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Proline excretion from proline overproducing strains of E. coli K12 has been studied as a model chemical production system. We have isolated proline overproducing mutants of E. coli and have shown that uncontrolled synthesis is not sufficient to cause excretion of this amino acid. An episomal mutation causing proline over production has been introduced into a series of otherwise isogenic strains that bear well defined, chromosomal lesions affecting the active uptake and catabolism of L-proline. A syntropism test reveals that L-proline is excreted by overproducing strains only if transport and/or catabolism are impaired. Dansyl derivatization and chromatographic analysis of culture supernatants shows that proline is the only amino acid excreted. Batch cultures of an excreting strain in an amino acid production medium yield culture supernatants containing 1 g proline/L, whereas no proline is detectable in supernatants derived from cultures of an overproducing strain with normal transport and catabolic activities. These data reveal that genetic lesions eliminating active uptake can be used to specifically enhance metabolite excretion. 相似文献
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The quaternary structure of Escherichia coli K12 aspartokinase II--homoserine dehydrogenase II has been examined. This multifunctional protein has a molecular weight Mr = 176000. It is composed of two subunits having the same molecular weight and the same charge. The results obtained from the examination of tryptic maps, the number and amino acid composition of cysteine-containing peptides and the uniqueness of the N-terminal sequence, strongly suggest that the 2 subunits are identical. The properties of aspartokinase II--homoserine dehydrogenase II can be compared to those of the much better known protein aspartokinase I--homoserine dehydrogenase I. 相似文献
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Triazole and azaserine resistant mutants of E. coli K12 affecting cysK gene coding for O-acetylserine sulphydrylase were isolated. The cysK gene in E. coli is located in the same region of chromosome as the cycK gene in Salmonella typhimurium. All azaserine and some triazole resistant mutants require cysteine for growth at a normal rate. The cysK mutants have reduced sulphate uptake. Stability and transfer by conjugation of triazole resistant phenotype were checked. Differences in sulphate metabolism between closely related organisms E. coli and S. typhimurium are discussed. 相似文献
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Homoserine kinase was purified to apparent homogeneity from a derepressed strain of Escherichia coli K12, using standard fractionation techniques. It is a dimer (Mr = 60000) composed of apparently identical polypeptide chains (Mr = 29000). Its amino acid composition and N-terminal sequence have been determined. L-Threonine is a competitive inhibitor of the substrate L-homoserine; this inhibition is straighforward and shows no sign of co-operativity. Evidence is presented that homoserine and threonine bind to the same site of this non-allosteric enzyme. The binding of homoserine and threonine can also be studied by difference spectroscopy; the latter studies reveal an unexpected effect of magnesium ions, which might be the basis for the unusual high Mg2+ requirement for optimal enzyme reaction. 相似文献
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Phospho-N-acetylmuramoyl-pentapeptide-transferase (UDP-N-acetyl-muramoyl-L-alanyl-D-gamma-glutamyl-L-lysyl-D-alanyl-D-alanine:undecaprenoid-alcohol-phosphate-phospho-N-acetylmuramoyl-pentapeptide-transferase, EC 2.7.8.13) was solubilized by repeated freezing and thawing of crude envelopes of Escherichia coli K12. The solubilized enzyme was partially purified by gel filtration and ion-exchange chromatography. This preparation contained small amounts of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol but no endogenous lipid substrate, C55-isoprenyl phosphate, could be detected. Some catalytic properties (exchange reaction) of the solubilized enzyme were compared to those of membrane-bound transferase. The transfer activity of the partially purified transferase was restored by the addition of an aqueous lipid dispersion. All the transferase activity was found to become incorporated into the liposomes. Preincubation of the transferase preparation with phospholipase A2 or D strongly reduce both exchange and transfer activity. This suggests that phospholipids sensitive to phospholipases are necessary for the enzymatic reaction. Different effects of some neutral detergents on the exchange activity were reported. 相似文献
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Regulation of pyruvate dehydrogenase activity in Escherichia coli K12 总被引:12,自引:0,他引:12
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The gene for a new outer membrane-associated protease, designated OmpP, of Escherichia coli has been cloned and sequenced. The gene encodes a 315-residue precursor protein possessing a 23-residue signal sequence. Including conservative substitutions and omitting the signal peptides, OmpP is 87% identical to the outer membrane protease OmpT. OmpP possessed the same enzymatic activity as OmpT. Immuno-electron microscopy demonstrated the exposure of the protein at the cell surface. Digestion of intact cells with proteinase K removed 155 N-terminal residues of OmpP, while the C-terminal half remained protected. It is possible that much of this N-terminal part is cell surface exposed and carries the enzymatic activity. Synthesis of OmpP was found to be thermoregulated, as is the expression of ompT (i.e., there is a low rate of synthesis at low temperatures) and, in addition, was found to be controlled by the cyclic AMP system. 相似文献
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gamma-Aminobutyraldehyde dehydrogenase from Escherichia coli K-12 has been purified and characterized from cell mutants able to grow in putrescine as the sole carbon and nitrogen source. The enzyme has an Mr of 195,000 +/- 10,000 in its dimeric form with an Mr of 95,000 +/- 1,000 for each subunit, a pH optimum at 5.4 in sodium citrate buffer, and does not require bivalent cations for its activity. Km values are 31.3 +/- 6.8 microM and 53.8 +/- 7.4 microM for delta-1-pyrroline and NAD+, respectively. An inhibitory capacity for NADH is also shown using the purified enzyme. 相似文献
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The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the pyruvate dehydrogenase component 总被引:27,自引:0,他引:27
The nucleotide sequence of a 3780-base-pair segment of DNA containing the aceE gene encoding the pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain-termination method. The aceE structural gene comprises 2655 base pairs (885 codons, excluding the initiation codon AUG), it is preceded by a good ribosome binding site and several potential RNA polymerase binding sites. Its polarity and location in the restriction map of the corresponding segment of DNA are consistent with it being the proximal gene in the ace operon, as defined in previous genetic and post-infection labelling studies. The relative molecular mass (99474), composition (885 amino acids), amino-terminal residue and carboxy-terminal sequence predicted from the nucleotide sequence are in excellent agreement with published information obtained from studies with the purified pyruvate dehydrogenase component (E1). The nucleotide sequence also contains a second gene (gene A) situated upstream of the aceE gene. It appears to be an independent gene containing 708 base pairs (236 codons) and encoding a weakly expressed product (protein A; Mr = 27049) of unknown function. 相似文献
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A polyadenylase, degrading specifically poly(A) sequences was isolated from Escherichia coli K12. The enzyme was purified about 850 times to practically electrophoretic homogeneity. It was free of poly(A) polymerase activity, as well as of the well known E. coli RNAases I and II. It is stimulated by bivalent cations like Mg2+ and Mn2+ and splits poly(A) to 3'-AMP and therefore it can be considered as an exonuclease. The enzyme does not degrade any other ribohomopolymer or RNA. 相似文献
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The alpha-galactosidase from Escherichia coli K12 总被引:6,自引:0,他引:6