RESUMEN
The secondary metabolite phenazine-1-carboxylic acid (PCA) is an important component of the newly registered biopesticide Shenqinmycin. We used a combined method involving gene, promoter, and protein engineering to modify the central biosynthetic and secondary metabolic pathways in the PCA-producing Pseudomonas aeruginosa strain PA1201. The PCA yield of the resulting strain PA-IV was increased 54.6-fold via the following strategies: (1) blocking PCA conversion and enhancing PCA efflux pumping; (2) increasing metabolic flux towards the PCA biosynthetic pathway through the over-production of two DAHP synthases and blocking the synthesis of 21 secondary metabolites; (3) increasing the PCA precursor supply through the engineering of five chorismate-utilizing enzymes; (4) engineering the promoters of two PCA biosynthetic gene clusters. Strain PA-IV produced 9882 mg/L PCA in fed-batch fermentation, which is twice as much as that produced by the current industrial strain. Strain PA-IV was also genetically stable and comparable to Escherichia coli in cytotoxicity.
Asunto(s)
Vías Biosintéticas/genética , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , 3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , 3-Desoxi-7-Fosfoheptulonato Sintasa/genética , Animales , Corismato Mutasa/genética , Corismato Mutasa/metabolismo , Medios de Cultivo , Drosophila melanogaster , Fermentación , Familia de Multigenes/genética , Mutación/genética , Fenazinas/metabolismo , Regiones Promotoras Genéticas/genética , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidadRESUMEN
Previous research on Corynebacterium glutamicum revealed that 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DSCg, formerly DS2098) interacts with chorismate mutase (CMCg, formerly CM0819). In this study, we investigated the interaction by means of structure-guided mutation and enzymatic assays. Our results show that the interaction imparted a new mechanism for regulation of DAHP activity: In the absence of CMCg, DSCg activity was not regulated by prephenate, whereas in the presence of CMCg, prephenate markedly inhibited DSCg activity. Prephenate competed with the substrate phosphoenolpyruvate, and the inhibition constant (K i) was determined to be 0.945 mM. Modeling based on the structure of the complex formed between DAHP synthase and chorismate mutase of Mycobacterium tuberculosis predicted the interaction surfaces of the putative DSCg-CMCg complex. The amino acid residues and structural domains that contributed to the interaction surfaces were experimentally identified to be the (212)SPAGARYE(219) sequence of DSCg and the (60)SGGTR(64) loop and C-terminus ((97)RGKLG(101)) of CMCg.
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , Corismato Mutasa/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Regulación Enzimológica de la Expresión Génica , Ácidos Ciclohexanocarboxílicos/metabolismo , Ciclohexenos/metabolismo , Análisis Mutacional de ADN , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosfoenolpiruvato/metabolismo , Unión Proteica , Mapeo de Interacción de ProteínasRESUMEN
3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step in the shikimate pathway, the pathway responsible for the biosynthesis of the aromatic amino acids Trp, Phe, and Tyr. Unlike many other organisms that produce up to three isozymes, each feedback-regulated by one of the aromatic amino acid pathway end products, Mycobacterium tuberculosis expresses a single DAH7PS enzyme that can be controlled by combinations of aromatic amino acids. This study shows that the synergistic inhibition of this enzyme by a combination of Trp and Phe can be significantly augmented by the addition of Tyr. We used X-ray crystallography, mutagenesis, and isothermal titration calorimetry studies to show that DAH7PS from M. tuberculosis possesses a Tyr-selective site in addition to the Trp and Phe sites, revealing an unusual and highly sophisticated network of three synergistic allosteric sites on one enzyme. This ternary inhibitory response, by a combination of all three aromatic amino acids, allows a tunable response of the protein to changing metabolic demands.
