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)

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

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

Amplification of the operon homdr-thrB encoding a feedback-insensitive homoserine dehydrogenase and a wild-type homoserine kinase in a Corynebacterium lactofermentum lysine-producing strain resulted in both homoserine and threonine accumulation, with some residual lysine production. A plasmid enabling separate transcriptional control of each gene was constructed to determine the effect of various enzyme activity ratios on metabolite accumulation. By increasing the activity of homoserine kinase relative to homoserine dehydrogenase activity, homoserine accumulation in the medium was essentially eliminated and the final threonine titer was increased by about 120%. Furthermore, a fortuitous result of the cloning strategy was an unexplained increase in homoserine dehydrogenase activity. This resulted in a further decrease in lysine production along with a concomitant increase in threonine accumulation.
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PMID:Effect of inducible thrB expression on amino acid production in Corynebacterium lactofermentum ATCC 21799. 788 27

We have cloned the homoserine dehydrogenase genes (hom) from the gram-negative obligate methylotrophs Methylobacillus glycogenes ATCC 21276 and ATCC 21371 by complementation of an Escherichia coli homoserine dehydrogenase-deficient mutant. The 4.15-kb DNA fragment cloned from M. glycogenes ATCC 21371 also complemented an E. coli threonine synthase-deficient mutant, suggesting the DNA fragment contained the thrC gene in addition to the hom gene. The homoserine dehydrogenases expressed in the E. coli recombinants were hardly inhibited by L-threonine, L-phenylalanine, or L-methionine. However, they became sensitive to the amino acids after storage at 4 degrees C for 4 days as in M. glycogenes. The structures of the homoserine dehydrogenases overexpressed in E. coli were thought to be different from those in M. glycogenes, probably in subunit numbers of the enzyme, and were thought to have converted to the correct structures during the storage. The nucleotide sequences of the hom and thrC genes were determined. The hom genes of M. glycogenes ATCC 21276 and ATCC 21371 encode peptides with M(r)s of 48,225 and 44,815, respectively. The thrC genes were located 50 bp downstream of the hom genes. The thrC gene of ATCC 21371 encodes a peptide with an M(r) of 52,111, and the gene product of ATCC 21276 was truncated. Northern (RNA) blot analysis suggests that the hom and thrC genes are organized in an operon. Significant homology between the predicted amino acid sequences of the hom and thrC genes and those from other microorganisms was found.
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PMID:Cloning and nucleotide sequences of the homoserine dehydrogenase genes (hom) and the threonine synthase genes (thrC) of the gram-negative obligate methylotroph Methylobacillus glycogenes. 811 70

The first and the third steps in the aspartate biosynthesis pathway in Streptococcus bovis are catalyzed by two different forms of aspartokinase and a single homoserine dehydrogenase, respectively. These enzymes can be separated by ammonium sulfate fractionation and gel filtration on Sephadex G-200. The two aspartokinase isozymes differ in molecular weights and are subject to differential regulation. The aspartokinase system of S. bovis is characterized by the absence of specific negative allosteric effectors among the end products of the synthesis of amino acids of the aspartic family. Homoserine dehydrogenase, which catalyzes the third step of the aspartic family amino acid synthesis, also has such negative effectors as threonine and methionine. The aspartokinase isozymes do not form multienzyme complexes with homoserine hydrogenase in S. bovis.
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PMID:[Analysis of key enzyme activities involved in aspartate amino acid biosynthesis in Streptococcus bovis]. 811 40

Isotope exchange kinetics at chemical equilibrium were used to probe the mechanisms of substrate binding and regulatory behavior of homoserine dehydrogenase-I from Escherichia coli. At pH 9.0, 37 degrees C, Keq = 100 (+/- 20) for the catalyzed reaction: L-aspartate-beta-semialdehyde + NADPH + H+ = L-homoserine + NADP+. Saturation curves for the exchange reactions, [14C]L-homoserine <--> L-aspartate-beta-semialdehyde and [3H]NADP+ <--> NADPH were observed as a function of different reactant-product pairs, varied in constant ratio at equilibrium. The NADP+ <--> NADPH exchange rate was inhibited upon variation of pairs involving L-aspartate-beta-semialdehyde and L-homoserine, consistent with preferred order random binding of cofactors before amino acids. Optimal rate constants, derived by simulations of equilibrium isotope exchange kinetics data with the ISOBI program, indicate faster dissociation of amino acids than cofactors from the central complexes but nearly equal rates for association of cofactors and amino acids to free enzyme. Rate limitation of net turnover in both directions is determined by dissociation of cofactor from the E-cofactor complex. The allosteric modifier, L-threonine, produces distinctive perturbations of the saturation curves for isotope exchange, which were analyzed systematically with the ISOBI program. The best fit to the data was obtained by L-threonine inhibiting catalysis between the central complexes without altering substrate association-dissociation rates. Simulations also showed that rate-limiting catalysis suppresses the kinetic inhibition effects that are characteristic of preferred order substrate binding, producing patterns typical for a (rapid equilibrium) random kinetic scheme.
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PMID:Kinetic and regulatory mechanisms for (Escherichia coli) homoserine dehydrogenase-I. Equilibrium isotope exchange kinetics. 844 66

