Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.1.1.3 (HSD)
3,464 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ethionine reduced both the growth rate and the final growth level of Serratia marcescens Sr41. Growth inhibition was completely reversed by methionine. Strain D-315, defective in homoserine dehydrogenase I, was more sensitive to ethionine-mediated growth inhibition than was the wild-type strain. Ethionine-resistant mutants were isolated from cultures of strain D-316, which was derived from strain D-315 as a threonine deaminase-deficient mutant. Of 60 resistant colonies, 7 excreted threonine on minimal agar plates. One threonine-excreting strain, ETr17, was highly resistant to ethionine and, moreover, insensitive to methionine-mediated growth inhibition, whereas the parent strain was sensitive. When cultured in minimal medium with or without excess methionine, strain ETr17 had a higher homoserine dehydrogenase level than did strain D-316. The homoserine dehydrogenase activity was not inhibited by threonine or methionine. Transductional analysis revealed that the ethionine-resistant (etr-1) mutation carried by strain ETr17 was located in the metBM-argE region and caused the derepressed synthesis of homoserine dehydrogenase II. Strain ETr17 had a higher aspartokinase level than did the parent strain. By transductional cross with the argE+ marker, the etr-1 mutation was transferred into strain D-562 which was derived from D-505, a strain defective in aspartokinases I and III. The constructed strain had a higher aspartokinase level than did strain D-505 in medium with or without excess methionine, indicating that the etr-1 mutation led to the derepressed synthesis of aspartokinase II. Strain ETr17 produced about 8 mg of threonine per ml of medium containing sucrose and urea.
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PMID:Threonine production by ethionine-resistant mutants of Serratia marcescens. 640 83

Aspartokinase I-homoserine dehydrogenase I (EC 2.7.2.4 and EC 1.1.1.3) a bifunctional and allosteric enzyme, has been renatured from its unfolded and separated polypeptide chains. Folding was measured by the reappearance of each of the two enzymatic activities, kinase and dehydrogenase, and of their allosteric inhibition by the same effector, threonine. The various observed properties yield different kinetics of folding, which shows the presence of intermediates having only some of the functional features of the native enzyme. Apparently, three successive steps can be detected during the folding of aspartokinase I-homoserine dehydrogenase I: first, a monomolecular step leads to a monomeric species with the kinase activity; then an association step leads to a dimeric species with the kinase and dehydrogenase activities, and a threonine-sensitive dehydrogenase; finally, a second association step leads to a tetrameric species with the two activities, both sensitive to threonine. The folding of this large protein appears as a sequential process during which the functional properties are regained successively, as the protein structure becomes more complex. During this process, the two regions of each polypeptide chain respectively responsible for the kinase and dehydrogenase activities seem to acquire their native conformation rather independently of each other.
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PMID:Sequential folding of a bifunctional allosteric protein. 677 37

A set of hybrid plasmids carrying Escherichia coli threonine genes was obtained and cloned. The plasmid pBR322 was used as a vehicle. The genetic and restriction analyses showed that genes thrA and thrB were placed between SalGI and EcoRI sites on the 2.6 megadaltons DNA region. The transcription of threonine operon genes inserted in the hybrid plasmids is under the control of its own promoter. The copy number of hybrid plasmids was reverse proportional to their molecular weight and did not depend on the replicon number. Amplification of genes of threonine operon by hybrid plasmids led to 20-25-fold increase of homoserine dehydrogenase activity, encoded by thrA gene. The expression of this gene, incorporated in hybrid plasmids, was repressed by the addition of threonine and isoleucine in the culture medium.
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PMID:[Cloning of threonine operon genes in Escherichia coli cells]. 677 49

