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R Hensel

Publications and source records attributed to R Hensel.

At least 37 records · Page 2Linked to original sources
PubMed1996

The 3-phosphoglycerate kinase of the hyperthermophilic archaeum Pyrococcus woesei produced in Escherichia coli: loss of heat resistance due to internal translation initiation and its restoration by site-directed mutagenesis.

Heterologous expression of the gene coding for 3-phosphoglycerate kinase (PGK) of the hyperthermophilic archaeum, Pyrococcus woesei (Pw), in Escherichia coli (Ec) yielded only low recovery of recombinant PGK (re-PGK) in heat-precipitated crude extracts. Moreover, we noticed contamination with a 28-kDa protein, from which PGK could hardly be separated, even under stringent conditions after tagging the re-PGK with a His6-tag. The preparations contaminated with the 28-kDa protein showed an unexpectedly low thermal stability. Under the same conditions (85 degrees C, 30 min), however, the enzyme from the original organism was completely resistant to heat inactivation. As shown by size-exclusion chromatography, re-PGK forms tight associations with the 28-kDa protein, which was found to represent a C-terminal fragment of PGK and to arise as a product of internal translation initiation within the pgk gene. Mutations changing the internal ribosome-binding site effectively suppressed the production of the 28-kDa protein and restored the thermal stability of the Pw re-PGK.

Archaea↗
PubMed1996

Tetrameric triosephosphate isomerase from hyperthermophilic Archaea.

Triosephosphate isomerase (TIM) of the hyperthermophilic Archaea Pyrococcus woesei and Methanothermus fervidus have been purified to homogeneity. The enzymes from the two hyperthermophiles represent homo-tetramers of 100 kDa, contrary to all known bacterial and eukaryotic TIMs, which are dimers of 48-60 kDa. Molecular size determination of the TIM from the mesophilic methanogen Methanobacterium bryantii yielded the usual molecular mass of only 57 kDa, indicating that the tetrameric aggregation state does not represent an archaeal feature but rather correlates with thermoadaptation. A similar preference for higher protein aggregates in hyperthermophilic Archaea has previously been demonstrated for 3-phosphoglycerate kinases. The gene of the P. woesei TIM was cloned and sequenced. The archaeal TIM proved to be homologous to its bacterial and eukaryotic pendants. Most strikingly, the deduced protein sequence comprises only 224 residues and thus represents the shortest TIM sequence known as yet. Taking the three-dimensional structure of the eucaryal TIM as a basis, from the shortenings of the chain considerable rearrangements at the bottom of the alpha/beta barrel and at its functionally inactive flank are expected, which are interpreted in terms of the formation of new subunit contacts.

Amino Acid Sequence↗
PubMed1996

Disposition of a novel recombinant antithrombotic agent, RG 12986, in cynomolgus monkeys.

RG 12986, a novel antagonist of platelet aggregation, is a recombinant peptide based on the sequence in von Willebrand factor, which contains the GP1b binding site. Disposition of the peptide in cynomolgus monkeys was determined using nonlabeled and 35S-labeled product. After iv administration, the peptide underwent a triphasic decay in the plasma. The first phase of elimination, after distribution, had a t1/2 (approximately 20 min) similar to that observed for inhibition of platelet aggregation (approximately 25 min). The correlation between the logarithm of the plasma peptide concentration and activity was r = 0.9989. The effective duration of pharmacological activity was approximately 2 hr. After this period, a slower terminal phase of plasma elimination was observed (t1/2 approximately 2 hr). Plasma clearance (7-15 ml/min/kg) and volume of distribution at steady-state (0.4-0.9 L/kg) estimates appeared to have a slight dose dependency, but the scope of the investigation did not allow this to be verified. There was a linear correlation between dose and AUC (r2 = 0.9998), but for each 4-fold increase in dose there was a greater than 4-fold increase in AUC. Immediately after iv administration, significant fragmentation of the peptide was observed with polyacrylamide gel electrophoresis analysis of the plasma. This initial rate of metabolism was subsequently slowed to t1/2 estimates of 2 hr, followed by a very long terminal half-life of plasma radioactivity of 12 days. It is likely that this terminal half-life represents metabolic recycling of 35S. Elimination of the label was primarily via the kidneys.

