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PubMed1991

Nucleotide sequence of trpE, anthranilate synthase I gene, of Bacillus caldotenax.

trpE gene of Bacillus caldotenax was cloned by complementation of an Escherichia coli tryptophan auxotroph. The trpE encoding a 508 amino acid polypeptide is followed by trpD. S1-nuclease mapping of a trpE in vivo transcript suggested that a promoter preceding the trpE was utilized by E. coli. The deduced amino acid sequence of anthranilate synthase I (ASI) was compared with that of five other bacterials ASIs.

Amino Acid Sequence↗
PubMed1991

Identification of amino acid residues involved in feedback regulation of the anthranilate synthase complex from Salmonella typhimurium. Evidence for an amino-terminal regulatory site.

The anthranilate synthase-phosphoribosyl transferase complex, a heterotetrameric enzyme made up of the TrpE and TrpD polypeptides, catalyzes three reactions comprising the first two steps of tryptophan biosynthesis in Salmonella typhimurium. All three activities of the complex are subject to feedback inhibition by tryptophan, which results from allosteric effects associated with the binding of one molecule of inhibitor to each of the TrpE subunits of the complex. Random in vitro chemical mutagenesis of the trpE gene was used to generate a collection of mutant forms of the complex which displayed varying degrees of resistance to feedback inhibition. Single amino acid substitutions, identified by DNA sequencing, were found at 14 different residues within the TrpE polypeptide. The residues were distributed throughout TrpE, but those that appeared to be most critical for regulation were found in two clusters, one at the extreme amino-terminal end, including residues Glu-39, Ser-40, and Ala-41, and the other in the middle of the polypeptide, including residues Asn-288, Pro-289, Met-293, Phe-294, and Gly-305. Kinetic and binding studies of the purified mutant complexes demonstrated that 9 of the 14 had a marked decrease in affinity for tryptophan with little or no change in substrate affinity or catalytic capacity. The remaining five enzymes exhibited more subtle changes, having small decreases in inhibitor affinity coupled with small increases in substrate affinity. Mutant enzymes that were not totally feed-back-resistant had a decreased kinetic response to tryptophan binding. All enzymes exhibited alterations in tryptophan-induced conformational changes as monitored by dye-ligand chromatography.

Allosteric Regulation↗
PubMed1989

Uptake of 3,5,3'-triiodothyronine by cultured rat hepatoma cells is inhibitable by nonbile acid cholephils, diphenylhydantoin, and nonsteroidal antiinflammatory drugs.

Cellular uptake of T3 was examined using rat H4 hepatoma cells. Uptake of [125I]T3 (10(-11) M) from serum-free medium was measured as the cell-associated counts retained by washed cells (2 X 10(6) per well). Displaceable uptake was 84% of total uptake at 2 min (2.9% of total counts). T4, tetraiodothyroacetic acid, triiodothyroacetic acid, rT3, and D-T3 were 2-5% as effective as T3 in displacing uptake. Nonequilibrium kinetics indicated a half-maximal uptake at 680 nM T3 with approximately 7 million sites per cell. Displaceable uptake was time and temperature dependent and was 73% inhibited by 2 mM KCN and 52% by 10 mM bacitracin but not by 2 mM ouabain or 10 microM cytochalasin B. Phloretin, 100 microM, inhibited uptake by 66%. T3 uptake was directly related to the free T3 concentration over the range of albumin concentrations, 0-10 g/liter. The nonbile acid cholephil compounds, bromosulfophthalein, iopanoic acid, and indocyanine green (all 100 microM) inhibited T3 uptake to 62%, 17%, and 5% of control, respectively. Taurocholate, methylaminoisobutyric acid, and oleic acid were noninhibitory. The half-inhibitory concentrations of reactive nonsteroidal antiinflammatory drugs were: meclofenamic acid (25 microM), mefenamic acid (45 microM), fenclofenac (69 microM), flufenamic acid (100 microM), and diclofenac (230 microM). Aspirin, ibuprofen, oxyphenbutazone, and phenylbutazone (all 100 microM) were noninhibitory. Diphenylhydantoin inhibited uptake to 50% at 75 microM. These findings suggest that T3 uptake by cultured rat hepatocytes is by an energy-dependent, saturable, stereo-selective mechanism that is dependent on cell membrane proteins. This mechanism appears to be shared by a number of other ligands, including nonbile acid cholephils and several nonsteroidal antiinflammatory drugs of the anthranilic and phenylacetic acid classes, as well as diphenylhydantoin. The bile acid taurocholate, oleic acid, and a probe for type A amino acid uptake were inactive. The extent to which these effects may modify expression of thyroid hormone action remains to be established.

