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Sedef Tunca - One of the best experts on this subject based on the ideXlab platform.
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cloning characterization and heterologous expression of the Aspartokinase and aspartate semialdehyde dehydrogenase genes of cephamycin c producer streptomyces clavuligerus
Research in Microbiology, 2004Co-Authors: Paloma Liras, Sedef Tunca, Ebru I Yilmaz, Jacqueline Piret, Gulay OzcengizAbstract:Carbon flow through the lysine branch of the aspartate biosynthetic pathway is a rate-limiting step in the formation of cephamycin C, a broad spectrum beta-lactam antibiotic produced by Streptomyces clavuligerus. In this study, genes which encode the enzymes catalyzing the first two steps of the aspartate pathway, ask (Aspartokinase) and asd (aspartate semialdehyde dehydrogenase), in S. clavuligerus NRRL 3585 were cloned and sequenced. Nucleotide sequencing and codon preference analysis revealed three complete open reading frames (ORFs). ORF2 starts within ORF1 and terminates by utilizing the same stop codon as ORF1, an arrangement typical of many ask genes. ORF3 is located 2 nucleotides downstream of ORF1,2. Database comparisons with these proteins identified ORF1 as the large (alpha) subunit of Aspartokinase, ORF2 as the small (beta) subunit of Aspartokinase and ORF3 as the aspartate semialdehyde dehydrogenase. The cloned genes were functionally expressed in auxotrophic Escherichia coli strains, CGSC5074 (ask(-)) and E. coli CGSC5080 (asd(-)), the two enzymes were partially purified from E. coli cell extracts and their kinetic parameters were determined. The effects of end product amino acids and diaminopimelic acid on the activity of Ask and Asd enzymes were also described.
Benjamin F. Matthews - One of the best experts on this subject based on the ideXlab platform.
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Identification and expression of a cDNA from Daucus carota encoding a bifunctional Aspartokinase-homoserine dehydrogenase
Plant molecular biology, 1993Co-Authors: Jane M. Weisemann, Benjamin F. MatthewsAbstract:Aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) catalyze steps in the pathway for the synthesis of lysine, threonine, and methionine from aspartate. Homoserine dehydrogenase was purified from carrot (Daucus carota L.) cell cultures and portions of it were subjected to amino acid sequencing. Oligonucleotides deduced from the amino acid sequences were used as primers in a polymerase chain reaction to amplify a DNA fragment using DNA derived from carrot cell culture mRNA as template. The amplification product was radiolabelled and used as a probe to identify cDNA clones from libraries derived from carrot cell culture and root RNA. Two overlapping clones were isolated. Together the cDNA clones delineate a 3089 bp long sequence encompassing an open reading frame encoding 921 amino acids, including the mature protein and a long chloroplast transit peptide. The deduced amino acid sequence has high homology with the Escherichia coli proteins Aspartokinase I-homoserine dehydrogenase I and Aspartokinase II-homoserine dehydrogenase II. Like the E. coli genes the isolated carrot cDNA appears to encode a bifunctional Aspartokinase-homoserine dehydrogenase enzyme.
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Bifunctional Protein in Carrot Contains Both Aspartokinase and Homoserine Dehydrogenase Activities
Plant physiology, 1991Co-Authors: Barbara J. Wilson, Ann C. Gray, Benjamin F. MatthewsAbstract:We have purified homoserine dehydrogenase to homogeneity and subjected polypeptide fragments derived from digests of the protein to amino acid sequencing. The amino acid sequence of homoserine dehydrogenase from carrot (Daucus carota) indicates that in carrot both Aspartokinase and homoserine dehydrogenase activities reside on the same protein. Additional evidence that Aspartokinase and homoserine dehydrogenase reside on a bifunctional protein is provided by coelution of activities during purification steps and by enzyme-specific gel staining techniques. Highly purified fractions containing Aspartokinase activity were stained for Aspartokinase activity, homoserine dehydrogenase activity, and protein. These gels confirmed that Aspartokinase activity and homoserine dehydrogenase activity were present on the same protein. This arrangement of Aspartokinase and homoserine dehydrogenase activities residing on the same protein is also found in Escherichia coli, which has two bifunctional enzymes, Aspartokinase I-homoserine dehydrogenase I and Aspartokinase II-homoserine dehydrogenase II. The amino acid sequence of the major form of homoserine dehydrogenase from carrot cell suspension cultures most closely resembles that of the E. coli ThrA gene product Aspartokinase I-homoserine dehydrogenase I.
Gulay Ozcengiz - One of the best experts on this subject based on the ideXlab platform.