Animals↗
PubMed1995

Dimeric 3-phosphoglycerate kinases from hyperthermophilic Archaea. Cloning, sequencing and expression of the 3-phosphoglycerate kinase gene of Pyrococcus woesei in Escherichia coli and characterization of the protein. Structural and functional comparison with the 3-phosphoglycerate kinase of Methanothermus fervidus.

The gene coding for the 3-phosphoglycerate kinase (EC 2.7.2.3) of Pyrococcus woesei was cloned and sequenced. The gene sequence comprises 1230 bp coding for a polypeptide with the theoretical M(r) of 46,195. The deduced protein sequence exhibits a high similarity (46.1% and 46.6% identity) to the other known archaeal 3-phosphoglycerate kinases of Methanobacterium bryantii and Methanothermus fervidus [Fabry, S., Heppner, P., Dietmaier, W. & Hensel, R. (1990) Gene 91, 19-25]. By comparing the 3-phosphoglycerate kinase sequences of the mesophilic and the two thermophilic Archaea, trends in thermoadaptation were confirmed that could be deduced from comparisons of glyceraldehyde-3-phosphate dehydrogenase sequences from the same organisms [Zwickl, P., Fabry, S., Bogedain, C., Haas, A. & Hensel, R. (1990) J. Bacteriol. 172, 4329-4338]. With increasing temperature the average hydrophobicity and the portion of aromatic residues increases, whereas the chain flexibility as well as the content in chemically labile residues (Asn, Cys) decreases. To study the phenotypic properties of the 3-phosphoglycerate kinases from thermophilic Archaea in more detail, the 3-phosphoglycerate kinase genes from P. woesei and M. fervidus were expressed in Escherichia coli. Comparisons of kinetic and molecular properties of the enzymes from the original organisms and from E. coli indicate that the proteins expressed in the mesophilic host are folded correctly. Besides their higher thermostability according to their origin from hyperthermophilic organisms, both enzymes differ from their bacterial and eucaryotic homologues mainly in two respects. (a) The 3-phosphoglycerate kinases from P. woesei and M. fervidus are homomeric dimers in their native state contrary to all other known 3-phosphoglycerate kinases, which are monomers including the enzyme from the mesophilic Archaeum M. bryantii. (b) Monovalent cations are essential for the activity of both archaeal enzymes with K+ being significantly more efficient than Na+. For the P. woesei enzyme, non-cooperative K+ binding with an apparent Kd (K+) of 88 mM could be determined by kinetic analysis, whereas for the M. fervidus 3-phosphoglycerate kinase the K+ binding is rather complex: from the fitting of the saturation data, non-cooperative binding sites with low selectivity for K+ and Na+ (apparent Kd = 270 mM) and at least three cooperative and highly specific K+ binding sites/subunit are deduced. At the optimum growth temperature of P. woesei (100 degrees C) and M. fervidus (83 degrees C), the 3-phosphoglycerate kinases show half-lives of inactivation of only 28 min and 44 min, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Cloning, sequencing and expression of the gene encoding 2-phosphoglycerate kinase from Methanothermus fervidus.

The gene encoding 2-phosphoglycerate kinase (2PGK), which catalyses the first step in the biosynthesis of cyclic 2,3-diphosphoglycerate in methanogens, was cloned and sequenced from the hyperthermophilic Methanothermus fervidus. The 2pgk gene codes for 304 amino acids, corresponding to a relative molecular mass of 35040. The 2pgk mRNA was estimated to be 1600 nucleotides in size. Putative transcription signals and the ribosome-binding site of 2pgk are discussed. Production of 2PGK from M. fervidus in Es-cherichia coli reveals the same apparent molecular weights for the native enzyme and its denatured subunit as those shown by the 2PGK purified from M. fervidus. Also the kinetic parameters of 2PKG produced in E. coli correspond well with those from the enzyme isolated from the natural host M. fervidus.

