site-directed mutagenesis, expression and biological activity of e. coli 5-enolpyruvylshikimate 3-phosphate synthase gene
نویسندگان
چکیده
site-directed mutagenesis (sdm) as a powerful technique was used to change two important and conserved amino acids in 5-enolpyruvylshikimate 3- phosphate synthase (epsps) gene of e. coli. the mutations changed glycine 96 to alanine and alanine 183 to threonine. these two amino acids are very important for intraction of the wide spectrum herbicide, glyphosate, to epsp synthase enzymes. by designing mutagen primers and overlapping extension method, three kinds of altered bacterial epsps enzymes with first, second and both mutations were produced. these modified enzymes are expected to show decreased affinity for herbicide, with least alteration in their enzymatic activity. these altered genes were cloned under the control of chemically inducible t7 promoter and over expressed in e. coli. biological activity analyses in the presence of glyphosate show that the bacteria containing the mutated enzymes, especially the enzyme with two mutations, were more tolerant to glyphosate.
منابع مشابه
Site-Directed Mutagenesis, Expression and Biological Activity of E. coli 5-Enolpyruvylshikimate 3-Phosphate Synthase Gene
Site-directed mutagenesis (SDM) as a powerful technique was used to change two important and conserved amino acids in 5-enolpyruvylshikimate 3- phosphate synthase (EPSPS) gene of E. coli. The mutations changed glycine 96 to alanine and alanine 183 to threonine. These two amino acids are very important for intraction of the wide spectrum herbicide, glyphosate, to EPSP synthase enzymes. By design...
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5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase is an enzyme of the shikimate pathway which is located in the chloroplasts in higher plants. This enzyme is the target of the nonselective herbicide glyphosate. We have isolated and sequenced cDNA clones encoding EPSP synthase from petunia and tomato. The deduced amino acid sequences of the two enzyme precursors show 93% identity in the mature ...
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Phosphate closes the solution structure of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Mycobacterium tuberculosis.
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متن کاملChorismate synthase. Pre-steady-state kinetics of phosphate release from 5-enolpyruvylshikimate 3-phosphate.
The pre-steady-state kinetics of phosphate formation from 5-enolpyruvylshikimate 3-phosphate catalysed by Escherichia coli chorismate synthase (EC 4.6.1.4) were studied by a rapid-acid-quench technique at 25 degrees C at pH 7.5. No pre-steady-state 'burst' or 'lag' phase was observed, showing that phosphate is released concomitant with the rate-limiting step of the enzyme. The implications of t...
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عنوان ژورنال:
iranian journal of biotechnologyناشر: national institute of genetic engineering and biotechnology
ISSN 1728-3043
دوره 4
شماره 4 2006
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