Aminoisobutyric Acids↗
PubMedDate not supplied

Induction of anthranilate synthase activity by elicitors in oats.

Oat phytoalexins, avenanthramides, are a series of substituted hydroxycinnamic acid amides with anthranilate. The anthranilate in avenanthramides is biosynthesized by anthranilate synthase (AS, EC 4.1.3.27). Induction of anthranilate synthase activity was investigated in oat leaves treated with oligo-N-acetylchitooligosaccharide elicitors. AS activity increased transiently, peaking 6 h after the elicitation. The induction of activity was dependent on the concentration and the degree of polymerization of the oligo-N-acetylchitooligosaccharide elicitor. These findings indicate that the induction is part of a concerted biochemical change required for avenanthramide production. The elicitor-inducible AS activity was strongly inhibited by L-tryptophan and its analogues including 5-methyl-DL-tryptophan, and 5- and 6-fluoro-DL-tryptophan, while the activity was not affected by D-tryptophan. The accumulation of avenanthramide A was also inhibited by treatment of elicited leaves with these AS inhibitors, indicating that a feedback-sensitive AS is responsible for the avenanthramide production. In elicited leaves, the content of free anthranilate remained at a steady, low level during avenanthramide production. Moreover, administration of anthranilate to elicited oat leaves resulted in an enhanced avenanthramide accumulation. AS may play a role as a rate-limiting enzyme in the biosynthesis of avenanthramides.

Anthranilate Synthase↗
PubMed1989

A cysteine-histidine-aspartate catalytic triad is involved in glutamine amide transfer function in purF-type glutamine amidotransferases.

A family of four glutamine amidotransferases has a homologous glutamine amide transfer domain, designated purF-type, that is named after purF-encoded glutamine phosphoribosylpyrophosphate amidotransferase. The glutamine amide transfer domain of approximately 194 amino acid residues is at the NH2 terminus of the protein chain. Site-directed mutagenesis was used to replace several of the 9 invariant amino acids in the glutamine amide transfer domain of glutamine phosphoribosylpyrophosphate amidotransferase. The results indicate that a Cys1-His101-Asp29 catalytic triad is involved in the glutamine amide transfer function of this enzyme. The evidence suggests that His101 functions to increase the nucleophilicity of Cys1, which is used to form a glutamine-enzyme covalent intermediate. Asp29 has a role subsequent to formation of the covalent intermediate. The Cys-His-Asp catalytic triad is implicated in the glutamine amide transfer function of purF-type amidotransferases.

Amino Acid Sequence↗
PubMed1987

Chorismate aminations: partial purification of Escherichia coli PABA synthase and mechanistic comparison with anthranilate synthase.

