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)

In order to isolate transcriptional regulatory proteins involved in L-methionine-dependent repression in Corynebacterium glutamicum, proteins binding to the putative promoter region upstream of the metY gene were isolated by DNA affinity chromatography. One of the isolated proteins was identified as a putative transcriptional repressor of the TetR-family by a mass spectrometry fingerprint technique based on the complete C. glutamicum genome sequence. The respective gene, designated mcbR, was deleted in the mutant strain C. glutamicum DR1. Using 2D-PAGE, the protein contents of the C. glutamicum wild type and the mutant strain DR1 grown in media with or without L-methionine supplementation were compared and a set of six proteins was identified. Their abundance was drastically enhanced in the mutant strain and no longer influenced by L-methionine added to the growth medium. The corresponding genes were identified by mass spectrometry fingerprint analysis. They included metY encoding O-acetyl-L-homoserine sulfhydrylase, metK encoding S-adenosyl-methionine synthethase, hom encoding homoserine dehydrogenase, cysK encoding L-cysteine synthase, cysI encoding an NADPH dependant sulfite reductase, and ssuD encoding an alkanesulfonate monooxygenase. Evidently, the putative transcriptional repressor McbR is involved in the regulation of the metabolic network directing the synthesis of L-methionine in C. glutamicum. The C. glutamicum mcbR mutant can be considered to represent a first step in the construction of an L-methionine production strain.
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PMID:The putative transcriptional repressor McbR, member of the TetR-family, is involved in the regulation of the metabolic network directing the synthesis of sulfur containing amino acids in Corynebacterium glutamicum. 1277 May 4

The genes hom, thrB and thrC, encoding homoserine dehydrogenase, homoserine kinase (HK) and threonine synthase, respectively, involved in the last steps of threonine biosynthesis, have been studied in Streptomyces sp. NRRL 5331, the producer of the ethylene synthetase inhibitor aminoethoxyvinylglycine (AVG), in order to determine their role in the biosynthesis of AVG. Different null mutants were obtained by plasmid-mediated disruption of each of the three genes. thrC gene disruption had no effect on AVG production, while the disruption of thrB blocked HK activity and substantially reduced the yield of this metabolite, probably due to the accumulation of homoserine and/or methionine which have a negative effect on AVG biosynthesis. Disruption of hom (thrA) completely blocked AVG biosynthesis, indicating that homoserine lies at the branching point of the aspartic-acid-derived biosynthetic route that leads to AVG. The four carbon atoms of the vinylglycine moiety of AVG derive, therefore, from homoserine.
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PMID:Role of homoserine and threonine pathway intermediates as precursors for the biosynthesis of aminoethoxyvinylglycine in Streptomyces sp. NRRL 5331. 1513 8

Candidate attenuators were identified that regulate operons responsible for biosynthesis of branched amino acids, histidine, threonine, tryptophan, and phenylalanine in gamma- and alpha-proteobacteria, and in some cases in low-GC Gram-positive bacteria, Thermotogales and Bacteroidetes/Chlorobi. This allowed us not only to describe the evolutionary dynamics of regulation by attenuation of transcription, but also to annotate a number of hypothetical genes. In particular, orthologs of ygeA of Escherichia coli were assigned the branched chain amino acid racemase function. Three new families of histidine transporters were predicted, orthologs of yuiF and yvsH of Bacillus subtilis, and lysQ of Lactococcus lactis. In Pasteurellales, the single bifunctional aspartate kinase/homoserine dehydrogenase gene thrA was predicted to be regulated not only by threonine and isoleucine, as in E. coli, but also by methionine. In alpha-proteobacteria, the single acetolactate synthase operon ilvIH was predicted to be regulated by branched amino acids-dependent attenuators. Histidine biosynthetic operons his were predicted to be regulated by histidine-dependent attenuators in Bacillus cereus and Clostridium difficile, and by histidine T-boxes in L. lactis and Streptococcus mutans.
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PMID:Attenuation regulation of amino acid biosynthetic operons in proteobacteria: comparative genomics analysis. 1513 44

A relatively unexploited potential target for antimicrobial agents is the biosynthesis of essential amino acids. Homoserine dehydrogenase, which reduces aspartate semi-aldehyde to homoserine in a NAD(P)H-dependent reaction, is one such target that is required for the biosynthesis of Met, Thr, and Ile from Asp. We report a small molecule screen of yeast homoserine dehydrogenase that has identified a new class of phenolic inhibitors of this class of enzyme. X-ray crystal structural analysis of one of the inhibitors in complex with homoserine dehydrogenase reveals that these molecules bind in the amino acid binding region of the active site and that the phenolic hydroxyl group interacts specifically with the backbone amide of Gly175. These results provide the first nonamino acid inhibitors of this class of enzyme and have the potential to be exploited as leads in antifungal compound design.
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PMID:New phenolic inhibitors of yeast homoserine dehydrogenase. 1521 Jan 49

