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K Sauer

Publications and source records attributed to K Sauer.

At least 37 records · Page 2Linked to original sources
PubMed2000

Methyl-coenzyme M formation in methanogenic archaea. Involvement of zinc in coenzyme M activation.

Methyl-coenzyme M (2-methylthioethane sulfonate) is the key intermediate of methane formation in methanogenic archaea. It is generated from coenzyme M (2-mercaptoethane sulfonate) in methyl transfer reactions catalyzed by proteins containing zinc. Here, we report that, for methyltransferase MtaA from Methanosarcina barkeri, the zinc is involved in coenzyme M activation. For the experiments an inactive MtaA apoprotein was obtained by heterologous overproduction in Escherichia coli grown in the presence of 2 mM EDTA. The apoprotein was found to react with zinc or cobalt to the fully active holoenzyme. Appoximately 1 mol of transition metal was bound per mol of protein. Upon incubation of the holoenzyme with coenzyme M approximately 1 mol of proton was released per mol of zinc or cobalt. Protons were not released upon incubation of the apoprotein with coenzyme M or of the holoprotein with other thiol compounds or with methyl-coenzyme M. The findings are interpreted as indicating that the role of the transition metal in MtaA is to lower the microscopic pKa of the thiol group of coenzyme M by coordination to the zinc, and thus to increase its nucleophilicity for methyl group attack. The pKZn2+ of MtaA was re-determined and found to be > 15 and not 9.6 as previously reported by us.

Enzyme Activation↗
PubMed1999

Methanol:coenzyme M methyltransferase from Methanosarcina barkeri -- substitution of the corrinoid harbouring subunit MtaC by free cob(I)alamin.

Methyl-coenzyme M formation from coenzyme M and methanol in Methanosarcina barkeri is catalysed by an enzyme system composed of three polypeptides MtaA, MtaB and MtaC, the latter of which harbours a corrinoid prosthetic group. We report here that MtaC can be substituted by free cob(I)alamin which is methylated with methanol in an MtaB-catalysed reaction and demethylated with coenzyme M in an MtaA-catalysed reaction. Methyl transfer from methanol to coenzyme M was found to proceed at a relatively high specific activity at micromolar concentrations of cob(I)alamin. This finding was surprising because the methylation of cob(I)alamin catalysed by MtaB alone and the demethylation of methylcob(III)alamin catalysed by MtaA alone exhibit apparent Km for cob(I)alamin and methylcob(III)alamin of above 1 mm. A possible explanation is that MtaA positively affects the MtaB catalytic efficiency and vice versa by decreasing the apparent Km for their corrinoid substrates. Activation of MtaA by MtaB was methanol-dependent. In the assay for methanol:coenzyme M methyltransferase activity cob(I)alamin could be substituted by cob(I)inamide which is devoid of the nucleotide loop. Substitution was, however, only possible when the assays were supplemented with imidazole: approximately 1 mm imidazole being required for half-maximal activity. Methylation of cob(I)inamide with methanol was found to be dependent on imidazole but not on the demethylation of methylcob(III)inamide with coenzyme M. The demethylation reaction was even inhibited by imidazole. The structure and catalytic mechanism of the MtaABC complex are compared with the cobalamin-dependent methionine synthase.

Catalysis↗
PubMed1998

His84 rather than His35 is the active site histidine in the corrinoid protein MrtA of the energy conserving methyltransferase complex from Methanobacterium thermoautotrophicum.

The energy conserving corrinoid containing MtrA-H complex from Methanobacterium thermoautotrophicum is composed of eight different subunits of which MtrA harbors the corrinoid prosthetic group, the corrinoid being bound in the base-off/His-on configuration. Based on sequence comparisons it was recently proposed that His35 of MtrA is the active site histidine. We report here that His84 rather than His35 is the axial ligand to the cobamide in MtrA.

Amino Acid Sequence↗
PubMed1998

Sulfur K-edge x-ray absorption spectroscopy: a spectroscopic tool to examine the redox state of S-containing metabolites in vivo.

