Anthranilate synthetase, an enzyme specified by the tryptophan operon of Escherichia coli: purification and characterization of component I

A procedure employed in the purification of anthranilate synthetase component I of Escherichia coli is described. The purified component appears homogeneous by starch gel electrophoresis and by sedimentation analysis. A molecular weight of 60,000 was estimated by gel filtration of Sephadex G-100. This value is consistent with the molecular weight estimated from the sedimentation and diffusion coefficients. Purified anthranilate synthetase component I cannot use glutamine as substrate and thus has no activity in the reaction of chorismate + l-glutamine --> anthranilate; however, it is active when ammonium sulfate is provided as amino donor. Sucrose density gradient analyses showed that ammonium sulfate does not affect the sedimentation velocity of component I. The ultraviolet absorption and fluorescence spectra of the purified component indicated that it contains tryptophan. Peptide pattern and extract complementation evidence suggested that the protein is a single polypeptide chain. Enzyme activity measurements indicated that wild-type E. coli produces equimolar amounts of at least four of the five polypeptides specified by the operon. Purified anthranilate synthetase component I is inhibited by l-tryptophan.     

Overproduction of tryptophanyl-tRNA synthetase relieves transcription termination at the Escherichia coli tryptophan operon attenuator

Three spontaneous derivatives of Streptococcus faecium ATCC 9790, originally isolated as conditionally Triton X-100 detergent-resistant at 25 degrees C, displayed normal penicillin-induced rates of lysis at 37 degrees C but substantially reduced rates of lysis and killing at 25 degrees C. The addition of exogenous unsaturated fatty acids at 25 degrees C restored wild-type penicillin lysis rates.     

Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli.

Escherichia coli forms three permeases that can transport the amino acid tryptophan: Mtr, AroP, and TnaB. The structural genes for these permeases reside in separate operons that are subject to different mechanisms of regulation. We have exploited the fact that the tryptophanase (tna) operon is induced by tryptophan to infer how tryptophan transport is influenced by the growth medium and by mutations that inactivate each of the permease proteins. In an acid-hydrolyzed casein medium, high levels of tryptophan are ordinarily required to obtain maximum tna operon induction. High levels are necessary because much of the added tryptophan is degraded by tryptophanase. An alternate inducer that is poorly cleaved by tryptophanase, 1-methyltryptophan, induces efficiently at low concentrations in both tna+ strains and tna mutants. In an acid-hydrolyzed casein medium, the TnaB permease is most critical for tryptophan uptake; i.e., only mutations in tnaB reduce tryptophanase induction. However, when 1-methyltryptophan replaces tryptophan as the inducer in this medium, mutations in both mtr and tnaB are required to prevent maximum induction. In this medium, AroP does not contribute to tryptophan uptake. However, in a medium lacking phenylalanine and tyrosine the AroP permease is active in tryptophan transport; under these conditions it is necessary to inactivate the three permeases to eliminate tna operon induction. The Mtr permease is principally responsible for transporting indole, the degradation product of tryptophan produced by tryptophanase action. The TnaB permease is essential for growth on tryptophan as the sole carbon source. When cells with high levels of tryptophanase are transferred to tryptophan-free growth medium, the expression of the tryptophan (trp) operon is elevated. This observation suggests that the tryptophanase present in these cells degrades some of the synthesized tryptophan, thereby creating a mild tryptophan deficiency. Our studies assign roles to the three permeases in tryptophan transport under different physiological conditions.     

Escherichia coli phenylalanyl-tRNA synthetase operon: transcription studies of wild-type and mutated operons on multicopy plasmids

The construction of three lambda bacteriophages containing parts of the structural gene for threonyl-tRNA synthetase, thrS, and those for the two subunits of phenylalanyl-tRNA synthetases, pheS and pheT, is described. These phages were used as hybridization probes to measure the in vivo levels of mRNA specific to these three genes. Plasmid pB1 carries the three genes thrS, pheS, and pheT, and strains carrying the plasmid show enhanced levels of mRNA corresponding to these genes. Although the steady-state levels of threonyl-tRNA synthetase and phenylalanyl-tRNA synthetase produced by the presence of the plasmid differed by a factor of 10, their pulse-labeled mRNA levels were about the same. Mutant derivatives of pB1 were also analyzed. Firstly, a cis-acting insertion located before the structural genes for phenylalanyl-tRNA synthetase caused a major decrease in both pheS and pheT mRNA. Secondly, mutations affecting either structural gene pheS or pheT caused a reduction in the mRNA levels for both pheS and pheT. This observation suggests that autoregulation plays a role in the expression of phenylalanyl-tRNA synthetase.     

