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A Kos J Kuijvenhoven K Wernars C J Bos H W van den Broek P H Pouwels C A van den Hondel 《Gene》1985,39(2-3):231-238
The Aspergillus niger trpC gene was isolated by complementation experiments with an Escherichia coli trpC mutant. Plasmid DNA containing the A. niger trpC gene transforms an Aspergillus nidulans mutant strain, defective in all three enzymatic activities of the trpC gene, to Trp+, indicating the presence of a complete and functional trpC gene. Southern blot analysis of DNA from these Trp+ transformants showed that plasmid DNA was present but that this DNA was not integrated at the site of the chromosomal trpC locus. The A. niger trpC gene was localized on the cloned fragment by heterologous hybridization experiments and sequence analysis. These experiments suggest that the organization of the A. niger trpC gene is identical to that of the analogous A. nidulans trpC and the Neurospora crassa trp-1 genes. 相似文献
3.
An evolutionary comparison of Acinetobacter calcoaceticus trpF with trpF genes of several organisms 总被引:4,自引:0,他引:4
The deduced amino acid sequence of Acinetobacter calcoaceticus N-(5'-phosphoribosyl) anthranilate isomerase (PRAI), which is coded by trpF, was compared with TrpF of Caulobacter crescentus, Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Neurospora crassa, and Aspergillus nidulans. Sixty percent of identical or similar amino acids were located in alpha/beta TIM (triose-phosphate isomerase) barrels and in residues important in substrate binding and catalysis. In addition, the analysis of trpF genes presented here supports a model by which fusion between separate trpC and trpF genes arose in some cases by in-frame deletions. 相似文献
4.
Forty single gene mutations in Chlamydomonas reinhardtii were isolated based on resistance to the compound 5'-methyl anthranilic acid (5-MAA). In other organisms, 5-MAA is converted to 5'-methyltryptophan (5-MT) and 5-MT is a potent inhibitor of anthranilate synthase, which catalyzes the first committed step in tryptophan biosynthesis. The mutant strains fall into two phenotypic classes based on the rate of cell division in the absence of 5-MAA. Strains with class I mutations divide more slowly than wild-type cells. These 17 mutations map to seven loci, which are designated MAA1 to MAA7. Strains with class II mutations have generation times indistinguishable from wild-type cells, and 7 of these 23 mutations map to loci defined by class I mutations. The remainder of the class II mutations map to 9 other loci, which are designated MAA8-MAA16. The maa5-1 mutant strain excretes high levels of anthranilate and phenylalanine into the medium. In this strain, four enzymatic activities in the tryptophan biosynthetic pathway are increased at least twofold. These include the combined activities of anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase, indoleglycerol phosphate synthetase and anthranilate synthase. The slow growth phenotypes of strains with class I mutations are not rescued by the addition of tryptophan, but the slow growth phenotype of the maa6-1 mutant strain is partially rescued by the addition of indole. The maa6-1 mutant strain excretes a fluorescent compound into the medium, and cell extracts have no combined anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase and indoleglycerol phosphate synthetase activity. The MAA6 locus is likely to encode a tryptophan biosynthetic enzyme. None of the other class I mutations affected these enzyme activities. Based on the phenotypes of double mutant strains, epistatic relationships among the class I mutations have been determined. 相似文献
5.