FKBP12 is a conserved member of the prolyl-isomerase enzyme family and serves as the intracellular receptor for FK506 that mediates immunosuppression in mammals and antimicrobial actions in fungi. To investigate the cellular functions of FKBP12 in Saccharomyces cerevisiae, we employed a high-throughput assay to identify mutations that are synthetically lethal with a mutation in the FPR1 gene, which encodes FKBP12. This screen identified a mutation in the HOM6 gene, which encodes homoserine dehydrogenase, the enzyme catalyzing the last step in conversion of aspartic acid into homoserine, the common precursor in threonine and methionine synthesis. Lethality of fpr1 hom6 double mutants was suppressed by null mutations in HOM3 or HOM2, encoding aspartokinase and aspartate beta-semialdehyde dehydrogenase, respectively, supporting the hypothesis that fpr1 hom6 double mutants are inviable because of toxic accumulation of aspartate beta-semialdehyde, the substrate of homoserine dehydrogenase. Our findings also indicate that mutation or inhibition of FKBP12 dysregulates the homoserine synthetic pathway by perturbing aspartokinase feedback inhibition by threonine. Because this pathway is conserved in fungi but not in mammals, our findings suggest a facile route to synergistic antifungal drug development via concomitant inhibition of FKBP12 and Hom6.
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PMID:FKBP12 controls aspartate pathway flux in Saccharomyces cerevisiae to prevent toxic intermediate accumulation. 1547 Feb 57

The Arabidopsis genome contains two genes predicted to code for bifunctional aspartate kinase-homoserine dehydrogenase enzymes (isoforms I and II). These two activities catalyze the first and the third steps toward the synthesis of the essential amino acids threonine, isoleucine, and methionine. We first characterized the kinetic and regulatory properties of the recombinant enzymes, showing that they mainly differ with respect to the inhibition of the homoserine dehydrogenase activity by threonine. A systematic search for other allosteric effectors allowed us to identify an additional inhibitor (leucine) and 5 activators (alanine, cysteine, isoleucine, serine, and valine) equally efficient on aspartate kinase I activity (4-fold activation). The six effectors of aspartate kinase I were all activators of aspartate kinase II activity (13-fold activation) and displayed a similar specificity for the enzyme. No synergy between different effectors could be observed. The activation, which resulted from a decrease in the Km values for the substrates, was detected using low substrates concentrations. Amino acid quantification revealed that alanine and threonine were much more abundant than the other effectors in Arabidopsis leaf chloroplasts. In vitro kinetics in the presence of physiological concentrations of the seven allosteric effectors confirmed that aspartate kinase I and II activities were highly sensitive to changes in alanine and threonine concentrations. Thus, physiological context rather than enzyme structure sets the specificity of the allosteric control. Stimulation by alanine may play the role of a feed forward activation of the aspartate-derived amino acid pathway in plant.
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PMID:Identification of six novel allosteric effectors of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase isoforms. Physiological context sets the specificity. 1621 75

Comprehensive studies were made with Lemna paucicostata Hegelm. 6746 of the effects of combinations of lysine, methionine, and threonine on growth rates, soluble amino acid contents, aspartokinase activities, and fluxes of 4-carbon moieties from aspartate through the aspartokinase step into the amino acids of the aspartate family. These studies show that flux in vitro through the aspartokinase step is insensitive to inhibition by lysine or threonine, and confirm previous in vitro data in establishing that aspartokinase in vivo is present in two orders of magnitude excess of its requirements. No evidence of channeling of the products of the lysine- and threonine-sensitive aspartokinases was obtained, either form of the enzyme alone being more than adequate for the combined in vivo flux through the aspartokinase step. The marked insensitivity of flux through the aspartokinase step to inhibition by lysine or threonine strongly suggests that inhibition of aspartokinase by these amino acids is not normally a major factor in regulation of entry of 4-carbon units into the aspartate family of amino acids. Direct measurement of fluxes of 4-carbon units demonstrated that: (a) Lysine strongly feedback regulates its own synthesis, probably at the step catalyzed by dihydrodipicolinate synthase. (b) Threonine alone does not regulate its own synthesis in vivo, thereby confirming previous studies of the metabolism of [(14)C]threonine and [(14)C]homoserine in Lemna. This finding excludes not only aspartokinases as an important regulatory determinant of threonine synthesis, but also two other enzymes (homoserine dehydrogenase and threonine synthase) suggested to fulfill this role. Complete inhibition of threonine synthesis was observed only in the combined presence of accumulated threonine and lysine. The physiological significance of this single example of apparent regulation of flux at the aspartokinase step, albeit under unusually stringent conditions of aspartokinase inhibition, remains to be determined. (c) Isoleucine strongly inhibits its own synthesis, probably at threonine dehydratase, without causing compensatory reduction in threonine synthesis. A fundamentally changed scheme for regulation of synthesis of the aspartate family of amino acids is presented that has important implications for improvement of the nutritional contents of these amino acids in plants.
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PMID:Regulatory Structure of the Biosynthetic Pathway for the Aspartate Family of Amino Acids in Lemna paucicostata Hegelm. 6746, with Special Reference to the Role of Aspartokinase. 1666 69