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/antagonistas & inhibidores , 3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , Aminoácidos Aromáticos/biosíntesis , Mycobacterium tuberculosis/química , 3-Desoxi-7-Fosfoheptulonato Sintasa/genética , Regulación Alostérica/genética , Sitio Alostérico/genética , Aminoácidos Aromáticos/farmacología , Cristalografía por Rayos X , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Regulación hacia Arriba/genéticaRESUMEN
3-Deoxy-D-arabino-heptulonate-7-phosphate (DAHP) synthetase is one of the key enzymes, which catalyzes the first step in the aromatic amino acid biosynthetic pathway and yields the three amino acids tyrosine, tryptophan, and phenylalanine. In Escherichia coli (E. coli), three differently regulated DAHP synthases carry out the first regulated step in the aromatic amino acid biosynthetic pathway. The three DAHP synthases encoded by the genes aroG, aroF and aroH are inhibited by phenylalanine, tyrosine and tryptophan, respectively. In this work, the aroG gene was cloned and mutated by site-directed mutagenesis using splicing overlap extension PCR (SOE-PCR) technique. The feedback-resistant DAHP synthase encoded by aroG was achieved by replacing the residue Leu175 of aroG with Asp as to increase net carbon flow down the common pathway. SDS-PAGE which was used to access the protein expression level of aroGM showed the strain harbored mutated aroGM gene achieve over-expression compared to strain contain empty plasmid pET-28b (+).
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/genética , Escherichia coli K12/enzimología , Escherichia coli K12/genética , Mutagénesis Sitio-Dirigida , 3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , 3-Desoxi-7-Fosfoheptulonato Sintasa/aislamiento & purificación , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Vectores Genéticos/genéticaRESUMEN
3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS), (EC 2.5.1.54) catalyzes the first step of the shikimate pathway, the route for the biosynthesis of aromatic compounds in plants and microbes. In Actinosynnema pretiosum, the aroF gene (GenBank: AF056968.1) encodes DAHPS to condensate phosphoenolpyruvate (PEP) and d-erythrose 4-phosphate (E4P) to generate DAHP. In this study, a recombinant pET28a-aroF plasmid was constructed and A. pretiosum DAHPS was successfully expressed in soluble form by co-expression with chaperonins GroEL/GroES in Escherichia coli. The purification and kinetic characterization of the expressed protein were then investigated. The DAHPS originated from A. pretiosum demonstrated a pronounced substrate inhibition by PEP but was not sensitive to E4P. The purified enzyme was completely inactivated by EDTA but potently activated by several bivalent metal ions, especially Mn(2+) and Co(2+).
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , Actinomycetales/enzimología , Proteínas Bacterianas/biosíntesis , Chaperonina 10/biosíntesis , Chaperonina 60/biosíntesis , Escherichia coli , 3-Desoxi-7-Fosfoheptulonato Sintasa/química , 3-Desoxi-7-Fosfoheptulonato Sintasa/aislamiento & purificación , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Cationes Bivalentes/química , Quelantes/química , Ácido Edético/química , Expresión Génica , Cinética , Datos de Secuencia Molecular , Filogenia , Proteínas Recombinantes/biosíntesis , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , SolubilidadRESUMEN
Metabolic engineering has achieved encouraging success in producing foreign metabolites in a variety of hosts. However, common strategies for engineering metabolic pathways focus on amplifying the desired enzymes and deregulating cellular controls. As a result, uncontrolled or deregulated metabolic pathways lead to metabolic imbalance and suboptimal productivity. Here we have demonstrated the second stage of metabolic engineering effort by designing and engineering a regulatory circuit to control gene expression in response to intracellular metabolic states. Specifically, we recruited and altered one of the global regulatory systems in Escherichia coli, the Ntr regulon, to control the engineered lycopene biosynthesis pathway. The artificially engineered regulon, stimulated by excess glycolytic flux through sensing of an intracellular metabolite, acetyl phosphate, controls the expression of two key enzymes in lycopene synthesis in response to flux dynamics. This intracellular control loop significantly enhanced lycopene production while reducing the negative impact caused by metabolic imbalance. Although we demonstrated this strategy for metabolite production, it can be extended into other fields where gene expression must be closely controlled by intracellular physiology, such as gene therapy.