A host/vector expression system based on an Arxula adeninivorans Delta atrp1 gene disruption mutant has been constructed. For this purpose the ATRP1 gene encoding a phosphoribosyl anthranilate isomerase was isolated from the yeast A. adeninivorans and its genome locus was characterized. The Delta atrp1 mutant was generated applying an amplified DNA fragment containing the ALEU2m gene flanked by ATRP1 gene sequences of some 750 bp. The generated auxotrophic host strain was transformed with the plasmid pAL-ATRP1-amyA, which contains the ATRP1 gene as selection marker and the 25S rDNA for targeting. For expression assessment, the plasmid was equipped with an expression cassette consisting of the Bacillus amyloliquefaciens-derived amyA gene fused to the constitutive A. adeninivorans-derived TEF1 promoter and Saccharomyces cerevisiae-derived PHO5 terminator. Transformants contained a single chromosomal copy of the heterologous DNA and were found to be mitotically stable. In initial fermentation trials on a 200 ml shake flask scale maximal alpha-amylase product levels of ca. 300 nkat ml(-1) were observed after 72 h of cultivation with more than 95% of the recombinant alpha-amylase accumulated in the culture medium. 相似文献
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Auxotrophs of Acinetobacter calcoaceticus blocked in each reaction of the synthetic pathway from chorismic acid to tryptophan were obtained after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. One novel class was found to be blocked in both anthranilate and p-aminobenzoate synthesis; these mutants (trpG) require p-aminobenzoate or folate as well as tryptophan (or anthranilate) for growth. The loci of six other auxotrophic classes requiring only tryptophan were defined by growth, accumulation, and enzymatic analysis where appropriate. The trp mutations map in three chromosomal locations. One group contains trpC and trpD (indoleglycerol phosphate synthetase and phosphoribosyl transferase) in addition to trpG mutations; this group is closely linked to a locus conferring a glutamate requirement. Another cluster contains trpA and trpB, coding for the two tryptophan synthetase (EC 4.2.1.20) subunits, along with trpF (phosphoribosylanthranilate isomerase); this group is weakly linked to a his marker. The trpE gene, coding for the large subunit of anthranilate synthetase, is unlinked to any of the above. This chromosomal distribution of the trp genes has not been observed in other organisms. 相似文献
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Isolation and characterization of two tryptophan biosynthetic enzymes, indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, from Bacillus subtilis.
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S O Hoch 《Journal of bacteriology》1979,139(2):362-368
Two of the enzymes responsible for tryptophan biosynthesis in Bacillus subtilis have been extensively purified. These proteins are indole-3-glycerol phosphate synthase and N-(5'-phosphoribosyl) anthranilate isomerase. By comparison to the non-differentiating enteric bacteria in which these two enzymes are fused into a single polypeptide, the isolation of the indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase from B. subtilis has demonstrated that the two proteins are separate species in this organism. The two enzymes were clearly separable by anion-exchange chromatography without any significant loss of activity. Molecular weights were determined for both enzymes by gel filtration and sodium dodecyl sulfate-slab gel electrophoresis, and indicated that the indoleglycerol phosphate synthase is the slightly larger of the two proteins. The minimum molecular weight for indoleglycerol phosphate synthase was 23,500, and that for phosphoribosyl anthranilate isomerase was 21,800. Both enzymes have been examined as to conditions necessary to achieve maximal activity of their individual functions and to maintain that activity. 相似文献
9.
(Beta alpha)8-barrel proteins of tryptophan biosynthesis in the hyperthermophile Thermotoga maritima. 总被引:2,自引:0,他引:2
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To better understand the evolution of a key metabolic pathway, we have sequenced the trpCFBA gene cluster of the hyperthermophilic bacterium Thermotoga maritima. The genes were cloned by complementation in vivo of trp deletion strains of Escherichia coli. The new sequences, together with earlier findings, establish that the trp operon of T.maritima has the order trpE(G.D)CFBA, which might represent the ancestral organization of the tryptophan operon. Heterologous expression of the trp(G.D) and trpC genes in E.coli and N-terminal sequencing of their polypeptide products showed that their translation is initiated at the rate start codons TTG and ATC, respectively. Consequently, the N-terminus of the trp(G.D) fusion protein is 43 residues shorter than previously postulated. Amino acid composition and sequence analyses of the protein products of T.maritima trpC (indoleglycerol phosphate synthase), trpF (phosphoribosyl anthranilate isomerase) and trpA (alpha-subunit of tryptophan synthase) suggest that these thermostable (beta alpha)8-barrel proteins may be stabilized by additional salt bridges, compared with the mesostable forms. Another notable feature is the predicted lack of the N-terminal helix alpha 0 in the alpha-subunit of tryptophan synthase. 相似文献
10.
