Structure and regulation of the anthranilate synthase genes in Pseudomonas aeruginosa: I. Sequence of trpG encoding the glutamine amidotransferase subunit

We have determined the DNA sequence of the distal 148 codons of trpE and all of trpG in Pseudomonas aeruginosa. These genes encode, respectively, the large and small (glutamine amidotransferase) subunits of anthranilate synthase, the first enzyme in the tryptophan synthetic pathway. The sequenced region of trpE is homologous with the distal portion of E. coli and Bacillus subtilis trpE, whereas the trpG sequence is homologous to the glutamine amidotransferase subunit genes of a number of bacterial and fungal anthranilate synthases. The two coding sequences overlap by 23 bp. Codon usage in these Pseudomonas genes shows a marked preference for codons ending in G or C, thereby resembling that of trpB, trpA, and several other chromosomal loci from this species and others with a high G + C content in their DNA. The deduced amino acid sequence for the P. aeruginosa trpG gene product differs to a surprising extent from the directly determined amino acid sequence of the glutamine amidotransferase subunit of P. putida anthranilate synthase (Kawamura et al. 1978). This suggests that these two proteins are encoded by loci that duplicated much earlier in the phylogeny of these organisms but have recently assumed the same function. We have also determined 490 bp of DNA sequence distal to trpG but have not ascertained the function of this segment, though it is rich in dyad symmetries.      

Evolutionary differences in chromosomal locations of four early genes of the tryptophan pathway in fluorescent pseudomonads: DNA sequences and characterization of Pseudomonas putida trpE and trpGDC.

Pseudomonas putida possesses seven structural genes for enzymes of the tryptophan pathway. All but one, trpG, which encodes the small (beta) subunit of anthranilate synthase, have been mapped on the circular chromosome. This report describes the cloning and sequencing of P. putida trpE, trpG, trpD, and trpC. In P. putida and Pseudomonas aeruginosa, DNA sequence analysis as well as growth and enzyme assays of insertionally inactivated strains indicated that trpG is the first gene in a three-gene operon that also contains trpD and trpC. In P. putida, trpE is 2.2 kilobases upstream from the trpGDC cluster, whereas in P. aeruginosa, they are separated by at least 25 kilobases (T. Shinomiya, S. Shiga, and M. Kageyama, Mol. Gen. Genet., 189:382-389, 1983). The DNA sequence in P. putida shows an open reading frame on the opposite strand between trpE and trpGDC; this putative gene was not characterized. Evidence is also presented for sequence similarities in the 5' untranslated regions of trpE and trpGDC in both pseudomonads; the function of these regions is unknown, but it is possible that they play some role in regulation of these genes, since all the genes respond to repression by tryptophan. The sequences of the anthranilate synthase genes in the fluorescent pseudomonads resemble those of p-aminobenzoate synthase genes of the enteric bacteria more closely than the anthranilate synthase genes of those organisms; however, no requirement for p-aminobenzoate was found in the Pseudomonas mutants created in this study.     

Structure and regulation of the anthranilate synthase genes in Pseudomonas aeruginosa: II. Cloning and expression in Escherichia coli

The genes for the large and small subunits of anthranilate synthase (trpE and trpG, respectively) have been cloned from Pseudomonas aeruginosa PAC174 into E. coli by R-prime formation with the broad-host- range plasmid R68.44. Sequential subcloning into plasmid vectors reduced the active Pseudomonas DNA fragment to a length of 3.1 kb. We obtained evidence that this region contains the promoter for its own expression and retains a vestigial regulatory response to tryptophan scarcity or excess.      

Molecular cloning and nucleotide sequence of Thermus thermophilus HB8 trpE and trpG

The trpE gene of Thermus thermophilus HB8 was cloned by complementation of an Escherichia coli tryptophan auxotroph. The E. coli harboring the cloned gene produced the anthranilate synthase I, which was heat-stable and enzymatically active at higher temperature. The nucleotide sequence of the trpE gene and its flanking regions was determined. The trpE gene was preceded by an attenuator-like structure and followed by the trpG gene, with a short gap between them. No other gene essential for tryptophan biosynthesis was observed after the trpG gene. The amino-acid sequences of the T. themophilus anthranilate synthase I and II deduced from the nucleotide sequence were compared with those of other organisms.     

Nucleotide sequence and analysis of a gene encoding anthranilate synthase component I in Spirochaeta aurantia.