Chorismate is converted by regiospecific amination/aromatization sequences to o-aminobenzoate and p-aminobenzoate (PABA) by anthranilate synthase (AS) and PABA synthase (PABS), respectively. We report here the first partial purification of the large subunit of Escherichia coli PABA synthase, previously reported to be quantitatively inactivated in purification attempts. The subunit encoded by the pabB gene was overexpressed from a T7 promoter and purified 9-fold to 25-30% homogeneity. The pabB subunit appears unusually sensitive to inactivation by glycerol so this cosolvent is contraindicated. The Km for chorismate is 42 microM in the ammonia-dependent conversion to PABA, and we estimate a turnover number of 2.6 min-1. A variety of chorismate analogues have been prepared and examined. Of these compounds, cycloheptadienyl analogue 11 has been found to be the most potent inhibitor of Serratia marcescens anthranilate synthase (Ki = 30 microM for an RS mixture) and of the E. coli pabB subunit of PABA synthase (Ki = 226 microM). Modifications in the substituents at C-3 [enolpyruyl ether, (R)- or (S)-lactyl ether, glycolyl ether] or C-4 (O-methyl) of chorismate lead to alternate substrates. The Vmax values for (R)- and (S)-lactyl ethers are down 10-20-fold for each enzyme, and V/K analyses show the (S)-lactyl chorismate analogue to be preferred by 12/1 over (R)-lactyl for anthranilate synthase while a 3/1 preference was observed for (R)-/(S)-lactyl analogues by PABA synthase. The glycolyl ether analogue of chorismate shows 15% Vmax vs. chorismate for anthranilate synthase but is actually a faster substrate (140%) than chorismate with PABA synthase, suggesting the elimination/aromatization step from an aminocyclohexadienyl species may be rate limiting with AS but not with PABS. Indeed, studies with (R)-lactyl analogue 14 and anthranilate synthase led to accumulation of an intermediate, isolable by high-performance liquid chromatography and characterized by NMR and UV-visible spectroscopy as 6-amino-5-[(1-carboxyethyl)oxy]-1,3-cyclohexadiene-1-carboxylic acid (17). This is the anticipated intermediate predicted by our previous work with conversion of synthetic trans-6-amino-5-[(1-carboxyethenyl)oxy]-1,3-cyclohexadiene-1-carbo xylic acid (2) to anthranilate by the enzyme. Compound 17 is quantitatively converted to anthranilate on reincubation with enzyme, but at a 1.3-10-fold lower Vmax than starting lactyl substrate 14 under the conditions investigated; the basis for this kinetic variation is not yet determined.

Anthranilate Synthase↗
PubMed1978

Anthranilate synthetase component II from Pseudomonas putida. Covalent structure and identification of the cysteine residue involved in catalysis.

The complete amino acid sequence of carboxamidomethylated anthranilate synthetase component II (AS II) from Pseudomonas putida has been determined by analysis of cyanogen bromide fragments, tryptic peptides from the citraconylated protein, and by analysis of subdigests of these peptides. AS II is a single polypeptide chain of 197 residues having a calculated molecular weight of 21,684. Previous studies (Goto, Y., Keim, P. S., Zalkin, H., and Heinrikson, R. L. (1976) J. Biol. Chem, 251, 941-949) identified a cysteine residue required for the formation of an acyl-enzyme intermediate. The protein has 3 cysteine residues at positions 54, 79, and 140. Cysteine-79 was alkylated selectively by iodoacetamide and by the glutamine affinity analogue L-2-amino-4-oxo-5-chloropentanoic acid. Based on this evidence cysteine-79 is the active site residue involved in formation of the acyl-enzyme intermediate. Comparison of the P. putida AS II sequence with that of the NH2-terminal 60 residues of the enzyme from Escherichia coli shows 38% sequence identity.

Amino Acid Sequence↗
PubMed1989

Molecular cloning and the nucleotide sequence of the Clostridium thermocellum trpE gene.