The Saccharomyces cerevisiae HOM6 gene, encoding homoserine dehydrogenase (EC 1.1.1.3) was cloned and its nucleotide sequence determined. The yeast homoserine dehydrogenase shows extensive homology to the homoserine dehydrogenase domains of the two aspartokinase-homoserine dehydrogenases from Escherichia coli as well as to the homoserine dehydrogenases from Gram positive bacteria. Sequence alignment reveals that the yeast enzyme is the smallest homoserine dehydrogenase known, owing to the absence of a C-terminal domain endowed with the L-threonine allosteric response in Gram positive bacteria. Accordingly, the S. cerevisiae enzyme appears to be a naturally occurring feedback resistant homoserine dehydrogenase. Our results indicate that homoserine dehydrogenase was originally an unregulated enzyme and that feedback control acquisition occurred twice during evolution after the divergence between Gram positive and Gram negative bacteria.
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PMID:Evolutionary relationships between yeast and bacterial homoserine dehydrogenases. 850 Jun 24

The gram-negative bacterium Corynebacterium glutamicum is used for the industrial production of amino acids, for example, of L-glutamate and L-lysine. By cloning and expressing the various genes of the L-lysine pathway in C. glutamicum, we would demonstrate that an increase of the flux of L-aspartate semialdehyde to L-lysine could be obtained in strains with increased dihydrodipicolinate synthase activity. Recently we detected that in C. glutamicum two pathways exist for synthesis of D,L-diaminopimelate and L-lysine. Mutants defective in one pathway are still able to synthesize enough L-lysine for growth, but the L-lysine secretion is reduced to 50 to 70%. Using NMR spectroscopy, we could calculate how much of the L-lysine secreted into the medium is synthesized via either one or the other pathway. Amplification of the feedback inhibition insensitive homoserine dehydrogenase and homoserine kinase in a high L-lysine-overproducing strain enabled channeling of the carbon flow from the intermediate aspartate semialdehyde towards homoserine, resulting in a high accumulation of L-threonine. For a further flux from L-threonine to L-isoleucine, the allosteric control of threonine dehydratase was eliminated.
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PMID:Construction of L-lysine-, L-threonine-, and L-isoleucine-overproducing strains of Corynebacterium glutamicum. 865 1

Two genes, hom and thrB, involved in threonine biosynthesis in Lactococcus lactis MG1614, were cloned and sequenced. These genes, which encode homoserine dehydrogenase and homoserine kinase, were initially identified by the homology of their gene products with known homoserine dehydrogenases and homoserine kinases from other organisms. The identification was supported by construction of a mutant containing a deletion in hom and thrB that was unable to grow in a defined medium lacking threonine. Transcriptional analysis showed that the two genes were located in a bicistronic operon with the order 5' hom-thrB 3' and that transcription started 66 bp upstream of the translational start codon of the hom gene. A putative -10 promoter region (TATAAT) was located 6 bp upstream of the transcriptional start point, but no putative -35 region was identified. A DNA fragment covering 155 bp upstream of the hom translational start site was functional in pAK80, an L. lactis promoter probe vector. In addition, transcriptional studies showed no threonine-dependent regulation of hom-thrB transcription.
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PMID:Cloning and transcriptional analysis of two threonine biosynthetic genes from Lactococcus lactis MG1614. 868 67

Recombinant homoserine dehydrogenase from Saccharomyces cerevisiae has been crystallized in three different forms. Crystals of the apo-enzyme belong to the tetragonal space group P4 and have unit-cell-dimensions a = b = 130 and c = 240 A. The resolution limit for these crystals is 3.9 A. Crystals of homoserine dehydrogenase grown in the presence of the co-factor NAD+ have the tetragonal space group P41212 or its enantiomorph P43212. The unit-cell dimensions for these crystals are a = b = 80.4 and c = 250.2 A, and the observed resolution limit is 2.2 A. Protein crystals grown in the presence of the product L-homoserine and the inert NAD+ analogue 3-aminopyridine adenine dinucleotide belong to the monoclinic space group P21 with unit-cell parameters a = 58.8, b = 104.2, c = 120.7 A, beta = 91.9 degrees. This last crystal form has a diffraction limit of 2.7 A resolution.
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PMID:Crystallization and preliminary X-ray diffraction studies of homoserine dehydrogenase from Saccharomyces cerevisiae. 976 13


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