A Spirochaeta aurantia DNA fragment containing the trpE gene and flanking chromosomal DNA was cloned, and the sequence of the trpE structural gene plus 870 bp upstream and 1,257 bp downstream of trpE was determined. The S. aurantia trpE gene codes for a polypeptide of 482 amino acid residues with a predicted molecular weight of 53,629 that showed sequence similarity to TrpE proteins from other organisms. The S. aurantia TrpE polypeptide is not more closely related to the other published spirochete TrpE sequence (that of Leptospira biflexa) than to TrpE polypeptides of other bacteria. Two additional complete open reading frames and one partial open reading frame were identified in the sequenced DNA. One of the complete open reading frames and the partial open reading frame are upstream of trpE and are encoded on the DNA strand opposite that containing trpE. The other open reading frame is downstream of trpE and on the same DNA strand as trpE. On the basis of the results of a protein sequence data base search, it appears that trpE is the only tryptophan biosynthesis gene in the sequenced DNA. This is in contrast to L. biflexa, in which trpE is separated from trpG by only 64 bp.     

DNA sequences and characterization of four early genes of the tryptophan pathway in Pseudomonas aeruginosa.

Two pairs of related but easily distinguishable genes for the two subunits of anthranilate synthase have been identified in Pseudomonas aeruginosa. These were cloned, sequenced, inactivated in vitro by insertion of an antibiotic resistance cassette, and returned to the P. aeruginosa chromosome, replacing the wild-type gene. Gene replacement implicated only one of the pairs in tryptophan biosynthesis. This report describes the cloning and sequencing of the tryptophan-related gene pair, designated trpE and trpG, and presents experiments implicating their gene products in tryptophan production. DNA sequence analysis as well as growth and enzyme assays of insertionally inactivated strains indicated that trpG is the first gene in a three-gene operon that also includes trpD and trpC. Complementation of Trp auxotrophs by R-prime plasmids (T. Shinomiya, S. Shiga, and M. Kageyama, Mol. Gen. Genet., 189:382-389, 1983) has shown that a large cluster of pyocin R2 genes is flanked at one end by trpE and the other end by trpDC; the physical map that was obtained shows the distance between trpE and trpDC to be about 25 kilobases. Our restriction map of the trpE and trpGDC regions agrees with data presented by Shinomiya et al.     

Genetic control of tryptophan hypersynthesis in regulatory mutants of Pseudomonas putida

The 6-fluorotryptophan resistant MR1 mutant was obtained from Pseudomonas putida M30 (Tyr- Phe-) strain. The mutant was able to excrete tryptophan (60 micrograms/ml) and has derepressed aroF gene encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase. The mutation isolated was identified as aroR with the help of cloning early aroF gene of P. putida. On the next step of selection, regulatory mutant MR2 was obtained producing 240 micrograms/ml of tryptophan. The MR2 has derepressed unlinked trpE and trpDC genes and represents a mutant of the trpR type. Expression of the trpE gene of P. putida MR2 weakened in the presence of tryptophan excess in the medium, which points to attenuation of this gene. From the prototrophic variant of P. putida MR2 the MRP3 mutant producing 850 micrograms/ml of tryptophan was obtained. This mutant was characterized by twofold increase in the activity of the anthranilate synthase encoded by the trpE gene. The assay of the activity of tryptophanyl-tRNA synthase in P. putida MRP3 demonstrated that the mutant has TrpS+ phenotype.     

Biochemical genetics of tryptophan synthesis in Pseudomonas acidovorans.

Sixty independent tryptophan auxotrophs of Pseudomonas acidovorans were isolated and characterized for nutritional response to intermediates of the pathway, accumulation of intermediates, and levels of tryptophan-synthetic enzymes. Mutants for each of the seven proteins catalyzing the five steps of tryptophan synthesis were obtained. Transductional analysis established three unlinked chromosomal regions: trpE, trpGDC, and trpFBA. The order of the genes within the two clusters was not determined. The levels and enzymatic activities of wild-type and mutant strains indicated that trpE and trpGDC were repressed by tryptophan. In contrast, trpFBA was not derepressed significantly by starvation for tryptophan. The trpG mutants had an additional requirement for p-aminobenzoate, which suggested that anthranilate synthase subunit II also served as glutamine-binding protein in the analogous reaction catalyzed by p-aminobenzoate synthase. In addition, trpD mutants revealed the ability of P. acidovorans to degrade anthranilate via the beta-ketoadipate pathway.     