The sulfur K-edge x-ray absorption spectra for the amino acids cysteine and methionine and their corresponding oxidized forms cystine and methionine sulfoxide are presented. Distinct differences in the shape of the edge and the inflection point energy for cysteine and cystine are observed. For methionine sulfoxide the inflection point energy is 2.8 eV higher compared with methionine. Glutathione, the most abundant thiol in animal cells, also has been investigated. The x-ray absorption near-edge structure spectrum of reduced glutathione resembles that of cysteine, whereas the spectrum of oxidized glutathione resembles that of cystine. The characteristic differences between the thiol and disulfide spectra enable one to determine the redox status (thiol to disulfide ratio) in intact biological systems, such as unbroken cells, where glutathione and cyst(e)ine are the two major sulfur-containing components. The sulfur K-edge spectra for whole human blood, plasma, and erythrocytes are shown. The erythrocyte sulfur K-edge spectrum is similar to that of fully reduced glutathione. Simulation of the plasma spectrum indicated 32% thiol and 68% disulfide sulfur. The whole blood spectrum can be simulated by a combination of 46% disulfide and 54% thiol sulfur.

Erythrocytes↗
PubMed1998

Methanol:coenzyme M methyltransferase from Methanosarcina barkeri--identification of the active-site histidine in the corrinoid-harboring subunit MtaC by site-directed mutagenesis.

The enzyme system catalyzing the formation of methyl-coenzyme M from methanol and coenzyme M in Methanosarcina barkeri is composed of the three different polypeptides MtaA, MtaB and MtaC of which MtaC harbors a corrinoid prosthetic group. The heterologous expression of mtaA and mtaB in Escherichia coli has been described previously. We report here on the overproduction of the apoprotein of MtaC in E. coli, on its reconstitution to the active holoprotein with either cob(II)alamin or methyl-cob(III)alamin, and on the properties of the reconstituted corrinoid protein. Reconstituted MtaC was found to contain 1 mol bound cobamide/mol. EPR spectroscopic evidence is presented for a His residue as an axial ligand to Co2+ of the bound corrinoid. This active-site His was identified by site-directed mutagenesis as His136 in the MtaC sequence that contains four His residues. The reconstituted MtaC, in the cob(I)amide oxidation state, was methylated with methanol in the presence of MtaB and demethylated with coenzyme M in the presence of MtaA. In the presence of both MtaB and MtaA, methyl-coenzyme M was formed from methanol and coenzyme M at specific rates comparable to those determined for the enzyme system purified from M. barkeri. M. barkeri contains an isoenzyme of MtaA designated MtbA. The isoenzyme reacted with MtaC with only 2.5% of the activity of MtaA.

Adenosine Triphosphatases↗
PubMed1998

Tetramethylammonium:coenzyme M methyltransferase system from methanococcoides sp

A methanogen (strain NaT1) that belongs to the family of Methanosarcinaceae and that can grow on tetramethylammonium as the sole energy source has recently been isolated. We report here that cell extracts of the archaeon catalyze the formation of methyl-coenzyme M from coenzyme M and tetramethylammonium. The activity was dependent on the presence of Ti(III) citrate and ATP, and was rapidly lost under oxic conditions. Anoxic chromatography on DEAE-Sepharose revealed that two fractions, fractions 3 and 4, were required for activity. A 50-kDa protein that together with fraction 3 catalyzed methyl-coenzyme M formation from tetramethylammonium and coenzyme M was purified from fraction 4. From fraction 3, a 22-kDa corrinoid protein and a 40-kDa protein exhibiting methylcobalamin:coenzyme M methyltransferase (MT2) activity were purified. The N-terminal amino acid sequences of these purified proteins were determined. The 40-kDa protein showed sequence similarity to MT2 isoenzymes from Methanosarcina barkeri. Cell extract of strain NaT1 grown on trimethylamine rather than on tetramethylammonium did not exhibit tetramethylammonium:coenzyme M methyltransferase activity. The strain was identified as belonging to the genus of Methanococcoides, its closest relative being Methanococcoides methylutens.

Journal Article↗
PubMed1998

Enhancement of synaptic excitation by GABAA receptor antagonists in rat embryonic midbrain culture.

Alterations of synaptic excitation induced by exposure to gamma-aminobutyric acid-A (GABAA) receptor antagonists were investigated employing tight-seal whole cell recording from single neurons or pairs of neurons in rat embryonic midbrain culture. Application of GABAA receptor antagonists led to sustained depolarizations followed by synchronous paroxysmal depolarization shifts (PDSs). PDSs induced a transient increase in miniature excitatory postsynaptic currents in the presence as well as in the absence of a N-methyl-aspartate receptor antagonist. The increase in glutamate release supports the excitatory drive required to reinitiate PDSs from the quiescent interburst intervals. After washout of GABAA receptor antagonists, synaptic activity remained grouped, regardless of the presence or absence of PDS blockade by tetrodotoxin (TTX). Impediment of action potential-triggered transmitter release by Cd2+ or TTX also induced grouped activity. We conclude that changes in synaptic excitation are produced by the impaired GABAA inhibition per se and by the initiation of PDSs.

