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D von Wettstein

Publications and source records attributed to D von Wettstein.

At least 19 recordsLinked to original sources
PubMed1992

The role of Lys272 in the pyridoxal 5-phosphate active site of Synechococcus glutamate-1-semialdehyde aminotransferase.

Glutamate-1-semialdehyde (GSA) aminotransferase catalyzes transfer of the C2 amino group of glutamate 1-semialdehyde to the C1 position to yield the tetrapyrrole precursor 5-aminolevulinate. Based on spectrophotometric and steady-state data, GSA aminotransferase is a B6-containing enzyme which uses a ping-pong bi-bi mechanism described for other aminotransferases. A putative active-site lysine at position 272 of Synechococcus GSA aminotransferase was replaced by Arg, Ile or Glu, and genes encoding the corresponding three site directed mutants were expressed in Escherichia coli. The catalytic competence of the resulting enzymes was determined. The similarity of the absorbance spectra of pyridoxal-P-treated forms of Lys272----Arg, Lys272----Ile, Lys272----Glu with free pyridoxal-P indicates that enzyme-bound pyridoxal-P does not form an internal aldimine in in these three site-directed mutants. Whereas Lys----Ile and Lys----Glu form only stable ketimines and aldimines with GSA and its analogues, addition of these compounds to the pyridoxamine-P and pyridoxal-P forms of Lys----Arg induces slow spectral changes, indicating the catalysis of a half-reaction with GSA, 4,5-dioxovalerate and 4,5-diaminovalerate. 5-Aminolevulinate apparently binds with both coenzyme forms of Lys272----Arg, however significant tautomeric rearrangement is only observed with the pyridoxal-P form. It is suggested that Lys272 is the covalent pyridoxal-P-binding site and that this catalytically active lysine residue channels the overall transamination reaction towards 5-aminolevulinate. The second-half reaction (4,5-diaminovalerate in equilibrium with 5-aminolevulinate) is possibly supported by the formation of an internal aldimine which correctly positions the C4 amino group of 4,5-diaminovalerate relative to the enzyme-bound pyridoxal-P.

Base Sequence↗
PubMed1992

Expression and secretion of pea-seed lipoxygenase isoenzymes in Saccharomyces cerevisiae.

Lipoxygenases (EC 1.13.11.12) catalyse the oxygenation of polyunsaturated fatty acids such as linoleic and arachidonic acid into reactive cis/trans hydroperoxidiene intermediates, which then serve as substrates for other enzymes leading to the production of a variety of secondary metabolites. In order to explore the characteristics of the individual lipoxygenase isoenzymes in more detail larger amounts of the pure enzymes are needed and their production in a heterologous host is therefore desirable. Full-length cDNAs encoding pea-seed lipoxygenase isoenzymes 2 and 3 were expressed in Saccharomyces cerevisiae with the aid of yeast-Escherichia coli shuttle vectors. Expression of the cDNA for lipoxygenase 2 under the control of the constitutive phosphoglycerate kinase (PGK) gene promoter yielded significant amounts of active enzyme inside the cell, both with yeast transformants carrying the cDNA gene on high-copy-number plasmids or integrated in chromosome V. Addition of the yeast invertase signal sequence in front of the pea lipoxygenase 3 yielded secreted active pea-seed lipoxygenase in the medium, but large amounts of inactive lipoxygenase 3 remained inside the yeast cell. Expression of the LOX3 cDNA can be achieved either constitutively with the PGK promoter or inducibly with the GAL1 promoter.

Base Sequence↗
PubMed1991

Characterization of glutamate-1-semialdehyde aminotransferase of Synechococcus. Steady-state kinetic analysis.