Molecular cloning of the genes for anthranilate synthetase from Streptomyces venezuelae ISP 5230

Fragments of genomic DNA from Streptomyces venezuelae ISP5230 were cloned in the Escherichia coli expression vector pTZ18R and the plasmids were used to transform E. coli JA194 (trpE). The transformants included a prototrophic strain containing a recombinant plasmid, pDQ181, with an approximately 6.8-kb insert. Subcloning located the trpE-complementing DNA in a 2.4-kb segment. Transformation of E. coli ED23 (lacking both trpE and trpG functions) with plasmids containing the 2.4-kb DNA segment gave prototrophic strains exhibiting both the ASI and ASII activities of anthranilate synthetase. The results indicated that trpE and trpG are clustered in S. venezuelae. Regions hybridizing to the pDQ181 insert were present in the genomic DNA of other streptomycetes.     

Nucleotide sequence of Saccharomyces cerevisiae genes TRP2 and TRP3 encoding bifunctional anthranilate synthase: indole-3-glycerol phosphate synthase

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.     

Molecular analysis of genes encoding phenazine biosynthesis in the biological control bacterium Pseudomonas aureofaciens 30–84

Abstract The DNA sequence of five contiguous open reading frames encoding enzymes for phenazine biosynthesis in the biological control bacterium Pseudomonas aureofaciens 30–84 was determined. These open reading frames were named phzF, phzA, phzB, phzC and phzD . Protein PhzF is similar to 3-deoxy-D-arabino-heptulosonate-7-phosphate synthases of solanaceous plants. PhzA is similar to 2,3-dihydro-2,3-dihydroxybenzoate synthase (EntB) of Escherichia coli . PhzB shares similarity with both subunits of anthranilate synthase and the phzB open reading frame complemented an E. coli trpE mutant deficient in anthranilate synthase activity. Although phzC shares little similarity to known genes, its product is responsible for the conversion of phenazine-1-carboxylic acid to 2-hydroxy-phenazine-1-carboxylic acid. PhzD is similar to pyridoxamine phosphate oxidases. These results indicate that phenazine biosynthesis in P. aureofaciens shares similarities with the shikimic acid, enterochelin, and tryptophan biosynthetic pathways.     

Constitutively active glutaminase variants provide insights into the activation mechanism of anthranilate synthase

The glutamine amidotransferase (GATase) family comprises enzyme complexes which consist of glutaminase and synthase subunits that catalyze in a concerted reaction the incorporation of nitrogen within various metabolic pathways. An important feature of GATases is the strong stimulation of glutaminase activity by the associated synthase. To understand the mechanism of this tight activity regulation, we probed by site-directed mutagenesis four residues of the glutaminase subunit TrpG from anthranilate synthase that are located between the catalytic Cys-His-Glu triad and the synthase subunit TrpE. In order to minimize structural perturbations induced by the introduced exchanges, the amino acids from TrpG were substituted with the corresponding residues of the closely related glutaminase HisH from imidazole glycerol phosphate synthase. Steady-state kinetic characterization showed that, in contrast to wild-type TrpG, two TrpG variants with single exchanges constitutively hydrolyzed glutamine in the absence of TrpE. A reaction assay performed with hydroxylamine as a stronger nucleophile replacing water and a filter assay with radiolabeled glutamine indicated that the formation of the thioester intermediate is the rate-limiting step of constitutive glutamine hydrolysis. Molecular dynamics simulations with wild-type TrpG and constitutively active TrpG variants suggest that the introduced amino acid exchanges result in a distance reduction between the active site Cys-His pair, which facilitates the deprotonation of the sulfhydryl group of the catalytic cysteine and thus enables its nucleophilic attack onto the carboxamide group of the glutamine side chain. We propose that native TrpG in the anthranilate synthase complex is activated by a similar mechanism.     

Identification and nucleotide sequence of the Leptospira biflexa serovar patoc trpE and trpG genes.

Leptospira biflexa is a representative of an evolutionarily distinct group of eubacteria. In order to better understand the genetic organization and gene regulatory mechanisms of this species, we have chosen to study the genes required for tryptophan biosynthesis in this bacterium. The nucleotide sequence of the region of the L. biflexa serovar patoc chromosome encoding the trpE and trpG genes has been determined. Four open reading frames (ORFs) were identified in this region, but only three ORFs were translated into proteins when the cloned genes were introduced into Escherichia coli. Analysis of the predicted amino acid sequences of the proteins encoded by the ORFs allowed us to identify the trpE and trpG genes of L. biflexa. Enzyme assays confirmed the identity of these two ORFs. Anthranilate synthase from L. biflexa was found to be subject to feedback inhibition by tryptophan. Codon usage analysis showed that there was a bias in L. biflexa towards the use of codons rich in A and T, as would be expected from its G + C content of 37%. Comparison of the amino acid sequences of the trpE gene product and the trpG gene product with corresponding gene products from other bacteria showed regions of highly conserved sequence.