Action Potentials↗
PubMed1997

Methanol:coenzyme M methyltransferase from Methanosarcina barkeri. Zinc dependence and thermodynamics of the methanol:cob(I)alamin methyltransferase reaction.

In Methanosarcina barkeri, methanogenesis from methanol is initiated by the formation of methyl-coenzyme M from methanol and coenzyme M. This methyl transfer reaction is catalyzed by two enzymes, designated methyltransferases 1 (MT1) and 2 (MT2). Transferase MT1, which is composed of a 50-kDa subunit, MtaB, and a 27-kDa corrinoid-harbouring subunit, MtaC, has been shown recently to catalyze the methylation of free cob(I)alamin with methanol [Sauer, K., Harms, U. & Thauer, R. K. (1997) Eur. J. Biochem. 243, 670-677]. We report here that this reaction is catalyzed by subunit MtaB overproduced in Escherichia coli. MtaB also catalyzed the formation of methanol from methylcobalamin and H2O, the hydrolysis being associated with a free-energy change deltaG(o)' of approximately +7.0 kJ/mol. MtaB was found to contain 1 mol zinc, and its activity to be zinc dependent (pK(Zn2+) = 9.3). The zinc dependence of the MT2 (MtaA)-catalyzed reaction is also described (pK(Zn2+) = 9.6).

Archaeal Proteins↗
PubMed1997

Methanol:coenzyme M methyltransferase from Methanosarcina barkeri. Purification, properties and encoding genes of the corrinoid protein MT1.

In Methanosarcina barkeri, methanogenesis from methanol is initiated by the formation of methylcoenzyme M from methanol and coenzyme M. This methyl transfer reaction is catalyzed by two enzymes, designated MT1 and MT2. Transferase MT1 is a corrinoid protein. The purification, catalytic properties and encoding genes of MT2 (MtaA) have been described previously [Harms, U. and Thauer, R.K. (1996) Eur. J. Biochem. 235, 653-659]. We report here on the corresponding analysis of MT1. The corrinoid protein MT1 was purified to apparent homogeneity and showed a specific activity of 750 mumol min-1 mg-1. The enzyme catalyzed the methylation of its bound corrinoid in the cob(I)amide oxidation state by methanol. In addition to this automethylation, the purified enzyme was found to catalyze the methylation of free cob(I)alamin to methylcob(III)alamin. It was composed of two different subunits designated MtaB and MtaC, with apparent molecular masses of 49 kDa and 24 kDa, respectively. The subunit MtaC was shown to harbour the corrinoid prosthetic group. The genes mtaB and mtaC were cloned and sequenced. They were found to be juxtapositioned and to form a transcription unit mtaCB. The corrinoid-harbouring subunit MtaC exhibits 35% sequence similarity to the cobalamin-binding domain of methionine synthase from Escherichia coli.

Amino Acid Sequence↗
PubMed1996

Dacapo, a cyclin-dependent kinase inhibitor, stops cell proliferation during Drosophila development.

Most cell types in multicellular eukaryotes exit from the mitotic cell cycle before terminal differentiation. We show that the dacapo gene is required to arrest the epidermal cell proliferation at the correct developmental stage during Drosophila embryogenesis. dacapo encodes an inhibitor of cyclin E/cdk2 complexes with similarity to the vertebrate Cip/Kip inhibitors. dacapo is transiently expressed beginning late in the G2 phase preceding the terminal division (mitosis 16). Mutants unable to express the inhibitor fail to arrest cell proliferation after mitosis 16 and progress through an extra division cycle. Conversely, premature dacapo expression in transgenic embryos results in a precocious G1 arrest.

Amino Acid Sequence↗
PubMed1996

Oxidation states of the manganese cluster during the flash-induced S-state cycle of the photosynthetic oxygen-evolving complex.