2,3-Diphosphoglycerate↗
PubMed1992

Di-myo-inositol-1,1'-phosphate: a new inositol phosphate isolated from Pyrococcus woesei.

A new inositol derivative could be isolated from the Archaeum Pyrococcus woesei and identified as di-myo-inositol-1,1'-phosphate by 1H, 31P NMR spectroscopy, mass spectrometry and thin layer chromatography. In P. woesei, this inositol phosphate represents the dominant counterion of K+ which ranges from 500 to 600 mM. The role of the potassium salt of di-myo-inositol-1,1'-phosphate as thermostabilizer is discussed.

Archaea↗
PubMed1991

Analysis and nucleotide sequence of the genes encoding the surface-layer glycoproteins of the hyperthermophilic methanogens Methanothermus fervidus and Methanothermus sociabilis.

The genes (slgA) encoding the surface-layer glycoproteins of the hyperthermophilic methanogens Methano-thermus pervidus and Methanothermus sociabilis were cloned and sequenced. The nucleotide sequences of these genes differ at only nine positions, resulting in three amino acid differences. In both organisms, the transcription start site was localized by primer extension analyses. The DNA sequence at this site conforms to the promotor box B motif for promotors of archaea. 24 nucleotides upstream of the transcription start is an A + T-rich region, which closely resembles the consensus box A motif of promoters of methanogens. Ribosome binding sites are exactly complementary to the 3' end of the 16S rRNA of these methanogens. Both slgA genes encode for a precursor of the mature surface-layer protein containing 593 amino acid residues with a putative N-terminal signal sequence of 22 amino acid residues. The deduced protein sequences contain 20 sequon structures representing possible carbohydrate-binding sites. In comparison with other surface-layer proteins, these obtained from the two hyperthermophilic methanogens contain unusually high amounts of isoleucine, asparagine and cysteine residues. Predicted secondary structures have a high content of beta-sheet structure (44%) and only 7% alpha-helix structures.

Amino Acid Sequence↗
PubMed1990

Engineering thermostability in archaebacterial glyceraldehyde-3-phosphate dehydrogenase. Hints for the important role of interdomain contacts in stabilizing protein conformation.

Construction of hybrid enzymes between the glyceraldehyde-3-phosphate dehydrogenases from the mesophilic Methanobacterium bryantii and the thermophilic Methanothermus fervidus by recombinant DNA techniques revealed that a short C-terminal fragment of the Mt. fervidus enzyme contributes largely to its thermostability. This C-terminal region appears to be homologous to the alpha 3-helix of eubacterial and eukaryotic glyceraldehyde-3-phosphate dehydrogenases which is involved in the contacts between the two domains of the enzyme subunit. Site-directed mutagenesis experiments indicate that hydrophobic interactions play an important role in these contacts.

Amino Acid Sequence↗
PubMed1990

Biosynthesis of cyclic 2,3-diphosphoglycerate. Isolation and characterization of 2-phosphoglycerate kinase and cyclic 2,3-diphosphoglycerate synthetase from Methanothermus fervidus.

Starting from 2-phosphoglycerate the biosynthesis of cDPG comprises two steps: (i) the phosphorylation of 2-phosphoglycerate to 2,3-diphosphoglycerate and (ii) the intramolecular cyclization to cyclic 2,3-diphosphoglycerate. The involved enzymes, 2-phosphoglycerate kinase and cyclic 2,3-diphosphoglycerate synthetase, were purified form Methanothermus fervidus. Their molecular and catalytic properties were characterized.

2,3-Diphosphoglycerate↗
PubMed1990

Cloning and sequencing the gene encoding 3-phosphoglycerate kinase from mesophilic Methanobacterium bryantii and thermophilic Methanothermus fervidus.

