Purification of aspartase and aspartokinase-homoserine dehydrogenase I from Escherichia coli by dye-ligand chromatography

Karsten, William E.; Hunsley, James R.; Viola, Ronald E.

doi:10.1016/0003-2697(85)90280-5

Cited by 43 publications

(27 citation statements)

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“…A further improvement on this procedure utilized the published low pH/heat and ammonium sulfate fractionation steps from these earlier purification schemes, followed by ionexchange and dye-ligand chromatography as the key steps to obtain homogeneous enzyme. This improved scheme resulted in over 800-fold purification of aspartase in 50% yield from wild-type E. coli (Karsten et al, 1985). The asp4 gene, encoding for aspartase, had been mapped in the E. coli genome (Spencer et al, 1976).…”

Section: Purification and Characterizationmentioning

confidence: 99%

L ‐Aspartase: New Tricks from an Old Enzyme

Viola

2000

Advances in Enzymology - And Related Areas of Molecular Biology

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Section: Purification and Characterizationmentioning

confidence: 99%

L ‐Aspartase: New Tricks from an Old Enzyme

Viola

2000

Advances in Enzymology - And Related Areas of Molecular Biology

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“…This enzyme has been purified from several gram-negative bacteria, and its gene has been cloned and sequenced (17,(38)(39)(40). In Bacillus subtilis, aspartase is present in vegetatively growing cells, and its synthesis is induced by aspartate (13).…”

mentioning

confidence: 99%

Cloning, nucleotide sequence, and expression of the Bacillus subtilis ans operon, which codes for L-asparaginase and L-aspartase

Sun

Setlow

1991

J Bacteriol

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L-Aspartase was purified from Bacillus subtilis, its N-terminal amino acid sequence was determined to construct a probe for the aspartase gene, and the gene (termed ansB) was cloned and sequenced. A second gene (termed ansA) was found upstream of the ansB gene and coded for L-asparaginase. These two genes were in an operon designated the ans operon, which is 80% cotransformed with the previously mapped aspHI mutation at 2150. Primer extension analysis of in vivo ans mRNA revealed two transcription start sites, depending on the growth medium. In wild-type cells in log-phase growth in 2x YT medium (tryptone-yeast extract rich medium), the ans transcript began at -67 relative to the translation start site, while cells in log-phase growth or sporulating (t, to t4) in 2x SG medium (glucose nutrient broth-based moderately rich medium) had an ans transcript which began at -73. The level of the -67 transcript was greatly increased in an aspH mutant grown in 2x YT medium; the -67 transcript also predominated when this mutant was grown in 2x SG medium, although the -73 transcript was also present. In vitro transcription of the ans operon by RNA polymerase from log-phase cells grown in 2x YT medium and log-phase or sporulating cells grown in 2x SG medium yielded only the -67 transcript. Depending on the growth medium, the levels of asparaginase and aspartase were from 2-to 40-fold higher in an aspH mutant than in wild-type cells, and evidence was obtained indicating that the gene defined by the aspHI mutation codes for a trans-acting transcriptional regulatory factor. In wild-type cells grown in 2x SG medium, the levels of both aspartase and asparaginase decreased significantly by to of sporulation but then showed a small increase, which was mirrored by changes in the level of ,-galactosidase from an ansB-lacZ fusion. The increase in the activities of ans operon enzymes between t2 and t5 of sporulation was found primarily in the forespore, and the great majority of the increased was found in the mature spore. However, throughout sporulation the only ans transcript detected was the -73 form, and no sporulationspecific RNA polymerase tested yielded a -73 transcript in vitro.

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“…The specific activity of the purified cloned AspA of Y. pseudotuberculosis was 80 units mg 21 (Table 2), which is comparable to that of AspA from E. coli (Karsten et al, 1985). In contrast, the specific activity of the similarly prepared CRIM of Y. pestis was more than 300-fold lower (0.2 units mg…”

Section: Activities Of Aspa From Y Pseudotuberculosis Versus the Y mentioning

confidence: 86%

“…The resulting values were then evaluated against a standard curve, prepared immediately before each determination by assays using known concentrations of NH 4 Cl in samples of 10 ml containing the same concentrations of trichloroacetic acid and Nessler's reagent that were used to prepare samples for spectrophotometric analysis. AspA activity in purified preparations was measured by monitoring the appearance of fumarate at 240 nm (e52.53 mM 21 cm 21 ) (Karsten et al, 1985) in an assay buffer consisting of HEPES, pH 8.0 (50 mM), magnesium acetate (10 mM) and L-aspartate (20 mM). Enzyme units were defined as mmol product generated per minute.…”

Section: +mentioning

confidence: 99%

A missense mutation causes aspartase deficiency in Yersinia pestis

et al. 2008

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It is established that cells of Yersinia pestis, the causative agent of bubonic plague, excrete L-aspartic acid at the expense of exogenous L-glutamic acid during expression of the low-calcium response. Results of enzymic analysis provided here suggest that a previously defined deficiency of aspartase (AspA) accounts for this phenomenon rather than an elevated oxaloacetate pool. The only known distinction between most sequenced isolates of aspA from Y. pestis and the active gene in Yersinia pseudotuberculosis (the immediate progenitor of Y. pestis) is a single base transversion (G?CAT?A) causing replacement of leucine (encoded by UUG) for valine (encoded by GUG) at amino acid position 363. The gene from Y. pestis KIM possesses a unique second transversion (G?CAT?A) at amino acid 146 causing substitution of aspartic acid (encoded by GAU) with tyrosine (encoded by UAU). We show in this study that Y. pestis expresses aspA as cross-reacting immunological material (CRIM). Functional and inactive aspA of Y. pseudotuberculosis PB1 and Y. pestis KIM, respectively, were then cloned and expressed in AspA-deficient Escherichia coli. After purification to near homogeneity, the products were subjected to biochemical analysis and found to exhibit similar secondary, tertiary and quaternary (tetrameric) structures as well as comparable Michaelis constants for L-aspartic acid. However, the k cat of the Y. pestis CRIM of strain KIM is only about 0.1 % of that determined for the active AspA of Y. pseudotuberculosis. Return of valine for leucine at position 363 of the Y. pestis enzyme restored normal turnover (k cat 86±2 s "1 ) provided that the amino acid substitution at position 146 was also reversed. These observations have important implications for understanding the nature of the stringent low-calcium response of Y. pestis and its role in promoting acute disease.

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Purification of aspartase and aspartokinase-homoserine dehydrogenase I from Escherichia coli by dye-ligand chromatography

Cited by 43 publications

References 15 publications

L ‐Aspartase: New Tricks from an Old Enzyme

L ‐Aspartase: New Tricks from an Old Enzyme

Cloning, nucleotide sequence, and expression of the Bacillus subtilis ans operon, which codes for L-asparaginase and L-aspartase

A missense mutation causes aspartase deficiency in Yersinia pestis

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