Asunto(s)
Proteínas Bacterianas , Carotenoides/biosíntesis , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Ingeniería Genética/métodos , Transactivadores , Factores de Transcripción , 3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , 3-Desoxi-7-Fosfoheptulonato Sintasa/genética , Anticarcinógenos/metabolismo , Antioxidantes/metabolismo , Isomerasas de Doble Vínculo Carbono-Carbono/biosíntesis , Isomerasas de Doble Vínculo Carbono-Carbono/genética , Proteínas de Unión al ADN/genética , Escherichia coli/metabolismo , Retroalimentación , Dosificación de Gen , Glucólisis , Hemiterpenos , Licopeno , Metabolismo/genética , Nitrógeno/deficiencia , Organofosfatos/metabolismo , Proteínas PII Reguladoras del Nitrógeno , Fosfoproteínas Fosfatasas/genética , Fosfotransferasas (Aceptores Pareados)/biosíntesis , Fosfotransferasas (Aceptores Pareados)/genética , Proteínas Quinasas/genética , RegulónRESUMEN
An overexpression system (pCR105) for DAHP synthase (Tyr) was constructed by cloning the aroF gene at the NdeI site of the pET-22b(+) translation vector, a plasmid expression vector that contains the T7 lac promoter. The enzyme was overexpressed, purified to > 90% purity (by SDS-polyacrylamide gel electrophoresis), and characterized. The protein was overexpressed at a level of 58% the total soluble cell protein (based on enzymatic activities). About 244 mg of pure enzyme was obtained from a 2-liter cell culture. So far, this is the highest yield reported for the isozyme DAHP synthase (Tyr). The enzyme showed a bell-shaped pH-activity profile, with a pH optimum at pH 7.0-7.5 and pK values of 6.10 and 8.92. Inhibition of the enzyme by tyrosine was specific with 50% inhibition observed at 9 microM tyrosine, pH 7.0. The specific activity of the enzyme increased with added metal and metal sensitivity increased with purity of the enzyme. Only substoichiometric amounts of Cu, Fe, and Zn were found in the pure enzyme and this result is consistent with sensitivity of the enzyme to added metal. Although treatment with EDTA inactivated the enzyme almost completely, the activity of the apoenzyme was restored to differing extents by a variety of metals including Mn2+, Cd2+, Co2+, Fe2+, Cu2+, Mg2+, and Zn2+. Both Fe2+ and Cu2+ only partially reactivated EDTA-treated enzyme. Reconstitution of EDTA-treated enzyme with either Cd2+ or Mn2+ gave 1 mol of metal per mole of enzyme monomer. KCN inactivated the enzyme to only 80% and added metals reactivated the CN-treated enzyme only to a small extent. These results confirm the importance of the metal in the enzymatic reaction.
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , 3-Desoxi-7-Fosfoheptulonato Sintasa/aislamiento & purificación , Escherichia coli/genética , Tirosina/metabolismo , 3-Desoxi-7-Fosfoheptulonato Sintasa/antagonistas & inhibidores , 3-Desoxi-7-Fosfoheptulonato Sintasa/química , 3-Desoxi-7-Fosfoheptulonato Sintasa/genética , Clonación Molecular , Activación Enzimática , Escherichia coli/química , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
We have shown recently that the first stage of the common site for the aromatic pathway in Pseudomonas aeruginosa, P. putida, P. fluorescens, P. acidovorans, and P. testosteroni is controlled by repression of 3-deoxy-D-arabinoheptulosenate-7-phosphate-synthase synthesis with phenylalanine and tyrosine. This work is devoted to the study of genetic control of the remaining six stages of the common site for aromatic pathway in representatives of Pseudomonas genus using auxotrophic and regulatory mutants it was shown that genes aroB, aroD, aroE, aroL, aroA, and aroC operate constitutively in P. putida, P. mendocina, P. marginata, and P. acidovorans.