The nucleotide sequences of the gene (pgk) encoding 3-phosphoglycerate kinase (PGK) from the mesophilic archaebacterium, Methanobacterium bryantii, and from the closely related thermophile, Methanothermus fervidus, were determined. The deduced amino acid (aa) sequences show 61% identity with each other and 32-36% identity with the enzyme homologues from eubacteria and eukaryotes. As found for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-malate dehydrogenase, the relatedness between the archaebacterial aa sequences on the one hand and the eubacterial or eukaryotic sequences on the other is lower than that between the latter ones. Comparison of the aa sequence of PGK from mesophilic and thermophilic archaebacteria indicates an increase of the overall hydrophobicity and a decrease of the chain flexibility in the thermophilic enzyme, as already deduced from respective comparisons between GAPDH aa sequences of the same organisms. In addition, glycine residues are strikingly discriminated in the thermophilic PGK, which was also observed for GAPDH. Contrary to GAPDH, however, Lys and Arg residues are preferred in the thermophilic PGK. Lys to Arg substitutions are the most frequent cold-to-hot changes in PGK, whereas in GAPDH from the same organisms these changes do not occur.

Amino Acid Sequence↗
PubMed1990

Properties and primary structure of the L-malate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus.

L-Malate dehydrogenase from the extremely thermophilic mathanogen Methanothermus fervidus was isolated and its phenotypic properties were characterized. The primary structure of the protein was deducted from the coding gene. The enzyme is a homomeric dimer with a molecular mass of 70 kDa, possesses low specificity for NAD+ or NADP+ and catalyzes preferentially the reduction of oxalacetate. The temperature dependence of the activity as depicted in the Arrhenius and van't Hoff plots shows discontinuities near 52 degrees C, as was found for glyceraldehyde-3-phosphate dehydrogenase from the same organism. With respect to the primary structure, the archaebacterial L-malate dehydrogenase deviates strikingly from the eubacterial and eukaryotic enzymes. The sequence similarity is even lower than that between the L-malate dehydrogenases and L-lactate dehydrogenases of eubacteria and eukaryotes. The phylogenetic meaning of this relationship is discussed.

Amino Acid Sequence↗
PubMed1990

Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei: characterization of the enzyme, cloning and sequencing of the gene, and expression in Escherichia coli.

The glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei (optimal growth temperature, 100 to 103 degrees C) was purified to homogeneity. This enzyme was strictly phosphate dependent, utilized either NAD+ or NADP+, and was insensitive to pentalenolactone like the enzyme from the methanogenic archaebacterium Methanothermus fervidus. The enzyme exhibited a considerable thermostability, with a 44-min half-life at 100 degrees C. The amino acid sequence of the glyceraldehyde-3-phosphate dehydrogenase from P. woesei was deduced from the nucleotide sequence of the coding gene. Compared with the enzyme homologs from mesophilic archaebacteria (Methanobacterium bryantii, Methanobacterium formicicum) and an extremely thermophilic archaebacterium (Methanothermus fervidus), the primary structure of the P. woesei enzyme exhibited a strikingly high proportion of aromatic amino acid residues and a low proportion of sulfur-containing residues. The coding gene of P. woesei was expressed at a high level in Escherichia coli, thus providing an ideal basis for detailed structural and functional studies of that enzyme.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gene from the mesophilic methanogenic archaebacteria Methanobacterium bryantii and Methanobacterium formicicum. Comparison with the respective gene structure of the closely related extreme thermophile Methanothermus fervidus.