Synechococcus glutamate-1-semialdehyde aminotransferase was expressed in large amounts in transformed cells of Escherichia coli. The resulting purified enzyme has an absorption spectrum characteristic of B6-containing enzymes and could be converted to the pyridoxal-phosphate form with excess dioxovalerate (O2Val), and back to the pyridoxamine-phosphate form with diaminovalerate (A2Val). Both enzyme forms are similarly active in the conversion of glutamate 1-semialdehyde (GSA) to 5-aminolevulinate (ALev), suggesting that A2Val and O2Val are intermediates. Initial rates of ALev synthesis at various fixed concentrations of GSA followed typical Michaelis-Menten kinetics (Km of GSA for the pyridoxamine-phosphate form of GSA aminotransferase = 12 microM, kcat = 0.23 s-1). In submicromolar amounts A2Val stimulates ALev synthesis, and in a series of concentrations with various fixed concentrations of GSA, gives a family of parallel lines in Lineweaver-Burk plots (Km for A2Val = 1.0 microM). On the other hand, O2Val gives competitive inhibition of the pyridoxamine-phosphate form of GSA-aminotransferase and mixed-type inhibition of the pyridoxal-phosphate form (Ki for O2Val = 1.4 mM). In general the kinetics were typical of ping-pong bi-bi mechanisms in which A2Val is the second substrate (intermediate) and O2Val is an alternative first substrate. There is no compelling evidence that O2Val accepts an amino group at its C5 position resulting in the direct formation of ALev, or the reverse involving the apparent formation of O2Val from ALev. These results are consistent with the hypothesis that the mechanism of GSA aminotransferase mimics that of other aminotransferases and that A2Val is the intermediate.

Chromatography, Ion Exchange↗
PubMed1989

Hybrid bacillus endo-(1-3,1-4)-beta-glucanases: construction of recombinant genes and molecular properties of the gene products.

Hybrid beta-glucanase genes were constructed by the reciprocal exchange of the two halves of the isolated beta-glucanase genes from Bacillus amyloliquefaciens and B. macerans. The beta-glucanase hybrid enzyme 1 (H1) contains the 107 amino-terminal residues of mature B. amyloliquefaciens beta-glucanase and the 107 carboxyl-terminal amino acid residues of B. macerans beta-glucanase. The reciprocal beta-glucanase hybrid enzyme 2 (H2) consists of the 105 amino-terminal residues from the B. macerans enzyme and the carboxyl-terminal 107 amino acids from B. amyloliquefaciens. The biochemical properties of the two hybrid enzymes differ significantly from each other as well as from both parental beta-glucanases. Hybrid beta-glucanase H1 exhibits increased thermostability in comparison to other beta-glucanases, especially in an acidic environment. This hybrid enzyme has maximum activity between pH 5.6 and 6.6, whereas the pH-optimum for enzymatic activity of B. amyloliquefaciens beta-glucanase was found to be at pH 6 to 7 and for B. macerans at pH 6.0 to 7.5. Hybrid enzyme 1 being more heat stable than both parental enzymes represents a case of intragenic heterosis. Hybrid beta-glucanase 2 (H2) was found to be more thermolabile than the naturally occurring beta-glucanases it was derived from and the pH-optimum for enzymatic activity was determined to be between pH 7 and pH 8.

Amino Acid Sequence↗
PubMed1988

A cDNA clone encoding a 10.8 kDa photosystem I polypeptide of barley.

A cDNA clone encoding the barley photosystem I polypeptide which migrates with an apparent molecular mass of 16 kDa on SDS-polyacrylamide gels has been isolated. The 634 bp sequence of this clone has been determined and contains one large open reading frame coding for a 15,457 Da precursor polypeptide. The molecular mass of the mature polypeptide is 10,821 Da. The amino acid sequence of the transit peptide indicates that the polypeptide is routed towards the stroma side of the thylakoid membrane. The hydropathy plot of the polypeptide shows no membrane-spanning regions.

Amino Acid Sequence↗
PubMed1984

The synaptonemal complex and genetic segregation.