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/genética , Genes Bacterianos , Pseudomonas/genética , 3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , Mutación , Fenilalanina/fisiología , Pseudomonas/enzimología , Proteínas Represoras/fisiología , Tirosina/fisiologíaRESUMEN
The activity of the first enzyme of aromatic path 3-deoxy-D-arabino-heptuloso-7-phosphate-synthase (DAHP-synthase) is regulated by retro-inhibition and is a subject of repression. Analysis of partially purified preparations of the enzyme has revealed three isoenzymes: DAHP-synthase-Tyr, DAHP-synthase-Trp and DAHP-synthase-Phe, each of them being regulated by a corresponding amino acid. DAHP-synthase-Phe is a dominant isoenzyme presenting 70% of the enzyme activity, 30% inhibition of which is possible by 7.0 mkM of phenylalanine. DAHP-synthase-Tyr and DAHP-synthase-Trp are minor isoenzymes (sharing 15% of enzyme activity each) and are controlled by tyrosine and tryptophane correspondingly. 50% of inhibition of activity is possible by adding 0.7 and 0.8 mkM of corresponding amino acid. Regulation of the enzyme synthesis was studied in the Trp-, Phe- and Tyr- mutants. The enzyme activity was registered under the conditions of limiting and surplus of each aromatic amino acid. The synthesis of DAHP-synthase in M. flagellatum KT is repressed by tryptophane and tyrosine decreasing the synthesis 18.8 and 15.6 fold.
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , Euryarchaeota/enzimología , Isoenzimas/biosíntesis , 3-Desoxi-7-Fosfoheptulonato Sintasa/antagonistas & inhibidores , 3-Desoxi-7-Fosfoheptulonato Sintasa/genética , 3-Desoxi-7-Fosfoheptulonato Sintasa/metabolismo , Aminoácidos/farmacología , Cromatografía DEAE-Celulosa , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Metanol/metabolismo , MutaciónRESUMEN
Regulation of synthesis of 3-deoxy-D-arabinoheptulose-7-phosphate-synthase (DAHP-synthase) was analysed in 12 strains of Pseudomonas belonging to 10 species. Repression of synthesis of DAHP-synthase was registered in half of strains under study, this being controlled by tyrosin and phenilalanine in P. aeruginosa PAO1 and PAT 2152, P. fluorescens BKMB 896, P. acidovorans BKMB 1251 and P. testosteroni BKMB 1241 and by only phenilalanine in P. maltophilia BKMB 30. In the rest of cases (in P. putida AC29, P. stutzeri BKMB 148, P. mendocina BKMB 1299, P. marginata BKMB 1298, P. vesicularis BKMB 974 and P. fluorescens BKMB 35) the enzyme was synthesized constitutively.
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , Pseudomonas/enzimología , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Fenilalanina/fisiología , Especificidad de la Especie , Tirosina/fisiologíaRESUMEN
The Salmonella typhimurium aroF gene, encoding the tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, was localized to a chromosomal PstI fragment by Southern blotting with an Escherichia coli aroF probe. This fragment was cloned by screening a plasmid library for complementation of an E. coli aroF mutant. The nucleotide sequence of S. typhimurium aroF was determined and compared with its E. coli homolog. The nucleotide sequences are 85.1% identical, and the corresponding amino acid sequences are 96.1% identical. The E. coli genes encoding the three DAHP synthase isoenzymes are evolutionarily more distant from one another than are the homologous aroF genes of E. coli and S. typhimurium. The S. typhimurium aroF regulatory region contains three imperfect palindromes, two upstream of the promoter and one overlapping the promoter, that are nearly identical to operators aroFo1, aroFo2, and TyrR box 1 of E. coli. The aroFo1 and aroFo2 sequences of the two organisms are each separated by three turns of the DNA helix with no sequence similarity. The 5' ends of the aroF transcripts for both organisms contain untranslated regions with potential stem-loop structures. Translational fusions of the aroF regulatory regions to lacZ were constructed and then introduced in single copy into the E. coli chromosome. beta-Galactosidase assays for tyrR-mediated regulation of aroF-lacZ expression revealed that the E. coli TyrR repressor apparently recognizes the operators of both organisms with about equal efficiency.