A methionine-producing strain was derived from a lysine-producing Corynebacterium glutamicum through a process of genetic manipulation in order to assess its potential to synthesize and accumulate methionine during growth. The strain carries a deregulated hom gene (hom(FBR)) to abolish feedback inhibition of homoserine dehydrogenase by threonine and a deletion of the thrB gene (delta thrB) to abolish threonine synthesis. The constructed C. glutamicum MH20-22B/hom(FBR)/delta thrB strain accumulated 2.9 g/l of methionine by batch fermentation and showed resistance to methionine analogue ethionine at concentrations up to 30 mM. The growth of the strain was apparently impaired as a result of the accumulation of methionine biosynthetic intermediate, homocysteine. Production assays also revealed that the accumulation of methionine in the growth medium was transient and declined as the carbon source was depleted. During the period of methionine disappearance, the methionine biosynthetic genes were completely repressed in the engineered strains but not in the parental strain. After all, we have not only successfully constructed a methionine-producing C. glutamicum strain by genetic manipulation, but also revealed cellular constraints in attaining high yield and productivity.
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PMID:Characteristics of methionine production by an engineered Corynebacterium glutamicum strain. 1760 70

Aspartate kinase (AK, EC 2.7.2.4), homoserine dehydrogenase (HSDH, EC 1.1.1.3) and dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) were isolated and partially purified from immature Chenopodium quinoa Willd seeds. Enzyme activities were studied in the presence of the aspartate-derived amino acids lysine, threonine and methionine and also the lysine analogue S-2-aminoethyl-l-cysteine (AEC), at 1 mM and 5 mM. The results confirmed the existence of, at least, two AK isoenzymes, one inhibited by lysine and the other inhibited by threonine, the latter being predominant in quinoa seeds. HSDH activity was also shown to be partially inhibited by threonine, whereas some of the activity was resistant to the inhibitory effect, indicating the presence of two isoenzymes, one resistant and another sensitive to threonine inhibition. Only one DHDPS isoenzyme highly sensitive to lysine inhibition was detected. The results suggest that the high concentration of lysine observed in quinoa seeds is possibly due to a combined effect of increased lysine synthesis and accumulation in the soluble form and/or as protein lysine. Nitrogen assimilation was also investigated and based on nitrate content, nitrate reductase activity, amino acid distribution and ureide content, the leaves were identified as the predominant site of nitrate reduction in this plant species. The amino acid profile analysis in leaves and roots also indicated an important role of soluble glutamine as a nitrogen transporting compound.
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PMID:Lysine biosynthesis and nitrogen metabolism in quinoa (Chenopodium quinoa): study of enzymes and nitrogen-containing compounds. 1800 25

Aspartate kinase (AK) and homoserine dehydrogenase (HSD) function as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback-inhibited by threonine. In plants the biochemical features of AK and bifunctional AK-HSD enzymes have been characterized, but the molecular properties of the monofunctional HSD remain unexamined. To investigate the role of HSD, we have cloned the cDNA and gene encoding the monofunctional HSD (GmHSD) from soybean. Using heterologously expressed and purified GmHSD, initial velocity and product inhibition studies support an ordered bi bi kinetic mechanism in which nicotinamide cofactor binds first and leaves last in the reaction sequence. Threonine inhibition of GmHSD occurs at concentrations (K(i) = 160-240 mM) more than 1000-fold above physiological levels. This is in contrast to the two AK-HSD isoforms in soybean that are sensitive to threonine inhibition (K(i) approximately 150 microM). In addition, GmHSD is not inhibited by other aspartate-derived amino acids. The ratio of threonine-resistant to threonine-sensitive HSD activity in soybean tissues varies and likely reflects different demands for amino acid biosynthesis. This is the first cloning and detailed biochemical characterization of a monofunctional feedback-insensitive HSD from any plant. Threonine-resistant HSD offers a useful biotechnology tool for manipulating the aspartate amino acid pathway to increase threonine and methionine production in plants for improved nutritional content.
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PMID:Threonine-insensitive homoserine dehydrogenase from soybean: genomic organization, kinetic mechanism, and in vivo activity. 1989 76


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