Homoserine dehydrogenase in dialyzed cell extracts of Bacillus subtilis 168 was studied, particularly with regard to inhibition, repression, and level of activity as a function of stage of development (growth and sporulation). It was assayed in the "forward direction" using L-aspartic semialdehyde and NADPH as substrates. Of the potentials inhibitors tested, only cysteine and NADP were found to be effective. Both L- and D-cysteine were equally effective. Therefore, the physiological significance of cysteine as an inhibitor is somewhat questionable. Amino acids involved in repression of homoserine dehydrogenase included methionine, isoleucine, possibly threonine, and one or more unidentified components of Casamino acids. The specific activity of homoserine dehydrogenase was highest during the exponential phase of growth and declined steadily during the stationary phase of growth. The low specific activity during late sporulation may favor preferential funnelling of L-aspartic semialdehyde into the lysine pathway, where it is needed for synthesis of large amounts of dipicolinic acid and diaminopimelic acid.
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PMID:Sporulation and regulation of homoserine dehydrogenase in Bacillus subtilis. 678 15

During the derepression of threonine and isoleucine-valine operons the increase of activity of homoserine dehydrogenase and threonine deaminase respectively occurs only in relA+ strains of Escherichia coli K-12, while the activity of these enzymes in relA mutants does not change. The increase of the activity of homoserine dehydrogenase in relA+ strains corresponds to the increase of the fraction of the thr-mRNA, i.e. the expression of threonine operon is regulated at the level of transcription. After isoleucine is exhausted, only relA+ cells of threonine producer MG442 increase homoserine dehydrogenase activity and production of threonine. Thus, the regulation of transcription and translation of threonine and isoleucine-valine operons depends upon the allelic state of the relA gene.
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PMID:[Funtional study of gene relA in the expression of amino acid operons. III. The effect of the allelic state of gene relA on the derepression of the threonine and isoleucine-valine operons of Escherichia coli K-12]. 698 24

The thrA gene of Escherichia coli codes for a single polypeptide chain having two enzymatic activities required for the biosynthesis of threonine, aspartokinase I and homoserine dehydrogenase I. This gene was cloned in a bacterial plasmid and its complete nucleotide sequence was established. It contains 2460 base pairs that encode for a polypeptide chain of 820 amino acids. The previously determined partial amino acid sequence of this protein is in good agreement with that predicted from the nucleotide sequence. The gene contains an internal sequence that resembles the structure of bacterial ribosome-binding sites, with an AUG preceded by four triplets, each of which can be converted to a nonsense codon by a single mutation. This suggests that the single polypeptide chain was formed by the fusion of two genes and that initiation of translation may occur inside the gene to give a protein fragment having only the homoserine dehydrogenase activity.
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PMID:Nucleotide sequence of the thrA gene of Escherichia coli. 700 95

Gene disruption and replacement techniques were applied to block the biosynthesis of threonine and methionine and thus to construct genetically stable lysine producers in a glutamate-producing bacteria, Brevibacterium divaricatum. The homoserine dehydrogenase gene (hom), homoserine kinase gene (thrB) and hom-thrB operon were amplified as 1.8, 1.25 and 2.8 kb fragments from B. divaricatum by polymerase chain reaction (PCR) and cloned in an E. coli-coryneform bacteria shuttle vector, pSUMN18. These genes were disrupted by inserting a kanamycin resistant gene (kan) from pUC4-KISS into the structural genes. Integrative plasmids were constructed and transformed into B. divaricatum. Integrative transformants could be obtained only when the integrative plasmids were constructed from the plasmids which had escaped from the restriction barrier of the hosts. The resulting integrative transformants showed kanamycin resistance and contained no plasmids. About 1-10% of the transformants were auxotrophs. By checking the nutritional requirement, it was found that all of these transformants required threonine and/or homoserine as expected. Southern blot analysis confirmed the integrations, and both single and double crossover homologous recombination mechanisms were proposed to explain the integration and replacement. These auxotrophic integrative transformants which were derived from double crossover events accumulated 1-3% lysine in culture broth only when the added threonine was limited. Integrative transformants which were site-specifically inactivated in hom or hom-thrB genes produced more lysine than did those only inactivated at the thrB gene. These transformants were extremely stable, and the reversion frequency was below 10(-9) per generation. It is suggested that this technique will be useful in the construction of stable auxotrophic mutants.
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PMID:Construction of lysine-producing strains by gene disruption and replacement in Brevibacterium divaricatum. 762 44