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/genética , Aldehído-Liasas/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Salmonella typhimurium/genética , 3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , 3-Desoxi-7-Fosfoheptulonato Sintasa/aislamiento & purificación , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Colifagos/genética , Escherichia coli/enzimología , Genotipo , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Mapeo Restrictivo , Salmonella typhimurium/enzimología , Homología de Secuencia de Ácido Nucleico , Tirosina/farmacologíaRESUMEN
The effects of a cell wall fraction of the fungus Phytophthora megasperma on the enzymatic activities of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15) in extracts of cultured parsley cells (Petroselinum crispum) were examined. The specific activity of a plastidic form of DAHP synthase, designated DS-Mn by Ganson et al. [Ganson, R. J., d'Amato, T. A. & Jensen, R. A. (1986) Plant Physiol. 82, 203-210], was increased 2- to 3-fold in extracts of treated cells, with maximum induction occurring with 60 micrograms of fungal elicitor per ml after 6-8 hr. The cytosolic form of DAHP synthase, DS-Co, was unaffected by fungal elicitor. In the same experiments, phenylalanine ammonia-lyase activity (EC 4.3.1.5) increased, while no effect on isoforms of chorismate mutase (EC 5.4.99.5) was observed. The subcellular localization of the two DAHP synthase isoforms in parsley was confirmed by differential centrifugation. Prior treatment of cultures with actinomycin D or cycloheximide prevented the increase in DS-Mn activity, indicating transcriptional regulation.
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , Aldehído-Liasas/biosíntesis , Quitridiomicetos/fisiología , Proteínas Fúngicas/farmacología , Phytophthora/fisiología , Plantas/enzimología , Células Cultivadas , Cicloheximida/farmacología , Dactinomicina/farmacología , Inducción Enzimática , Proteínas Fúngicas/aislamiento & purificación , Cinética , Especificidad por SustratoRESUMEN
The ARO3 gene encodes one of two 3-deoxy-D-arabino-heptulosonate-7-phosphate isoenzymes in Saccharomyces cerevisiae catalyzing the first step in the biosynthesis of aromatic amino acids. The ARO3-encoded 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) is feedback inhibited by phenylalanine; its isoenzyme, the ARO4 gene product, is inhibited by tyrosine. Both genes ARO3 and ARO4 are strongly regulated under the general control regulatory system. Cells carrying only one intact isogene are phenotypically indistinguishable from a wild-type strain when grown on minimal medium. The complete functional ARO3 promoter comprises 231 base pairs and contains only one TGACTA binding site for the general control activator protein GCN4. Mutating this element to TTACTA inhibits binding of GCN4 and results in a decreased basal level of ARO3 gene product and slow growth of a strain defective in its isogene ARO4. In addition, ARO3 gene expression cannot be elevated under amino acid starvation conditions. An ARO3 aro4 strain with gcn4 genetic background has the same phenotype of low ARO3 gene expression and slow growth. The amount of GCN4 protein present in repressed wild-type cells therefore seems to contribute to a basal level of ARO3 gene expression. The general control activator GCN4 has thus two functions: (i) to maintain a basal level of ARO3 transcription (basal control) in the presence of amino acids and (ii) to derepress the ARO3 gene to a higher transcription rate under amino acid starvation (general control).