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cloning characterization and heterologous expression of the Aspartokinase and aspartate semialdehyde dehydrogenase genes of cephamycin c producer streptomyces clavuligerus
Research in Microbiology, 2004Co-Authors: Paloma Liras, Sedef Tunca, Ebru I Yilmaz, Jacqueline Piret, Gulay OzcengizAbstract:Carbon flow through the lysine branch of the aspartate biosynthetic pathway is a rate-limiting step in the formation of cephamycin C, a broad spectrum beta-lactam antibiotic produced by Streptomyces clavuligerus. In this study, genes which encode the enzymes catalyzing the first two steps of the aspartate pathway, ask (Aspartokinase) and asd (aspartate semialdehyde dehydrogenase), in S. clavuligerus NRRL 3585 were cloned and sequenced. Nucleotide sequencing and codon preference analysis revealed three complete open reading frames (ORFs). ORF2 starts within ORF1 and terminates by utilizing the same stop codon as ORF1, an arrangement typical of many ask genes. ORF3 is located 2 nucleotides downstream of ORF1,2. Database comparisons with these proteins identified ORF1 as the large (alpha) subunit of Aspartokinase, ORF2 as the small (beta) subunit of Aspartokinase and ORF3 as the aspartate semialdehyde dehydrogenase. The cloned genes were functionally expressed in auxotrophic Escherichia coli strains, CGSC5074 (ask(-)) and E. coli CGSC5080 (asd(-)), the two enzymes were partially purified from E. coli cell extracts and their kinetic parameters were determined. The effects of end product amino acids and diaminopimelic acid on the activity of Ask and Asd enzymes were also described.
Ebru I Yilmaz - One of the best experts on this subject based on the ideXlab platform.
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cloning characterization and heterologous expression of the Aspartokinase and aspartate semialdehyde dehydrogenase genes of cephamycin c producer streptomyces clavuligerus
Research in Microbiology, 2004Co-Authors: Paloma Liras, Sedef Tunca, Ebru I Yilmaz, Jacqueline Piret, Gulay OzcengizAbstract:Carbon flow through the lysine branch of the aspartate biosynthetic pathway is a rate-limiting step in the formation of cephamycin C, a broad spectrum beta-lactam antibiotic produced by Streptomyces clavuligerus. In this study, genes which encode the enzymes catalyzing the first two steps of the aspartate pathway, ask (Aspartokinase) and asd (aspartate semialdehyde dehydrogenase), in S. clavuligerus NRRL 3585 were cloned and sequenced. Nucleotide sequencing and codon preference analysis revealed three complete open reading frames (ORFs). ORF2 starts within ORF1 and terminates by utilizing the same stop codon as ORF1, an arrangement typical of many ask genes. ORF3 is located 2 nucleotides downstream of ORF1,2. Database comparisons with these proteins identified ORF1 as the large (alpha) subunit of Aspartokinase, ORF2 as the small (beta) subunit of Aspartokinase and ORF3 as the aspartate semialdehyde dehydrogenase. The cloned genes were functionally expressed in auxotrophic Escherichia coli strains, CGSC5074 (ask(-)) and E. coli CGSC5080 (asd(-)), the two enzymes were partially purified from E. coli cell extracts and their kinetic parameters were determined. The effects of end product amino acids and diaminopimelic acid on the activity of Ask and Asd enzymes were also described.
Jui Shen Chiao - One of the best experts on this subject based on the ideXlab platform.
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expression in escherichia coli purification and kinetic analysis of the Aspartokinase and aspartate semialdehyde dehydrogenase from the rifamycin sv producing amycolatopsis mediterranei u32
Applied Microbiology and Biotechnology, 2000Co-Authors: Weiwen Zhang, Yunliu Yang, Weihong Jiang, Guoping Zhao, Jui Shen ChiaoAbstract:The operon encoding Aspartokinase and aspartate semialdehyde dehydrogenase was cloned and sequenced from rifamycin-SV-producing Amycolatopsis mediterranei U32 previously. In the present work, these two genes were introduced into the auxotrophic Escherichia coli strain CGSC5074 (ask−) and E. coli X6118 (asd −), respectively. The A. mediterranei U32 Aspartokinase and aspartate semialdehyde dehydrogenase genes can be functionally expressed in E. coli and the gene products are able to substitute for the E. coli enzymes. Histidine-tagged Aspartokinase and aspartate semialdehyde dehydrogenase were partially purified from E. coli cellular extracts and their kinetic characteristics were studied. Both Aspartokinase and aspartate semialdehyde dehydrogenase showed typical Michaelis-Menten type substrate saturation patterns. Aspartokinase has Km values of 3.4 mM for aspartate and 2.3 mM for ATP, while aspartate semialdehyde dehydrogenase has Km values of 1.25 mM for dl-aspartate semialdehyde and 0.73 mM for NADP, respectively. Aspartokinase was inhibited by l-threonine, l-lysine, and l-methionine, but not by l-isoleucine and diaminopimelate. Aspartate semialdehyde dehydrogenase was not inhibited by any of the end-product amino acids at a concentration of less than 5 mM. Hill plot analysis suggested that Aspartokinase was subject to allosteric control by l-threonine. Repression of both Aspartokinase and aspartate semialdehyde dehydrogenase gene transcription in A. mediterranei U32 by l-lysine, l-methionine, l-threonine, and l-isoleucine were found. The network of regulation of Aspartokinase and aspartate semialdehyde dehydrogenase in rifamycin SV-producing A. mediterranei U32 is presented.