Overproduction of isoleucine, an essential amino acid, was achieved by amplification of the gene encoding threonine dehydratase, the first enzyme in the threonine to isoleucine pathway, in a Corynebacterium lactofermentum threonine producer. Threonine overproduction was previously achieved with C. lactofermentum ATCC 21799, a lysine-hyperproducing strain, by introduction of plasmid pGC42 containing the Corynebacterium homdr and thrB genes (encoding homoserine dehydrogenase and homoserine kinase respectively) under separate promoters. The pGC42 derivative, pGC77, also contains ilvA, which encodes threonine dehydratase. In a shake-flask fermentation, strain 21799(pGC77) produced 15 g/l isoleucine, along with small amounts of lysine and glycine. A molar carbon balance indicates that most of the carbon previously converted to threonine, lysine, glycine and isolecine was incorporated into isoleucine by the new strain. Thus, in our system, simple overexpression of wild-type ilvA sufficed to overcome the effects of feedback inhibition of threonine dehydratase by the end-product, isoleucine.
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PMID:Production of isoleucine by overexpression of ilvA in a Corynebacterium lactofermentum threonine producer. 763 98

The coding regions for the Escherichia coli gene for aspartokinase I/homoserine dehydrogenase I (thrA) and the Corynebacterium glutamicum gene for aspartic semialdehyde dehydrogenase (asd) have been subcloned into a Simian Virus 40 (SV40)-based mammalian expression vector. Both enzyme activities are expressed in mouse 3T3 cells after transfer of the corresponding chimaeric gene. The kinetic parameters are similar to those of the native bacterial enzymes, and aspartokinase I/homoserine dehydrogenase I retains its allosteric regulation by threonine. An extract of the cells expressing aspartokinase I/homoserine dehydrogenase I, mixed with one from cells expressing aspartic semialdehyde dehydrogenase, produced homoserine when the mixture was incubated with aspartic acid, ATP and NADPH. The thrA and asd expression cassettes were combined into a single plasmid which, when transfected into 3T3 cells, enabled them to produce homoserine from aspartic acid. Homoserine-producing 3T3 cells were transfected with the plasmid pSVthrB/C (homoserine kinase and threonine synthase) and selected for growth on homoserine. Cell lines isolated from these cells expressed the complete bacterial threonine pathway, were independent of threonine for growth and could be maintained in medium which contained no free threonine. The threonine in the proteins of these cells became enriched in 15N when the culture medium contained [15N]aspartic acid. The production of homoserine and the growth of cells was at a maximum when there was more than 2.5 mM aspartate in the medium. Below this concentration the high Km of aspartokinase limited the flux through the pathway. In the presence of additional aspartic acid the new pathway could sustain a cell cycle time close to that of the same cells cultured in threonine-containing medium.
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PMID:The biosynthesis of threonine by mammalian cells: expression of a complete bacterial biosynthetic pathway in an animal cell. 763 21

This paper deals with the application of the metabolic control theory, especially the measurement of control coefficients, to the threonine pathway in E. coli. The control coefficient of a step on a metabolic flux quantitatively assesses the flux response to the step variations. This concept is particularly relevant both in pathological situations (decrease in the activity of an enzymatic step in the metabolism) and in biotechnologies, where, on the contrary steps are amplified. Measurement of the control coefficients of the steps of a metabolic network makes it possible to know those whose amplification should lead to a simultaneous increase in the fluxes. We have applied these concepts to threonine biosynthesis from aspartate in E. coli. The threonine pathway starting from aspartate involves five steps catalyzed by five enzyme activities: aspartokinase (AK), aspartate-semialdehyde-dehydrogenase (ASA-DH), homoserine dehydrogenase (HDH), homoserine kinase (HK) and hreonine synthetase activity (TS). Measurement of the control coefficient of the first step (AK, insensitive to threonine inhibition in the studied strain) has shown that it controls threonine production weakly. Our study has revealed a hitherto unknown inhibition of homoserine kinase activity by lysine. Mathematical modeling of this metabolic pathway has been undertaken to better understand our experimental results.
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PMID:[Control of the metabolic pathway of threonine in E coli. Application of biotechnology]. 770 83


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