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , Aldehído-Liasas/biosíntesis , ADN Polimerasa I/biosíntesis , Proteínas Fúngicas/fisiología , Genes Fúngicos , Genes Reguladores , Regiones Promotoras Genéticas , Proteínas Quinasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción/fisiología , 3-Desoxi-7-Fosfoheptulonato Sintasa/genética , ADN/análisis , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Isoenzimas/biosíntesis , Isoenzimas/genética , Mutación , Plásmidos , Saccharomyces cerevisiae/enzimología , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The synthesis of 3-deoxy-D-arabinoheptulosonate-7-phosphate-synthase has been shown to be repressed by tyrosine and phenylalanine in the cells of facultative methylotrophic bacteria Pseudomonas sp. M. Activity of the enzyme is subjected to allosteric inhibition by tyrosine, tryptophane, anthranylate and phenylpyruvate.
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , Aldehído-Liasas/biosíntesis , Pseudomonas/enzimología , 3-Desoxi-7-Fosfoheptulonato Sintasa/antagonistas & inhibidores , Regulación Alostérica , Aminoácidos/farmacología , Cromatografía DEAE-Celulosa , Mutación , Pseudomonas/genéticaRESUMEN
Strains of Escherichia coli K-12 in which the transcription of lacZ is initiated from the tyrR promoter have been constructed by use of the Mu d (Apr lac) phage of Casadaban and Cohen (Proc. Natl. Acad. Sci. U.S.A. 76:4530-4533, 1979). These strains have been used to examine the regulation of expression from the tyrR promoter, with the synthesis of beta-galactosidase used as an index of expression. The specific activity of beta-galactosidase fell to 51% upon introduction of lambda (Tn10) tyrR+; to 39% upon introduction of F123, an F-prime carrying tyrR+; to 29% upon introduction of pMU309, a derivative of the plasmid RP4 carrying tyrR+; and to 13.6% upon introduction of pMU352, a derivative of the multicopy plasmid pBR322 carrying tyrR+. These results indicate that the tyrR gene product interacts with its own promoter-operator region, decreasing synthesis of beta galactosidase in the tyrR::Mu d (Apr lac) strains. The increasing extent of repression of beta-galactosidase synthesis with increasing tyrR+ gene dosage was accompanied by increasing repression of the synthesis of tyrosine- and phenylalanine-repressible 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetases. The interaction of the repressor with tyrRo appears unusual in the sense that aporepressor alone is probably one of the repressing species. The levels of beta-galactosidase synthesized in the tyrR::Mu d (Apr lac) strains indicate that tyrR has a relatively efficient promoter, the maximum levels representing on the order of a relatively efficient promoter, the maximum levels representing on the order of 1,000 monomers of beta-galactosidase per cell in the tyrR strain and about 500 monomers in the tyrR+ haploid strain.
Asunto(s)
Escherichia coli/genética , Regulación de la Expresión Génica , Genes Reguladores , Operón , Proteínas Represoras/genética , Factores de Transcripción/genética , Tirosina/biosíntesis , 3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , ADN Recombinante , beta-Galactosidasa/biosíntesisRESUMEN
Homogeneous preparations of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate lyase (pyruvate phosphorylating), EC 4.1.2.15] isolated as the enzyme-phosphoenolpyruvate complex from Escherichia coli are shown by atomic absorption analysis to contain approximately one mole of iron per mole of native enzyme. No cobalt was found, in contrast to suggestions of earlier workers. Pure enzyme preparations show a unique absorption maximum around 350 nm with an epsilon value of about 3500 M-1cm-1. The 350-nm band as well as the enzyme activity is lost when the enzyme is denatured with guanidine-hydrochloride, or when phosphoenolpyruvate, the first substrate to bind to the enzyme, is totally removed from the enzyme by incubation with an excess of erythrose 4-phosphate, the second substrate to bind to the enzyme. The iron remains bound to the enzyme when phosphoenolpyruvate is removed from the enzyme-phosphoenolpyruvate complex.