The trpC gene in the tryptophan biosynthetic pathway was isolated from an aflatoxigenic Aspergillus parasiticus by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase (PRAI) activity. The cloned gene complemented an E. coli trpC mutant deficient in indoleglycerolphosphate synthase (IGPS) activity as well as an Aspergillus nidulans mutant strain that was defective in all three enzymatic activities of the trpC gene (glutamine amidotransferase, IGPS, and PRAI), thus indicating the presence of a complete and functional trpC gene. The location and organization of the A. parasiticus trpC gene on the cloned DNA fragment were determined by deletion mapping and by hybridization to heterologous DNA probes that were prepared from cloned trpC genes of A. nidulans and Aspergillus niger. These experiments suggested that the A. parasiticus trpC gene encoded a trifunctional polypeptide with a functional domain structure organized identically to those of analogous genes from other filamentous fungi. The A. parasiticus trpC gene was expressed constitutively regardless of the nutritional status of the culture medium. This gene should be useful as a selectable marker in developing a DNA-mediated transformation system to analyze the aflatoxin biosynthetic pathway of A. parasiticus. 相似文献
11.
Cloning and characterization of the trpC gene from an aflatoxigenic strain of Aspergillus parasiticus. 总被引:5,自引:5,他引:0
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The trpC gene in the tryptophan biosynthetic pathway was isolated from an aflatoxigenic Aspergillus parasiticus by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase (PRAI) activity. The cloned gene complemented an E. coli trpC mutant deficient in indoleglycerolphosphate synthase (IGPS) activity as well as an Aspergillus nidulans mutant strain that was defective in all three enzymatic activities of the trpC gene (glutamine amidotransferase, IGPS, and PRAI), thus indicating the presence of a complete and functional trpC gene. The location and organization of the A. parasiticus trpC gene on the cloned DNA fragment were determined by deletion mapping and by hybridization to heterologous DNA probes that were prepared from cloned trpC genes of A. nidulans and Aspergillus niger. These experiments suggested that the A. parasiticus trpC gene encoded a trifunctional polypeptide with a functional domain structure organized identically to those of analogous genes from other filamentous fungi. The A. parasiticus trpC gene was expressed constitutively regardless of the nutritional status of the culture medium. This gene should be useful as a selectable marker in developing a DNA-mediated transformation system to analyze the aflatoxin biosynthetic pathway of A. parasiticus. 相似文献
12.
Partial Characterization of Phosphoribosyl Transferase, Phosphoribosyl Anthranilate Isomerase, and Indole Glycerol Phosphate Synthase from Serratia marcescens 总被引:1,自引:1,他引:0
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The molecular organization of the enzymes phosphoribosyl (PR) transferase, phosphoribosyl anthranilate (PRA) isomerase, and indole glycerol phosphate (InGP) synthase of the tryptophan biosynthetic pathway of Serratia marcescens was investigated and compared with that reported in other enteric bacteria. PRA isomerase and InGP synthase activities were found to reside in a single polypeptide chain, a situation analogous to that in Escherichia coli, Salmonella typhimurium, and Aerobacter aerogenes. This bifunctional enzyme was purified to near homogeneity. Its molecular weight was estimated to be 48,000. PR transferase was found unassociated with PRA isomerase and InGP synthase after gel filtration and ion-exchange chromatography. Whereas in other enteric organisms PR transferase has been reported to form an aggregate with anthranilate synthase, it is a distinct entity in S. marcescens. 相似文献
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Nucleotide sequence of Saccharomyces cerevisiae genes TRP2 and TRP3 encoding bifunctional anthranilate synthase: indole-3-glycerol phosphate synthase 总被引:30,自引:0,他引:30