The trpE gene of Clostridium thermocellum, a moderately thermophilic and absolutely anaerobic bacterium, was cloned by its ability to complement growth of an Escherichia coli tryptophan auxotroph (trpE) deficient in anthranilate synthase I. The nucleotide sequence of trpE and its flanking region was determined. The trpE gene overlapped at the termination codon with a putative initiation codon of trpG (trp[G]D), as deduced from the amino acid sequence homology with anthranilate synthase II of Serratia marcescens. S1-nuclease mapping of the trpE transcript produced in E. coli cells suggested that the promoter of C. thermocellum was utilized by E. coli. The amino acid sequence of anthranilate synthase I of C. thermocellum predicted from the nucleotide sequence is more similar to that of an extremely thermophilic bacterium, Thermus thermophilus HB8, than that of mesophilic bacteria.

Amino Acid Sequence↗
PubMed1990

Temperature-independent binding of [3H](+-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid in brain synaptic membranes treated by Triton X-100.

Specific binding of [3H](+-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), a highly selective antagonist for N-methyl-D-aspartic acid (NMDA) receptors, was examined in brain synaptic membranes treated with Triton X-100 by using a filtration assay method. Elevation of incubation temperature from 2 to 30 degrees C markedly diminished the binding. The binding reached a plateau within 5 min after the initiation of incubation at 2 degrees C, while the time required to attain an equilibrium was 1 min at 30 degrees C. The binding at 2 degrees C was rapidly dissociated by the addition of an excess of unlabeled CPP, NMDA and L-glutamic acid (L-Glu). The binding was also saturable with increasing concentrations of the ligand and displaced by various amino acids structurally related to L-Glu in a stereospecific manner. Competitive but not noncompetitive antagonists for the NMDA receptors invariably inhibited the binding. However, the binding was not prominently affected by agonists for the other subclasses of the brain excitatory amino acid receptors. Both reduced and oxidized forms of glutathione significantly displaced the binding. Scatchard analysis revealed that Triton treatment increased the affinity and density of binding sites which consisted of a single component. Among some endogenous tryptophan metabolites, kynurenic, anthranilic and quinolinic acids inhibited the binding. These results suggest that a filtration assay method is also useful to detect the binding of NMDA receptors in the brain.

Animals↗
PubMed1999

Enhanced detection of oligonucleotides in UV MALDI MS using the tetraamine spermine as a matrix additive.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been used to analyze oligonucleotides. However, success has been limited by cation adduction and high detection limits. Both of these problems are due to the high net negative charge that oligonucleotides carry on the phosphodiester backbone. Comatrixes such as ammonium salts with UV absorbers such as 3-hydroxypicolinic acid, 2,4,6-trihydroxyacetophenone, and 6-aza-2-thiothymine have been used to improve the spectral quality for oligonucleotides in MALDI MS. Organic bases have also been used as co-matrixes; however, the most popular matrix, 3-hydroxypicolinic acid, is not compatible with these additives. We have found that the tetraamine spermine as a matrix additive can successfully eliminate cation adduction and lower the detection limits for DNA in the MALDI experiment, without having to resort to desalting steps. The results suggest that multiply protonated spermine molecules function better than ammonium ions in neutralizing oligonucleotides and displacing alkali cations. Protonated spermine is chemically similar to ammonium ions since it binds to the phosphate backbone and releases protons to the phosphate groups. Spermine can be used successfully with the matrixes 6-aza-2-thiothymine and 80% anthranilic acid/20% nicotinic acid but not with 3-hydroxypicolinic acid. The additive also works well for the analysis of metalated DNA.

Base Sequence↗
PubMedDate not supplied

[Model of genes expression regulation in bacteria by means of formation of secondary RNA structures].

In this article a model, first, classical attenuation RNA regulation of gene expression by means of transcription termination is offered. The model bases on representation about a macrostate of secondary structure in RNA regulatory region between a ribosome and a RNA polymerase, on the formulas of a resonant type defining the value of deceleration of a RNA polymerase by a set of hairpins in the same region. The special attention is given to selection of parameters of model. To check of model the computer simulation is carried out and the dependences of transcription termination probability from the value of concentration charged tRNA are obtained, in particular, and from concentration of amino acid for many regulatory regions in genomes of bacteria (here data are presented for trpE genes in Streptomyces spp., Bradyrhizobium japonicum and Escherichia coli) and at various values of three parameters, which authors consider as the main. The obtained dependences are compounded with the accessible experimental data; including, under the form of the graphs concerning to activity of an enzyme depending on concentration of amino acid (for example, anthranilate synthase from tryptophan in S. venezuela). One possible usage: now attenuation is predicted usually by means of multiple alignment, it needs some sequences; the obtaining with the help of model on an individual sequence characteristic for attenuation or its absence of a curve at approaching parameters could be considered as argument for the benefit of presence or absence of attenuation.