The genes for glyceraldehyde-3-phosphate dehydrogenase (gap genes) from the mesophilic methanogenic archaebacteria Methanobacterium formicicum and Methanobacterium bryantii were cloned and sequenced. The deduced amino acid sequences show 95% identity to each other and about 70% identity to the glyceraldehyde-3-phosphate dehydrogenase from the thermophilic methanogenic archaebacterium Methanothermus fervidus. Although the sequence similarity between the archaebacterial glyceraldehyde-3-phosphate dehydrogenase and the homologous enzyme of eubacteria and eukaryotes is low, an equivalent secondary-structural arrangement can be deduced from the profiles of the physical parameters hydropathy, chain flexibility and amphipathy. In order to find possible thermophile-specific structural features of the enzyme from M. fervidus, a comparative primary-sequence analysis was performed. Amino acid exchanges leading, to a stabilization of the main-chain conformation, could be found throughout the sequence of the thermophile enzyme. Striking features of the thermophile sequence are the preference for isoleucine, especially in beta-sheets, and a low arginine/lysine ratio of 0.54.

Amino Acids↗
PubMed1989

Sequence comparison of glyceraldehyde-3-phosphate dehydrogenases from the three urkingdoms: evolutionary implication.

The primary structure of the glyceraldehyde-3-phosphate dehydrogenase from the archaebacteria shows striking deviation from the known sequences of eubacterial and eukaryotic sequences, despite unequivocal homologies in functionally important regions. Thus, the structural similarity between the eubacterial and eukaryotic enzymes is significantly higher than that between the archaebacterial enzymes and the eubacterial and eukaryotic enzymes. This preferred similarity of eubacterial and eukaryotic glyceraldehyde-3-phosphate dehydrogenase structures does not correspond to the phylogenetic distances among the three urkingdoms as deduced from comparisons of ribosomal ribonucleic acid sequences. Indications will be presented that the closer relationship of the eubacterial and eukaryotic glyceraldehyde-3-phosphate dehydrogenase resulted from a gene transfer from eubacteria to eukaryotes after the segregation of the three urkingdoms.

Amino Acid Sequence↗
PubMed1989

Effect of uremic toxins--guanidino compounds and creatinine--on proliferation of HL60 and K562 cell lines.

Growth of the promyelocytic cell line HL60 and the erythroleukemia cell line K562 is inhibited by 'uremic toxins': creatinine, guanidino propionic acid and guanidino succinic acid in a concentration range similar to that of uremic sera. Among the tested compounds, creatinine exhibits the strongest and most dose-dependent inhibitory effect on both kinds of cells. These results provide a better understanding of the mechanism involved in the anemia of uremic patients.

Cell Line↗
PubMed1988

Expression of the glyceraldehyde-3-phosphate dehydrogenase gene from the extremely thermophilic archaebacterium Methanothermus fervidus in E. coli. Enzyme purification, crystallization, and preliminary crystal data.

The gene of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the extremely thermophilic archaebacterium Methanothermus fervidus (growth optimum 82 degrees C) was cloned in vector pJF118EH and expressed in E. coli cells. As shown by molecular mass determination, protein sequencing, heat stability, and substrate saturation kinetics, the enzyme synthesized in E. coli is identical to the original enzyme from M. fervidus. The high thermostability of the E. coli-produced M. fervidus GAPDH allows rapid purification to homogeneity. From this enzyme protein crystals were grown which proved to be suitable for X-ray analysis. The crystals are of tetragonal space group P4(1)22 and contain a dimer per asymmetric unit.

Base Sequence↗
PubMed1988

Primary structure of glyceraldehyde-3-phosphate dehydrogenase deduced from the nucleotide sequence of the thermophilic archaebacterium Methanothermus fervidus.

The gene for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the thermophilic methanogenic archaebacterium Methanothermus fervidus (growth optimum at 84 degrees C) was cloned in Escherichia coli and the nucleotide sequence was determined. A striking preference for adenine and thymidine bases was found in the gene, which is in agreement with the low G + C content of the M. fervidus DNA. The deduced amino acid sequence indicates an Mr of 37,500 for the protein subunit. Alignment with the amino acid sequences of GAPDHs from other organisms shows that the archaebacterial GAPDH is homologous to the respective eubacterial and eukaryotic enzymes, but the similarity between the archaebacterial enzyme and the eubacterial or eukaryotic GAPDHs is much less than that between the latter two.

Amino Acid Sequence↗