The assembly, rearrangement and disassembly of the synaptonemal complex during the stages from leptotene to anaphase I of meiosis are described. The assembly of the lateral component of the synaptonemal complex between the sister chromatids of the leptotene chromosome causes the undivided appearance of the leptotene chromosome in the light microscope. During zygotene the lateral component mediates the assembly of the synaptonemal complex between homologous segments of homologous as well as homoeologous chromosomes. Chromosome and bivalent interlockings are a regular feature during chromosome pairing at zygotene. They are resolved by breakage and precise repair of the chromosomes. The phase of synaptonemal complex formation between homologous chromosome segments is succeeded by a phase of rearrangements. In this phase, chromosome homology is no longer required for synaptonemal complex assembly. This reduces the number of univalent chromosome sections. Rearrangement of chromosome pairing with the synaptonemal complex is not expected to take place at positions where a crossover has occurred. Disassembly of the synaptonemal complex at diplotene is initiated at specific regions along the bivalent arms. Chiasmata are formed from retained pieces of the synaptonemal complex in which a recombination nodule is or had been present. A limit to the number of available recombination nodules and a mechanism which ensures that at least one nodule is placed in each bivalent arm are sources for the positive interference observed in linkage studies employing three-point tests. The time of crossing over relative to the zygotene assembly and pachytene rearrangement phases of the synaptonemal complex determines if multivalents or exclusively bivalents are found at metaphase I in amphidiploids and autotetraploids. The synaptonemal complex provides both the structural basis for the regular disjunction of chromosomes at meiosis and gene recombination between homologous or homoeologous chromosome arms.

Animals↗
PubMed1982

Ultrastructural characterization of the meiotic prophase. A tool in the assessment of radiation damage in man.

The three-dimensional reconstruction of meiotic nuclei from serial sections micrographed in the electron microscope has provided information about man and several other organisms that is not obtainable by light microscopy or biochemical analysis. At zygotene, the previously unpaired chromosomes align and form synaptonemal complexes between homologous chromosome segments either by progressive initiation from the telomeres or by interstitial recognition. Chromosome and bivalent interlocking at zygotene is a regular phenomenon and occurs at a frequency of 0.7-4.0 per nucleus in samples of meiocytes analyzed from different organisms. This frequency is reduced to 0.1 per nucleus at pachytene. The interlockings are resolved by breakage and precise rejoining of the broken ends. This breakage and rejoining can also occur in the absence of the DNA nicking and repair involved in crossing-over. The synaptonemal complexes combining homologous chromosome segments are stabilized by recombination nodules, after which a second round of synaptonemal complex formation between as yet unpaired or unstably paired chromosome segments occurs, apparently for optimization of bivalent formation. Non-homologous pairing with the synaptonemal complex can take place in this phase of pachytene. Continuity between recombination nodules and chromatin chiasmata has been traced at the ultrastructural level but not all nodules lead to chiasmata. The distributions of recombination nodules among the bivalents and along the bivalents at successive stages of meiotic prophase show that the nodules are placed at random at early-zygotene after which bivalents without nodules have preference for the acquisition of these structures. Chiasma interference appears as a consequence of the limited number of recombination nodules available together with a decreased affinity of a bivalent arm with a nodule for additional ones. The relevance of these observations in the study of genetic damage by radiation is discussed.

Aneuploidy↗
PubMed1977

The assembly of the synaptinemal complex.

The assembly of the synaptinemal complex in the ascomycete Neottiella was studied by three-dimensional reconstruction of a late zygotene nucleus. A single banded lateral component is formed between the two sister chromatids of each homologous chromosome prior to their pairing. The central regions are pre-assembled in organized form in folds of the granular part of the nucleolus and then converted into an amorphous transport form. The latter appears to move through the nucleoplasm to sites between the lateral components of synapsing homologous chromosomes. The central region material is reorganized into blocks with a recognizable central component and attached to one lateral component. The last step in the completion of the synaptinemal complex is the association of the free surface of the organized central region with the corresponding segment of the homologous lateral component. The findings are discussed in relation to mechanisms of chromosome pairing and chiasma formation.

Ascomycota↗
PubMed1971

The synaptinemal complex and four-strand crossing over.

The synaptinemal complex provides a structural basis for four-strand crossing over: prior to chromosome pairing, both sister chromatids of each homologous chromosome participate in the genesis of one lateral component. During precise pairing, the two lateral components are combined into one synaptinemal complex per bivalent.

Ascomycota↗