The Mn K-edge x-ray absorption spectra for the pure S states of the tetranuclear Mn cluster of the oxygen-evolving complex of photosystem II during flash-induced S-state cycling have been determined. The relative S-state populations in samples given 0, 1, 2, 3, 4, or 5 flashes were determined from fitting the flash-induced electron paramagnetic resonance (EPR) multiline signal oscillation pattern to the Kok model. The edge spectra of samples given 0, 1, 2, or 3 flashes were combined with EPR information to calculate the pure S-state edge spectra. The edge positions (defined as the zero-crossing of the second derivatives) are 6550.1, 6551.7, 6553.5, and 6553.8 eV for S0, S1, S2, and S3, respectively. In addition to the shift in edge position, the S0--> S1 and S1--> S2 transitions are accompanied by characteristic changes in the shape of the edge, both indicative of Mn oxidation. The edge position shifts very little (0.3 eV) for the S2--> S3 transition, and the edge shape shows only subtle changes. We conclude that probably no direct Mn oxidation is involved in this transition. The proposed Mn oxidation state assignments are as follows: S0 (II, III, IV, IV) or (III, III, III, IV), S1 (III, III, IV, IV), S2 (III, IV, IV, IV), S3 (III, IV, IV, IV).

Journal Article↗
PubMed1996

Pigment-pigment interactions and energy transfer in the antenna complex of the photosynthetic bacterium Rhodopseudomonas acidophila.

BACKGROUND: Photosynthesis starts with the absorption of solar radiation by antenna pigment molecules. In purple bacteria these chromophores, (bacteriochlorophyll a and carotenoid) are embedded in the membrane; they are non-covalently bound to apoproteins which have the ability to modulate the chromophores' absorbing characteristics. The first structure of the bacterial antenna complex from Rhodopseudomonas acidophila, strain 10050, shows a ring of nonameric symmetry. Two concentric cylinders of apoproteins enclose the pigment molecules. The current resolution of the structure, to 2.5 A, allows us to begin to explore the mechanism of energy transfer among these pigments. RESULTS: The mechanism of energy transfer, from the short- to long-wavelength-absorbing pigments, is largely determined by the relative distances and orientations of the chromophores. In this paper we provide evidence that energy transfer between the B800 and B850 bacteriochlorophylls is largely via Förster induced dipole-dipole resonance. Strong Coulombic (exciton) coupling among the 18 short distanced chromophores in the B850 macrocycle is promoted by good alignment of the Qy dipoles. Singlet-singlet energy transfer from carotenoid to the B800 macrocycle appears to be minimal, with most of the energy transfer going to B850. The higher energy state of both chromophores dominates in more complex situations. CONCLUSIONS: The structure of the antenna complex not only shows Nature at its most aesthetic but also illustrates how clever and efficient the energy transfer mechanism has become, with singlet-singlet excitation being passed smoothly down the spectral gradient to the reaction centre.

Bacterial Proteins↗
PubMed1996

Novel members of the cdc2-related kinase family in Drosophila: cdk4/6, cdk5, PFTAIRE, and PITSLRE kinase.

In addition to the previously identified Drosophila cdc2 and cdc2c genes, we have identified four additional cdc2-related genes with low stringency and polymerase chain reaction approaches. Sequence comparisons suggest that the four putative kinases represent the Drosophila homologues of vertebrate cdk4/6, cdk5, PCTAIRE, and PITSLRE kinases. Although the similarity between human and Drosophila homologues is extensive in the case of cdk5, PCTAIRE, and PITSLRE kinases (78%, 58%, and 65% identity in the kinase domain), only limited conservation is observed for Drosophila cdk4/6 (47% identity). However, like vertebrate cdk4 and cdk6, Drosophila cdk4/6 binds also to a D-type cyclin according to the results of two-hybrid experiments in yeast. Northern blot analysis indicated that the four Drosophila kinases are expressed throughout embryogenesis. Expression in early embryogenesis appeared to be ubiquitous according to in situ hybridization. Abundant expression already at the start of embryogenesis and long before neuron differentiation was also observed in the case of cdk5 protein, which has been described as predominantly neuron specific in mice. Sequence conservation and expression pattern, therefore, suggest that all of these kinases perform important cellular functions.

Amino Acid Sequence↗
PubMed1995

Evidence for the proximity of calcium to the manganese cluster of photosystem II: determination by X-ray absorption spectroscopy.