Bacteria↗
PubMed1984

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.

Amino Acid Sequence↗
PubMed1986

Chemical enhancement of SA7 virus transformation of hamster embryo cells: evaluation by interlaboratory testing of diverse chemicals.

Twelve chemicals from diverse structural classes were tested under code for their capacity to enhance the transformation of Syrian hamster embryo cells by simian adenovirus SA7 in two independent laboratories. Pretreatment of hamster cells with eight of those chemicals (reserpine, dichlorvos, methapyrilene hydrochloride, benzidine dihydrochloride, diphenylhydantoin, cinnamyl anthranilate, 11-aminoundecanoic acid, and 4,4'-oxydianiline) produced repeatable enhancement of SA7 transformation at two or more consecutive dose levels, which constitutes clear evidence of enhancing activity in this assay. Both toxic and nontoxic doses of each of these chemicals caused enhancement of virus transformation. Two chemicals (2,6-dichloro-p-phenylenediamine and cinnamaldehyde) produced some evidence of enhancing activity (repeatable transformation enhancement at one dose). Dose ranges for cytotoxicity and enhancement of SA7 transformation were similar in both laboratories for all chemicals producing activity. The final two chemicals, chloramphenicol sodium succinate and ethylene thiourea, failed to reproducibly demonstrate either significant cytotoxicity or enhancement of SA7 transformation at concentrations up to 10-20 mM. The test results for these 12 chemicals were combined with the test results for 9 known carcinogens and noncarcinogens in order to evaluate relationships between activity, dose response, and lowest effective enhancing concentration for these compounds, as well as to correlate them with rodent carcinogenesis classifications. The Syrian hamster embryo cell-SA7 system demonstrated reproducible test responses in both intra- and interlaboratory studies and detected 13 out of 15 known rodent carcinogens.

Adenoviridae↗
PubMed1996

Epitope of 24G7 anti-bilirubin monoclonal antibody.

We examined an antigenic epitope recognized by an anti-bilirubin monoclonal antibody designated 24G7 (Shimizu, S., Izumi, Y., Yamazaki, M., Shimizu, K., Yamaguchi, T., and Nakajima, H. (1988) Biochim. Biophys. Acta 967, 255-260). The reactivity of bilirubin-IX alpha, its analogues (III alpha, XIII alpha, and mesobilirubin-IX alpha) and related azo compounds, with 24G7 was compared by means of an enzyme-linked immunosorbent assay (ELISA). The order of reactivity was as follows: (a) aniline azopigment > ethyl anthranilate azopigment > sulfanilic acid azopigment, (b) bilirubin-XIII alpha > bilirubin-IX alpha > bilirubin-III alpha, (c) bilirubin-IX alpha = mesobilirubin-IX alpha. These findings indicated that the epitope is present in (a) the dipyrrolic moiety of the endovinyl (correspond to N-21 and N-22 of bilirubin), or exovinyl types (N-23 and N-24); (b) the dipyrrolic moiety of the endovinyl type, which contains (c) a methyl group (C-2) but not a vinyl group (C-3) in the dipyrrolic moiety of endovinyl type. Therefore, we concluded that the epitope was the region containing the oxo group at C-1, the methyl group at C-2, C-(4,5,6,9), and the N-21 and -22 of bilirubin-IX alpha.

Antibodies, Monoclonal↗