Saito M Atkins MS Lloyd K Teske A 《Extremophiles : life under extreme conditions》2007,11(2):329-342
Growth and survival of hyperthermophilic archaea in their extreme hydrothermal vent and subsurface environments are controlled
by chemical and physical key parameters. This study examined the effects of elevated sulfide concentrations, temperature,
and acidic pH on growth and survival of two hydrothermal vent archaea (Pyrococcus strain GB-D and Thermococcus fumicolans) under high temperature and pressure regimes. These two strains are members of the Thermococcales, a family of hyperthermophilic,
heterotrophic, sulfur-reducing archaea that occur in high densities at vent sites. As actively growing cells, these two strains
tolerated regimes of pH, pressure, and temperature that were in most cases not tolerated under severe substrate limitation.
A moderate pH of 5.5–7 extends their survival and growth range over a wider range of sulfide concentrations, temperature and
pressure, relative to lower pH conditions. T. fumicolans and Pyrococcus strain GB-D grew under very high pressures that exceeded in-situ pressures typical of hydrothermal vent depths, and included
deep subsurface pressures. However, under the same conditions, but in the absence of carbon substrates and electron acceptors,
survival was generally lower, and decreased rapidly when low pH stress was combined with high pressure and high temperature.
Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. 相似文献
59.
Kuroita T Kanno T Kawai A Kawakami B Oka M Endo Y Tozawa Y 《Extremophiles : life under extreme conditions》2007,11(1):85-94
We have isolated and characterized a gene for a putative protein-disulfide oxidoreductase (phdsb) in the archaeon Pyrococcus horikoshii. The open reading frame of phdsb encodes a protein of 170 amino acids with an NH2-terminal extension similar to the bacterial signal peptides. The putative mature region of PhDsb includes a sequence motif,
Cys-Pro-His-Cys (CPHC), that is conserved in members of the bacterial DsbA family, but otherwise the archaeal and bacterial
sequences do not show substantial similarity. A recombinant protein corresponding to the predicted mature form of PhDsb behaved
as a monomer and manifested oxidoreductase activities in vitro similar to those of DsbA of Escherichia coli. The catalytic activity of PhDsb was thermostable and was shown by mutation analysis to depend on the NH2-terminal cysteine residue of the CPHC motif. Thus, in spite of their low overall sequence similarities, DsbA-like proteins
of archaea and bacteria appear to be highly similar in terms of function. 相似文献
60.
Kuratani M Kasai T Akasaka R Higashijima K Terada T Kigawa T Shinkai A Bessho Y Yokoyama S 《Proteins》2011,79(7):2065-2075
The hypermodified nucleoside N6‐threonylcarbamoyladenosine resides at position 37 of tRNA molecules bearing U at position 36 and maintains translational fidelity in the three kingdoms of life. The N6‐threonylcarbamoyl moiety is composed of L ‐threonine and bicarbonate, and its synthesis was genetically shown to require YrdC/Sua5. YrdC/Sua5 binds to tRNA and ATP. In this study, we analyzed the L ‐threonine‐binding mode of Sua5 from the archaeon Sulfolobus tokodaii. Isothermal titration calorimetry measurements revealed that S. tokodaii Sua5 binds L ‐threonine more strongly than L ‐serine and glycine. The Kd values of Sua5 for L ‐threonine and L ‐serine are 9.3 μM and 2.6 mM, respectively. We determined the crystal structure of S. tokodaii Sua5, complexed with AMPPNP and L ‐threonine, at 1.8 Å resolution. The L ‐threonine is bound next to AMPPNP in the same pocket of the N‐terminal domain. Thr118 and two water molecules form hydrogen bonds with AMPPNP in a unique manner for adenine‐specific recognition. The carboxyl group and the side‐chain hydroxyl and methyl groups of L ‐threonine are buried deep in the pocket, whereas the amino group faces AMPPNP. The L ‐threonine is located in a suitable position to react together with ATP for the synthesis of N6‐threonylcarbamoyladenosine. Proteins 2011. © 2011 Wiley‐Liss, Inc. 相似文献