The photosynthetic oxygen-evolving complex contains a cluster of four manganese atoms and requires both Ca and Cl for activity. The question of Ca proximity to the Mn cluster has been investigated by performing Mn X-ray absorption experiments on native samples of photosystem II (PS II) and on samples depleted of Ca and reconstituted by either Ca or Sr. Analysis of X-ray K-edge spectra demonstrates no significant differences in oxidation state or symmetry between Ca- and Sr-reactivated preparations. Differences are observed in the extended X-ray absorption fine structure (EXAFS). The amplitude of a Fourier transform peak due to scatters at distances greater than 3 A is larger for samples reactivated with strontium than for calcium-reactivated samples. Taking into account the stoichiometry of Mn and Ca atoms in PS II, and considering physically reasonable structures, curve-fitting analyses of the EXAFS data using FEFF5-calculated parameters favor a model where both manganese and calcium (or strontium) scatterers contribute to the Fourier peak at approximately 3 A. Other models for the approximately 3 A peak with multiple Mn-Mn interactions or multiple Mn-Ca(Sr) interactions can also be fit to the data, but are considered less likely. This result provides confirmation for the structural proximity of Ca to the Mn cluster suggested previously [Yachandra, V. K., et al. (1993) Science 260, 675-679]. Possible structural arrangements for a calcium-binding site are discussed.

Binding Sites↗
PubMed1995

Distinct modes of cyclin E/cdc2c kinase regulation and S-phase control in mitotic and endoreduplication cycles of Drosophila embryogenesis.

Drosophila cyclin E (DmcycE) is required in embryos for S phase of mitotic and endoreduplication cycles. Here, we describe regulatory differences characteristic for these two cell cycle types. While DmcycE transcript levels decline in DmcycE mutant cells programmed for mitotic proliferation, they are maintained and no longer restricted to transient pulses in DmcycE mutant cells programmed for endoreduplication. Moreover, DmcycE expression in endoreduplicating cells is down-regulated by ectopic expression of a heat-inducible cyclin E transgene. DmcycE expression in endoreduplicating tissues, therefore, is restricted by a negative feedback to the transient pulse triggering entry into S-phase. Conversely, during mitotic cycles, where S phase entry is not only dependent on cyclin E but also on progression through M phase, cyclin E and associated Dmcdc2c kinase activity are present throughout the cell cycle. Reinitiation of DNA replication during the G2 phase of the mitotic cell cycle, therefore, is prevented by cyclin E/Dmcdc2c kinase-independent regulation. Observations in cyclin A mutants implicate G2 cyclins in this regulation. Our results suggest molecular explanations for the different rules governing S phase during mitotic and endoreduplication cycles.

Animals↗
PubMed1995

Structural consequences of ammonia binding to the manganese center of the photosynthetic oxygen-evolving complex: an X-ray absorption spectroscopy study of isotropic and oriented photosystem II particles.

The structure and orientation of the manganese complex in NH3-treated photosystem II (PS II) membrane particles of spinach are being studied by X-ray absorption spectroscopy. On the basis of earlier work by our group, a structure for the tetranuclear manganese complex of PS II, which consists of two di-mu-oxo-bridged binuclear Mn units linked by a mono-mu-oxo group, has been proposed [Yachandra, V. K., et al. (1993) Science 260, 675-679]. The extended X-ray absorption fine structure (EXAFS) of the complex modified by NH3 binding in the S2-state is suggestive of an increase in the Mn-Mn distance of one of these units from 2.72 +/- 0.02 to 2.87 +/- 0.02 A, whereas the Mn-Mn distance of the second unit seems to be unaffected by NH3 treatment. The elongation of one binuclear center could result from the replacement of one bridging mu-oxo by an amido group. The lengthening of one Mn-Mn distance means that, by NH3 treatment, the distance degeneracy of the 2.7 A Mn-Mn EXAFS interaction is removed. Consequently, the orientation of individual binuclear units with respect to the membrane normal becomes resolvable by EXAFS spectroscopy of partially oriented PS II membrane particles. The angle between the normal of the PS II-containing membrane and the Mn-Mn vector is determined to be 67 degrees +/- 3 degrees for the 2.87 A distance and 55 degrees +/- 4 degrees for the 2.72 A distance. Only small effects on position, shape, and orientation dependence of Mn K-edge spectra result from NH3 treatment, indicating that the Mn oxidation state, the symmetry of the Mn ligand environment, and the orientation of the complex remain essentially unaffected in the annealed NH3 S2-state. Therefore, it seems likely that the angles determined for the ammonia-modified manganese complex are similar to the respective angles of the untreated complex. The structure of the manganese complex and its orientation in the membrane are discussed.

Ammonia↗