H Zalkin J L Paluh M van Cleemput W S Moye C Yanofsky 《The Journal of biological chemistry》1984,259(6):3985-3992
Saccharomyces cerevisiae anthranilate synthase:indole-3-glycerol phosphate synthase is a multifunctional hetero-oligomeric enzyme encoded by genes TRP2 and TRP3. TRP2, encoding anthranilate synthase Component I, was cloned by complementation of a yeast trp2 mutant. The nucleotide sequence of TRP2 as well as that of TRP3 were determined. The deduced anthranilate synthase Component I primary structure from yeast exhibits only limited similarity to that of the corresponding Escherichia coli subunit encoded by trpE. On the other hand, yeast anthranilate synthase Component II and indole-3-glycerol phosphate synthase amino acid sequences from TRP3 are clearly homologous with the corresponding sequences of the E. coli trpG and trpC polypeptide segments and thereby establish the bifunctional structure of TRP3 protein. Based on comparisons of TRP3 amino acid sequence with homologous sequences from E. coli and Neurospora crassa, an 11-amino acid residue connecting segment was identified which fuses the trpG and trpC functions of the bifunctional TRP3 protein chain. These comparisons support the conclusion that the amino acid sequence of connectors in homologous multifunctional enzymes need not be conserved. Connector function is thus not dependent on a specific sequence. Nuclease S1 mapping was used to identify mRNA 5' termini. Heterogeneous 5' termini were found for both TRP2 and TRP3 mRNA. TRP2 and TRP3 5'-flanking regions were analyzed for sequences that might function in regulation of these genes by the S. cerevisiae general amino acid control system. The 9 base pair direct repeat (Hinnebusch, A.G., and Fink, G.R. (1983) J. Biol. Chem. 258, 5238-5247) and inverted repeats were identified in the 5'-flanking sequences of TRP2 and TRP3. 相似文献
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The genomic TRP1 gene from basidiomycete Flammulina velutipes was cloned by complementation of yeast Saccharomyces cerevisiae trp1 mutation. Sequencing analysis revealed that the TRP1 gene encoded a single protein consisting of three catalytic functional domains; glutamine amidotransferase, indole-3-glycerol phosphate synthase ) and N-(5'-phosphoribosyl) anthranilate isomerase, in order of NH2-glutamine amidotransferase-indole-3-glycerol phosphate synthase N-(5'-phosphoribosyl) anthranilate isomerase-COOH. The coding sequence of the TRP1 gene was interrupted by a single intron of 48 bases, the position and flanking sequences of which were highly homologous to those of basidiomycete Phanerochaete chrysosporium trpC. 相似文献
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Transformation in Penicillium chrysogenum 总被引:3,自引:0,他引:3
An auxotrophic mutant of Penicillium chrysogenum with a DNA rearrangement that affects the trpC region has been transformed to the Trp+ phenotype by using a plasmid that contains the trifunctional wild-type gene. A frequency of 40-80 transformants per microgram of input DNA was usually achieved. A low frequency of plasmid integration at the recipient mutated trpC gene was detected; however, most of the transformants integrated the plasmid DNA elsewhere into the genome. Some of the transformants contain multiple rearranged copies of the vector integrated in a tandem fashion. 相似文献
16.
Flora Sánchez Angeles Touriño Susana Traseira Agustín Pérez-Aranda Víctor Rubio Miguel A. Peñalva 《Molecular & general genetics : MGG》1986,205(2):248-252
Summary We cloned the Penicillium chrysogenum trpC gene from a genomic library by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase activity. The gene ecodes a 2.7 kb poly(A)+ RNA. We localized the gene by sequence analysis in a 2.9 kb DNA insert found in the smallest plasmid selected from the library. Sequence data strongly suggest that the organization of the gene is similar to that described in other Ascomycetes. We found that a DNA fragment which codes only for the carboxy-terminal protion of the polypeptide is sufficient for complementation of the E. coli trpC9830 mutation. 相似文献
17.
