Anthranilate synthetase, an enzyme specified by the tryptophan operon of Escherichia coli: Comparative studies on the complex and the subunits

The properties of the anthranilate synthetase complex and its separated subunits were compared in catalyzing the anthranilate synthetase reaction, chorismate + l-glutamine or NH(4) (+) --> anthranilate, and the transferase reaction, anthranilate + 5'-phosphorylribosyl-1-pyrophosphate --> phosphoribosyl anthranilate. It is shown that anthranilate synthetase component I is activated by normal anthranilate synthetase component II, a component II(CRM) (CRM = immunologically cross-reacting material), and by a presumed fragment of component II produced by a deletion mutant. Significant differences between the complex and its subunits are demonstrated with respect to substrate affinity, thermostability, feedback inhibitor sensitivity, and activity in the presence of various divalent cations. Of particular interest are the findings that the transferase activity of component II is only inhibitable by l-tryptophan when the component is in the complex and that this inhibition does not appear to depend upon the feedback-sensitive site of component I.     

Overproduction, purification, and characterization of adenylosuccinate synthetase from Escherichia coli

Adenylosuccinate synthetase, encoded by the purA gene of Escherichia coli, catalyzes the first committed step toward AMP in the de novo purine biosynthetic pathway and plays an important role in the interconversion of purines. A 3.2-kb DNA fragment, which carries the purA gene, was cloned into the temperature-inducible, high-copy-number plasmid vector, pMOB45. Upon temperature induction, cells containing this plasmid produce adenylosuccinate synthetase at approximately 40 times the wild-type level. A scheme is presented for the purification of the overproduced adenylosuccinate synthetase to homogeneity in amounts sufficient for studies of its structure and mechanism. The wild-type and the overproduced adenylosuccinate synthetase enzyme preparations were judged to be identical by the following criteria. The amino acid sequence at the N-terminus of the overproduced enzyme proved identical to the corresponding sequence of the wild-type enzyme. Michaelis constants for both the wild-type and overproduced enzyme preparations were the same. And (iii) both proteins shared similar chromatographic behavior and the same mobility during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Results from size-exclusion chromatography and SDS-polyacrylamide gel electrophoresis suggest that adenylosuccinate synthetase exists as a dimer of identical, 48,000-Da, subunits.     

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.     

Escherichia coli phenylalanyl-tRNA synthetase operon: characterization of mutations isolated on multicopy plasmids

Plasmid pB1 carries the genes for threonyl-tRNA synthetase, phenylalanyl-tRNA synthetase, and translation initiation factor IF3. Strains carrying this plasmid overproduce phenylalanyl-tRNA synthetase about 100-fold. Spontaneous mutant plasmids were obtained which no longer caused the overproduction of the enzyme. Three classes of mutations were found. (i) Deletion mutations were found, some of which had the interesting property of fusing different genes together, e.g., putting phenylalanyl-tRNA synthetase under the control of the threonyl-tRNA synthetase promoter. (ii) Insertion mutations were found; one insertion in particular was studied. This insertion is located in front of the structural gene for phenylalanyl-tRNA synthetase and is shown to interrupt a cis-acting regulatory region. (iii) Mutations that showed no major change in DNA structure were found. One of these mutations is apparently purely structural, as it produces a small subunit of phenylalanyl-tRNA synthetase with a reduced molecular weight. This protein is less stable than the wild-type enzyme. These mutations represent useful tools to investigate how the phenylalanyl-tRNA synthetase operon is regulated.     

Escherichia coli DNA topoisomerase III: purification and characterization of a new type I enzyme

A new topoisomerase capable of relaxing negatively supercoiled DNA in Escherichia coli has been identified during chromatography on novobiocin-Sepharose. A simple and reproducible purification procedure is described to obtain this enzyme, called topoisomerase III (topo III), in a homogeneous form. The protein is a single polypeptide with a molecular weight of 74 000 +/- 2000 and is a type I topoisomerase, changing the linking number of DNA circles in steps of one. It is present in deletion strains lacking the topA gene and further differs from the well-studied topoisomerase I (omega protein; Eco topo I) in (1) its requirement for K+ in addition to Mg2+ to exhibit optimal activity and (2) its affinity to novobiocin-Sepharose. Positively supercoiled DNA is not relaxed during exposure to the enzyme. Topo III has no ATPase activity, and ATP does not show any discernible effect on the reduction of superhelical turns. The purified topoisomerase has no supercoiling activity and is unaffected by high concentrations of oxolinic acid and novobiocin in the relaxing reaction. Single-stranded DNA and spermidine strongly inhibit the topoisomerase activity.     

