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At least 19 recordsLinked to original sources
PubMed1988

Cloning and nucleotide sequence of the Bacillus subtilis hom gene coding for homoserine dehydrogenase. Structural and evolutionary relationships with Escherichia coli aspartokinases-homoserine dehydrogenases I and II.

The Bacillus subtilis hom gene, encoding homoserine dehydrogenase (L-homoserine:NADP+ oxidoreductase, EC 1.1.1.3) has been cloned and its nucleotide sequence determined. The B. subtilis enzyme expressed in Escherichia coli is sensitive by inhibition by threonine and allows complementation of a strain lacking homoserine dehydrogenases I and II. Nucleotide sequence analysis indicates that the hom stop codon overlaps the start codon of thrC (threonine synthase) suggesting that these genes, as well as thrB (homoserine kinase) located downstream from thrC, belong to the same transcription unit. The deduced amino acid sequence of the B. subtilis homoserine dehydrogenase shows extensive similarity with the C-terminal part of E. coli aspartokinases-homoserine dehydrogenases I and II; this similarity starts at the exact point where the similarity between E. coli or B. subtilis aspartokinases and E. coli aspartokinases-homoserine dehydrogenases stops. These data suggest that the E. coli bifunctional polypeptide could have resulted from the direct fusion of ancestral aspartokinase and homoserine dehydrogenase. The B. subtilis homoserine dehydrogenase has a C-terminal extension of about 100 residues (relative to the E. coli enzymes) that could be involved in the regulation of the enzyme activity.

Alcohol Oxidoreductases↗
PubMed2000

Enzyme-catalyzed acylation of homoserine: mechanistic characterization of the Haemophilus influenzae met2-encoded homoserine transacetylase.

The first unique step in bacterial and plant methionine biosynthesis involves the acylation of the gamma-hydroxyl of homoserine. In Haemophilus influenzae, acylation is accomplished via an acetyl-CoA-dependent acetylation catalyzed by homoserine transacetylase. The activity of this enzyme regulates flux of homoserine into multiple biosynthetic pathways and, therefore, represents a critical control point for cell growth and viability. We have cloned homoserine transacetylase from H. influenzae and present the first detailed enzymatic study of this enzyme. Steady-state kinetic experiments demonstrate that the enzyme utilizes a ping-pong kinetic mechanism in which the acetyl group of acetyl-CoA is initially transferred to an enzyme nucleophile before subsequent transfer to homoserine to form the final product, O-acetylhomoserine. The maximal velocity and V/K(homoserine) were independent of pH over the range of values tested, while V/K(acetyl)(-)(CoA) was dependent upon the ionization state of a single group exhibiting a pK value of 8.6, which was required to be protonated. Solvent kinetic isotope effect studies yielded inverse effects of 0.75 on V and 0.74 on V/K(CoA) on the reverse reaction and effects of 1.2 on V and 1.7 on V/K(homoserine) on the forward reaction. Direct evidence for the formation of an acetyl-enzyme intermediate was obtained using rapid-quench labeling studies. On the basis of these observations, we propose a chemical mechanism for this important member of the acyltransferase family and contrast its mechanism with that of homoserine transsuccinylase.

Acetyltransferases↗
PubMed1999

Enzyme-catalyzed acylation of homoserine: mechanistic characterization of the Escherichia coli metA-encoded homoserine transsuccinylase.

The first unique step in bacterial and plant methionine biosynthesis involves the activation of the gamma-hydroxyl of homoserine. In Escherichia coli, this activation is accomplished via a succinylation reaction catalyzed by homoserine transsuccinylase. The activity of this enzyme is closely regulated in vivo and therefore represents a critical control point for cell growth and viability. We have cloned homoserine transsuccinylase from E. coli and present the first detailed enzymatic study of this enzyme. Steady-state kinetic experiments demonstrate that the enzyme utilizes a ping-pong kinetic mechanism in which the succinyl group of succinyl-CoA is initially transferred to an enzyme nucleophile before subsequent transfer to homoserine to form the final product, O-succinylhomoserine. The maximal velocity, V/K(succinyl)(-)(CoA), and V/K(homoserine) all exhibited a bell-shaped pH dependence with apparent pK's of 6.6 and approximately 7.9. The enzyme was inhibited by iodoacetamide in a pH-dependent manner, with an apparent pK of the group being inactivated of 6.4. This suggests the presence of an active site cysteine which forms a succinyl-cysteine intermediate during enzymatic turnover. Solvent kinetic isotope effect studies yielded inverse effects of 0.7 on V and 0.61 on V/K in the reverse reaction only. On the basis of these observations, we propose a detailed chemical mechanism for this important member of the acyltransferase family.

Acylation↗
PubMed1977

Proteolysis of the bifunctional methionine-repressible aspartokinase II-homoserine dehydrogenase II of Escherichia coli K12. Production of an active homoserine dehydrogenase fragment.