Temperature-sensitive repression of the tryptophan operon in Escherichia coli

Mutants of Escherichia coli exhibiting temperature-sensitive repression of the tryptophan operon have been isolated among the revertants of a tryptophan auxotroph, trpS5, that produces an altered tryptophanyl transfer ribonucleic acid (tRNA) synthetase. Unlike the parental strain, these mutants grew in the absence of tryptophan at high but not at low temperature. When grown at 43.5 C with excess tryptophan (repression conditions), they produced 10 times more anthranilate synthetase than when grown at 36 C or lower temperatures. Similar, though less striking, temperature-sensitivity was observed with respect to the formation of tryptophan synthetase. Transduction mapping by phage P1 revealed that these mutants carry a mutation cotransducible with thr at 60 to 80%, in addition to trpS5, and that the former mutation is primarily responsible for the temperature-sensitive repression. These results suggest that the present mutants represent a novel type of mutation of the classical regulatory gene trpR, which probably determines the structure of a protein involved in repression of the tryptophan operon. In agreement with this conclusion, tRNA of several trpR mutants was found to be normal with respect to its tryptophan acceptability. It was also shown that the trpS5 allele, whether present in trpR or trpR(+) strains, produced appreciably higher amounts of anthranilate synthetase than the corresponding trpS(+) strains under repression conditions. This was particularly true at higher temperatures. These results provide further evidence for our previous conclusion that tryptophanyl-tRNA synthetase is somehow involved in repression of this operon.     

Pressure effects on the association of the and 2 subunits of tryptophan synthetase from Escherichia coli and Salmonella typhimurium

Sucrose density gradient centrifugation was employed to study the association of the α and β₂ subunits of the enzyme tryptophan synthetase from Escherichia coli and Salmonella typhimurium. In both cases, the fully associated enzyme (α₂β₂) showed a sedimentation coefficient of 6.4 S, in agreement with the values reported by other workers for the E. coli enzyme. The substrate, l-serine, and the cofactor, pyridoxal phosphate, were required for complex formation in both cases. Generation of moderately high pressures by increasing the centrifuge speed from 39,000 rpm to 50,000 rpm was found to interfere with complex formation in both species at 5 °C. This effect was reversed by a temperature increase from 5 °C to 20 °C or by low concentrations of a nonpolar solvent, ethanol, at 5 °C. These results indicate that hydrophobic bonding plays an important role in the formation of the active tryptophan synthetase α₂β₂ complex. Monovalent and divalent cations also interfered with the formation of the α₂β₂ complex, indicating the possibility that ionic bonds are also involved.     

Comparative study of subunits of phenylalanyl-tRNA synthetase from Escherichia coli and Thermus thermophilus

FPLC separation of - and β-subunits of phenylalanyl-tRNA synthetases from E. coli MRE-600 and Thermus thermophilus HB8 has been carried out in the presence of urea. Native -subunits of both enzymes were primarily ₂-dimers and tended to aggregate. Most E. coli enzyme β-subunits were monomeric and only a small fraction was represented by β₂-dimers. All thermophilic β-subunits were β-dimers. It was shown that monomers and all forms of homologous subunits had no catalytic activity in tRNA^Phe aminoacylation. For the enzymes and their subunits, titration curves were obtained and isoelectric points were determined. The comparison of the relative surface charges indicated similarity of the surfaces of entire enzymes and the corresponding β-subunits. -Subunits displayed a distinctly different pH dependence of the surface charge. A spatial model of the oligomeric structure and a putative mechanism for its formation are discussed.