The proteins encoded by the rbs operon of Escherichia coli: I. Overproduction, purification, characterization, and functional analysis of RbsA.

The nucleotide-binding component of the high-affinity ribose transport system of Escherichia coli, RbsA, was overproduced from a T7-7 expression vector, and the protein was purified. Biochemical analyses of the purified protein indicated that the ATP analogues, 5'-FSBA and 8-azido ATP, covalently labeled the protein, a reaction that was inhibited by ATP, but not by GTP or CTP. The pure protein exhibited low-level ATPase activity with a K(m) of about 140 microM. Analyses of bacterial strains carrying chromosomal deletions of rbsA and other rbs genes suggested that RbsA is important for the chemotaxis function, a surprising result that was not anticipated from previous studies. However, an inconsistency between the several results from deletion strains raises questions regarding the interpretations of the in vivo data.     

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.     

Duplication-translocations of tryptophan operon genes in Escherichia coli

Mutants of Escherichia coli were selected in which a single mutational event had both relieved the polar effect of an early trpE mutation on trpB and simultaneously released the expression of trpB from tryptophan repression. The frequency at which these mutations appeared was roughly equal to the frequency of point mutations. In each of these mutants, the mutation increased the function of trpB and also increased the activity of some, but not all, of the other four tryptophan operon genes. Genetic analysis showed that the mutations were not located within the trp operon since in each case the parental trp operon could be recovered from the mutants. Each mutant was shown to carry a duplication of a trp operon segment translocated to a new position near the trp operon. Polarity is relieved since the trpB duplication-translocation is not in the same operon as the trpE polar mutation. The duplicated and translocated segments are fused to operons not regulated by tryptophan, so trpB function is no longer subject to tryptophan repression. The properties of the mutants indicate that the length of the duplicated segment and the position to which it is translocated differ in each of the seven mutants studied. The duplications are unstable, but the segregation pattern observed is not consistent with a single crossover model for segregation. That such duplication-translocation events generate a variety of new genetic arrangements at a frequency comparable with point mutations suggests they may play an important role in evolution.     

Cloning and expression of recombinant human pineal tryptophan hydroxylase in Escherichia coli: purification and characterization of the cloned enzyme.

The first step in the biosynthesis of melatonin in the pineal gland is the hydroxylation of tryptophan to 5-hydroxytryptophan. A cDNA of human tryptophan hydroxylase (TPH) was cloned from a library of human pineal gland and expressed in Escherichia coli. This cDNA sequence is identical to the cDNA sequence published from the human carcinoid tissue [1]. This human pineal hydroxylase gene encodes a protein of 444 amino acids and a molecular mass of 51 kDa estimated for the purified enzyme. Tryptophan hydroxylase from human brainstem exhibits high sequence homology (93% identity) with the human pineal hydroxylase. The recombinant tryptophan hydroxylase exists in solution as tetramers. The expressed human pineal tryptophan hydroxylase has a specific activity of 600 nmol/min/mg when measured in the presence of tetrahydrobiopterin and L-tryptophan. The enzyme catalyzes the hydroxylation of tryptophan and phenylalanine at comparable rates. Phosphorylation of the hydroxylase by protein kinase A or calmodulin-dependent kinase II results in the incorporation of 1 mol of phosphate/mol of subunit, but this degree of phosphorylation leads to only a modest (30%) increase in BH(4)-dependent activity when assayed in the presence of 14-3-3. Rapid scanning ultraviolet spectroscopy has revealed the formation of the transient intermediate compound, 4alpha-hydroxytetrahydrobiopterin, during the hydroxylation of either tryptophan or phenylalanine catalyzed by the recombinant pineal TPH.