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , Aldehído-Liasas/biosíntesis , Hierro/metabolismo , Quelantes , Escherichia coli , Espectrofotometría AtómicaRESUMEN
A comparison was made of the repressibility of certain enzymes in the tyrosine, methionine, and lysine biosynthetic pathways in wild-type Salmonella typhimurium and a hisT mutant. The results show that (i) tyrosine represses the synthesis of the tyrosine-sensitive 3-deoxy-D-arabino-heptulsonic acid 7-phosphate synthetase and the tyrosine aminotransferase to the same extent in a hisT mutant as in wild type and (ii) there is no detectable alteration in the extent to which methionine represses O-succinylhomoserine synthetase or in the extent to which lysine represses the lysine-sensitive beta-aspartokinase as a result of the hisT mutation.
Asunto(s)
Lisina/biosíntesis , Metionina/biosíntesis , Mutación , Salmonella typhimurium/metabolismo , Tirosina/biosíntesis , 2-Isopropilmalato Sintasa/biosíntesis , 3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , Acetiltransferasas/biosíntesis , Aspartato Quinasa/biosíntesis , Represión Enzimática , Histidinol-Fosfatasa/biosíntesis , Lisina/farmacología , Metionina/farmacología , Salmonella typhimurium/enzimología , Tirosina/farmacología , Tirosina Transaminasa/biosíntesisRESUMEN
Synthesis of five of the enzymes of the common pathway of aromatic biosynthesis has been shown to be unaffected by either the aromatic amino acids--the product of the first reaction (3-deoxy-D-arabinoheptulosonic acid-7-phosphate) or the product of the last reaction (chorismate)--or by the state of regulator gene loci tyrR. On the other hand, the rate of synthesis of these enzymes, and of several other enzymes for which repression control was inactive because of mutations in relevant regulator genes, was found to change with growth rate. These changes were found to correlate at faster growth rates than those observed in glucose minimal medium with the alterations in the relative frequencies of the corresponding structural genes which occur at these growth rates. It was found that when wild-type cells were grown at these faster growth rates in medium lacking the aromatic amino acids, complete derepression of the tyrosine-inhibitable 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase occurred, in strong contrast to the situation when wild-type cells are grown in glucose minimal medium.
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , Oxidorreductasas de Alcohol/biosíntesis , Aldehído-Liasas/biosíntesis , Escherichia coli/enzimología , Genes Reguladores , Hidroliasas/biosíntesis , Fosfotransferasas/biosíntesis , Ácido Corísmico/farmacología , Inducción Enzimática , Represión Enzimática , Escherichia coli/crecimiento & desarrollo , Genes , CinéticaRESUMEN
Several types of 4-fluorophenylalanine resistant mutants were isolated. In one type of mutant DAHP synthetase (tyr) and prephenate dehydrogenase were coordinately derepressed. The mutation was linked to aroF and tyrA and was cis- dominant by merodiploid analysis, thus confirming that it is an operator constitutive mutation (tyrOc). A second type of mutation showed highly elevated levels of tyrosine pathway enzymes which were not repressed by L-tyrosine. It was unlinked to tyrA and aroF, and was trans-recessive in merodiploids. These properties were attributed to a mutation in a regulator gene, tyrR (linked to pyr F), that resulted in altered or non-functional aporepressor. Hence tyrO, tyrA, and aroF constitute an operon regulated by tyrR. In a third type of mutation chorismate mutase P-prephenate dehydratase was highly elevated. It was not linked to pheA, was located in the 95--100 min region of the Salmonella chromosome, and was recessive to the wild type gene in merodiploids. A mutation was, therefore, indicated in a regulatory gene, pheR, which specified an aporepressor for regulating pheA. DAHP synthetase (phe), specified by aroG, was not regulated by pheR, but was derepressed in one of the tyrR mutants, suggesting that as in Escherichia coli tyrR may regulate DAHP synthetase(phe) and DAHP synthetase (tyr) with the same aporepressor. A novel mutation in chorismate mutase is described.