The dimeric bifunctional enzyme aspartokinase II-homoserine dehydrogenase II (Mr = 2 X 88,000) of Escherichia coli K12 can be cleaved into two nonoverlapping fragments by limited proteolysis with subtilisin. These two fragments can be separated under nondenaturing conditions as dimeric species, which indicates that each fragment has retained some of the association areas involved in the conformation of the native protein. The smaller fragment (Mr = 2 X 24,000) is devoid of aspartokinase and homoserine dehydrogenase activity. The larger fragment (Mr = 2 X 37,000) is endowed with full homoserine dehydrogenase activity. These results show that the polypeptide chains of the native enzyme are organized in two different domains, that both domains participate in building up the native dimeric structure, and that one of these domains only is responsible for homoserine dehydrogenase activity. A model of aspartokinase II-homoserine dehydrogenase II is proposed, which accounts for the present results.

Alcohol Oxidoreductases↗
PubMed2004

Bacterial populations in the rhizosphere of tobacco plants producing the quorum-sensing signals hexanoyl-homoserine lactone and 3-oxo-hexanoyl-homoserine lactone.

A tobacco line genetically modified to produce two N-acyl homoserine lactones and its non-transformed parental line were grown in non-sterile soil. Microbial populations inhabiting the bulk soil, and those colonizing the root system of the two tobacco lines, were analyzed using cultivation-independent (phospholipid fatty acid and denaturing gradient gel electrophoresis) and cultivation-based assays. The cell density of total cultivable bacteria, fluorescent pseudomonads, sporulated, and thermotolerant bacteria was also determined in a time-course experiment (15 weeks). A possible "rhizosphere effect" related to the development of the plant was seen. However, no dissimilarities in cell population densities or population ratios of the microbial groups were detected in the rhizosphere of the two plant lines. Similarly, bacterial communities that either produced N-acyl homoserine lactone or degraded the signal hexanoyl homoserine lactone were enumerated from the two plant lines. No noticeable differences were evidenced from one plant genotype to the other. Whilst the transgenic plants released detectable amounts of the quorum-sensing signal molecules and efficiently cross-talked with the surrounding microbial populations, the bias generated by these signals in the reported experimental conditions therefore appears to remain weak, if not non-existent.

4-Butyrolactone↗
PubMed1990

[Development of the vector-host system in Corynebacterium. Cloning and expression of homoserine dehydrogenase and homoserine kinase genes in Corynebacterium cells].

Novel cloning vectors for glutamic acid producing bacteria have been constructed. The cryptic plasmid pBO1 (4.4 kb) from Brevibacterium sp. recombined with the plasmid pACYC184 (4.0 kb) from Escherichia coli was used to produce composite plasmid named pKA1. The plasmid could propagate and express the Cm-r phenotype in E. coli and coryneform glutamic acid producing bacteria Br. flavum, C. glutamicum, Br. lactofermentum. The pKA1 plasmid and its variants deleted within non-essential plasmid regions with unique restriction sites HindIII, SalGI, SphI were used in cloning experiments. The genes coding for threonine biosynthesis of C. glutamicum and Br. flavum were subcloned into shuttle vectors in C. glutamicum cells. Recombinant plasmids were introduced into protoplasts by polyethylenglycol-mediated transformation of plasmid DNAs. It was shown that the presence of plasmids containing the Br. flavum thrA2 gene in C. glutamicum (thrB) caused 10-fold increase in homoserine dehydrogenase activity, as compared to that of wild type strain, and in homoserine production.

Alcohol Oxidoreductases↗
PubMed1987

Effect of homoserine on growth of Mycobacterium smegmatis: inhibition of glutamate transport by homoserine.

Homoserine strongly inhibited growth of Mycobacterium smegmatis in medium containing glutamate as the sole source of nitrogen but was without effect when asparagine, alanine or glutamine was the sole nitrogen source. It was readily taken up by glutamate-grown cells, reaching an intracellular concentration of over 20 mM after 4 h incubation. The primary site of action of homoserine was deduced to be the non-competitive inhibition of glutamate transport.

Biological Transport↗
PubMed2005

Methionine and threonine synthesis are limited by homoserine availability and not the activity of homoserine kinase in Arabidopsis thaliana.

Homoserine kinase (HSK) produces O-phospho-l-homoserine (HserP) used by cystathionine gamma-synthase (CGS) for Met synthesis and threonine synthase (TS) for Thr synthesis. The effects of overexpressing Arabidopsis thaliana HSK, CGS, and Escherichia coli TS (eTS), each controlled by the 35S promoter, were compared. The results indicate that in Arabidopsis Hser supply is the major factor limiting the synthesis of HserP, Met and Thr. HSK is not limiting and CGS or TS control the partitioning of HserP. HSK overexpression had no effect on the level of soluble HserP, Met or Thr, however, when treated with Hser these plants produced far more HserP than wild type. Met and Thr also accumulated markedly after Hser treatment but the increase was similar in HSK overexpressing and wild-type plants. CGS overexpression was previously shown to increase Met content, but had no effect on Thr. After Hser treatment Met accumulation increased in CGS-overexpressing plants compared with wild type, whereas HserP declined and Thr was unaffected. Arabidopsis responded differentially to eTS expression depending on the level of the enzyme. At the highest eTS level the Thr content was not increased, but the phenotype was negatively affected and the T1 plants died before reproducing. Comparatively low eTS did not affect phenotype or Thr/Met level, however after Hser treatment HserP and Met accumulation were reduced compared with wild type and Thr was increased slightly. At intermediate eTS activity seedling growth was retarded unless Met was supplied and CGS expression was induced, indicating that eTS limited HserP availability for Met synthesis.