New approach to tryptophan production by Escherichia coli: genetic manipulation of composite plasmids in vitro. 总被引:6,自引:2,他引:6
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For the purpose of studying the production of L-tryptophan by Escherichia coli, the deletion mutants of the trp operon (trpAE1) were transformed with mutant plasmids carrying the trp operon whose anthranilate synthase and phosphoribosyl anthranilate transferase (anthranilate aggregate), respectively, had been desensitized to tryptophan inhibition. In addition to release of the anthranilate aggregate from the feedback inhibition required for plasmids such as pSC101 trp.I15, the properties of trp repression (trpR) and tryptophanase deficiency (tnaA) were both indispensable for host strains such as strain Tna (trpAE1 trpR tnaA). The gene dosage effects on tryptophan synthase activities and on production of tryptophan were assessed. A moderate plasmid copy number, approximately five per chromosome, was optimal for tryptophan production. Similarly, an appropriate release of the anthranilate aggregate from feedback inhibition was also a necessary step to ward off the metabolic anomaly. If the mutant plasmid pSC101 trp-I15 was further mutagenized (pSC101 trp.I15.14) and then transferred to Tna cells, an effective enhancement of tryptophan production was achieved. Although further improvement of the host-plasmid system is needed before commercial production of tryptophan can be realized by this means, a promising step toward this goal has been established. 相似文献
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Treatment of the multifunctional alpha 2 beta 2 anthranilate synthase complex of Neurospora crassa with elastase produced two fragments of the complex, one possessing anthranilate synthase activity and the other having both indole-3-glycerol phosphate (InGP) synthase and N-(5'-phosphoribosyl)anthranilate (PRA) isomerase activities. Sequencing the NH2 terminus of the InGP synthase-PRA isomerase fragment revealed that cleavage was between positions 237 and 238 of the beta-subunit within a segment of the polypeptide chain which links the glutamine-binding (G) domain with the InGP synthase-PRA isomerase domains. The fragment containing anthranilate synthase activity has a molecular weight of 98,000, as estimated by gel filtration, and is composed of an apparently intact alpha-subunit (70 kDa) associated with the G-domain fragment (29 kDa) derived from the beta-subunit. The alpha X G-domain complex was resistant to further degradation by elastase. When either the alpha 2 beta 2 complex or the alpha X G-domain complex was incubated with trypsin, the alpha-subunit was degraded to a 66-kDa alpha-fragment with reduced enzymatic activity, which was resistant to further cleavage. In contrast, incubation of alpha-subunit alone with either elastase or trypsin resulted in its complete degradation, indicating that association of the alpha-subunit with either G-domain or beta-subunit protected the alpha-subunit from this extensive degradation. A model for the anthranilate synthase complex is proposed in which the trifunctional beta-subunit forms a dimer by the self-association of the InGP synthase-PRA isomerase domains; the G-domain is connected to the InGP synthase-PRA isomerase domain by a relatively disordered region of the peptide chain which, in the alpha 2 beta 2 complex, remains susceptible to proteases; and neither alpha-subunit nor G-domain significantly self-associates. 相似文献
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Purification and characterization of the trifunctional beta-subunit of anthranilate synthase from Neurospora crassa 总被引:1,自引:0,他引:1
The trifunctional beta-subunit of anthranilate synthase complex of Neurospora crassa has been purified from a mutant which produces no detectable alpha-subunit. The isolated beta-subunit appeared to be a highly asymmetric dimer with a s20,w of 7.35 and an apparent molecular weight of 200,000 as determined by gel filtration on Sephacryl S-300 compared with a monomer molecular weight of approximately 84,000 Da as determined by sodium dodecyl sulfate-gel electrophoresis. The purified subunit was cleaved by elastase, trypsin, or chymotrypsin into fragments which retained the three enzyme activities. After elastase digestion, two active fragments were separated by gel filtration and ion exchange chromatography. A 30,000-Da fragment, which behaved as a monomer on gel filtration, interacted with free alpha-subunit to produce glutamine-dependent anthranilate synthase activity. A second 56,000-Da fragment, which behaved as an asymmetric dimer (apparent molecular weight 140,000) on gel filtration, retained both N-(5'-phosphoribosyl)anthranilate isomerase and indole-3-glycerol phosphate synthase activity. The failure to detect an NH2-terminal amino acid residue on either the intact beta-subunit or the 30,000-Da complementing fragment, while the 56,000-Da fragment possessed an NH2-terminal histidine residue, indicated that the complementing fragment was derived from the NH2-terminal sequence of the beta-subunit. 相似文献
20.
Víctor E Balderas-Hernández Andrea Sabido-Ramos Patricia Silva Natividad Cabrera-Valladares Georgina Hernández-Chávez José L Báez-Viveros Alfredo Martínez Francisco Bolívar Guillermo Gosset 《Microbial cell factories》2009,8(1):19-12