Arabidopsis↗
PubMed1975

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12. Specific inactivation of the homoserine dehydrogenase activity by the affinity label, 2-amino-4-oxo-5-chloropentanoic acid.

2-Amino-4-oxo-5-chloropentanoic acid inactivates specifically the homoserine dehydrogenase activity of the bifunctional enzyme, aspartokinase I--homoserine dehydrogenase I. The aspartokinase activity remains essentially untouched and retains its threonine sensitivity. The inactivation of the dehydrogenase requires the covalent binding of one equivalent of the analogue per subunit. Alkylation does not affect the tetrameric state of the protein. The alkylating agent, a substrate analogue, meets the qualitative and quantitative requirements of an affinity label.

Alcohol Oxidoreductases↗
PubMed1984

Homoserine kinase of Escherichia coli: kinetic mechanism and inhibition by L-aspartate semialdehyde.

The kinetic mechanism of homoserine kinase, purified to homogeneity from Escherichia coli, was examined by initial velocity techniques at pH 7.6. Whereas ATP displayed normal Michaelis-Menten saturation kinetics (Km = 0.2 mM), L-homoserine showed hyperbolic saturation kinetics only up to a concentration of 0.75 mM (Km = 0.15 mM). Above this concentration, L-homoserine caused marked but partial inhibition (Ki approximately 2 mM). The kinetic data indicated that the addition of substrates to homoserine kinase occurs by a preferred order random mechanism, with ATP preferentially binding before L-homoserine. When the ATP concentration was varied at several fixed inhibitory concentrations of L-homoserine, the resulting inhibition pattern indicated hyperbolic mixed inhibition. This suggested a second binding site for L-homoserine. L-Aspartate semialdehyde, an amino acid analog of L-homoserine, proved to be an alternative substrate of homoserine kinase (Km = 0.68 mM), and was subsequently used as a probe of its kinetic mechanism. In aqueous solution, at pH 7.5, this analog was found to exist predominantly (ca 85%) as its hydrated species. When examined as an inhibitor of the physiological reaction, L-aspartate semialdehyde showed mixed inhibition versus both L-homoserine and ATP. Although the pH profiles for the binding of L-homoserine as a substrate (Km) and as an inhibitor (Ki) were identical, the kinetic data were best fit to a two-site model, with separate catalytic and inhibitory sites for L-homoserine.

Adenosine Triphosphate↗
PubMed1994

Threonine synthesis from homoserine as a selectable marker in mammalian cells.

The plasmid pSVthrBC expresses the Escherichia coli thrB (homoserine kinase) and thrC (threonine synthase) genes in mouse cells and enables them to synthesize threonine from homoserine. After transfection with pSVthrBC and culture in medium containing homoserine, only cells that have incorporated pSVthrBC survive. Homoserine at concentrations greater than 1 mM is toxic to mammalian cells. Mouse cells selected from medium containing 5 mM homoserine had incorporated 20-100 copies of the plasmid per cell and had homoserine kinase activities of 0.001-0.012 nmol/min per mg of protein per copy. Cells selected from medium containing 10 mM homoserine had incorporated one or two copies of the plasmid per cell and had homoserine kinase activities of 0.06-0.39 nmol/min per mg of protein per copy. By using high concentrations of homoserine, it is possible to use pSVthrBC to select and isolate cell lines that have one or two copies of the plasmid incorporated into an active region of chromatin. CHO and HeLa cells have also been successfully transfected with pSVthrBC. COS-7 cells are naturally resistant to homoserine as they are able to metabolize homoserine.

3T3 Cells↗
PubMed1996

Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein.

Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesis of the V. fischeri signal. (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synthesis are not amino acids from biosynthetic pathways or fatty acid degradation products, but rather they are S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway. We purified a maltose binding protein-LuxI fusion polypeptide and showed that, when provided with the appropriate substrates, it catalyzes the synthesis of an acyl homoserine lactone. In V. fischeri, luxi directs the synthesis of N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone. The purified maltose binding protein-LuxI fusion protein catalyzes the synthesis of hexanoyl homoserine lactone from hexanoyl-ACP and SAM. There is a high level of specificity for hexanoyl-ACP over ACPs with differing acyl group lengths, and hexanoyl homoserine lactone was not synthesized when SAM was replaced with other amino acids, such as methionine, S-adenosylhomocysteine, homoserine, or homoserine lactone, or when hexanoyl-SAM was provided as the substrate. This provides direct evidence that the LuxI protein is an auto-inducer synthase that catalyzes the formation of an amide bond between SAM and a fatty acyl-ACP and then catalyzes the formation of the acyl homoserine lactone from the acyl-SAM intermediate.

4-Butyrolactone↗