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PubMed2000

Comparative analyses of secondary gene products of 3-deoxy-D-manno-oct-2-ulosonic acid transferases from Chlamydiaceae in Escherichia coli K-12.

The waaA gene encoding the essential, lipopolysaccharide (LPS)-specific 3-deoxy-Dmanno-oct-2-ulosonic acid (Kdo) transferase was inactivated in the chromosome of a heptosyltransferase I and II deficient Escherichia coli K-12 strain by insertion of gene expression cassettes encoding the waaA genes of Chlamydia trachomatis, Chlamydophila pneumoniae or Chlamydophila psittaci. The three chlamydial Kdo transferases were able to complement the knockout mutation without changing the growth or multiplication behaviour. The LPS of the mutants were serologically and structurally characterized in comparison to the LPS of the parent strain using compositional analyses, high performance anion exchange chromatography, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and specific monoclonal antibodies. The data show that chlamydial Kdo transferases can replace in E. coli K-12 the host's Kdo transferase and retain the product specificities described in their natural background. In addition, we unequivocally proved that WaaA from C. psittaci transfers predominantly four Kdo residues to lipid A, forming a branched tetrasaccharide with the structure alpha-Kdo-(2-->8)-[alpha-Kdo-(2-->4)]-alpha-Kdo-(2-->4)-alpha-Kdo.

Base Sequence↗
PubMed2006

Changes produced by bound tryptophan in the ribosome peptidyl transferase center in response to TnaC, a nascent leader peptide.

Studies in vitro have established that free tryptophan induces tna operon expression by binding to the ribosome that has just completed synthesis of TnaC-tRNA(Pro), the peptidyl-tRNA precursor of the leader peptide of this operon. Tryptophan acts by inhibiting Release Factor 2-mediated cleavage of this peptidyl-tRNA at the tnaC stop codon. Here we analyze the ribosomal location of free tryptophan, the changes it produces in the ribosome, and the role of the nascent TnaC-tRNA(Pro) peptide in facilitating tryptophan binding and induction. The positional changes of 23S rRNA nucleotides that occur during induction were detected by using methylation protection and binding/competition assays. The ribosome-TnaC-tRNA(Pro) complexes analyzed were formed in vitro; they contained either wild-type TnaC-tRNA(Pro) or its nonfunctional substitute, TnaC(W12R)-tRNA(Pro). Upon comparing these two peptidyl-tRNA-ribosome complexes, free tryptophan was found to block methylation of nucleotide A2572 of wild-type ribosome-TnaC-tRNA(Pro) complexes but not of ribosome-TnaC(W12R)-tRNA(Pro) complexes. Nucleotide A2572 is in the ribosomal peptidyl transferase center. Tryptophanol, a noninducing competitor of tryptophan, was ineffective in blocking A2572 methylation; however, it did reverse the protective effect of tryptophan. Free tryptophan inhibited puromycin cleavage of TnaC-tRNA(Pro); it also inhibited binding of the antibiotic sparsomycin. These effects were not observed with TnaC(W12R)-tRNA(Pro) mutant complexes. These findings establish that Trp-12 of TnaC-tRNA(Pro) is required for introducing specific changes in the peptidyl transferase center of the ribosome that activate free tryptophan binding, resulting in peptidyl transferase inhibition. Free tryptophan appears to act at or near the binding sites of several antibiotics in the peptidyl transferase center.

Amino Acid Substitution↗
PubMed1971

Hydrolysis of fMet-tRNA by peptidyl transferase.

Escherichia coli and rabbit reticulocyte (f[(3)H]Met-tRNA.AUG.ribosome) intermediates undergo hydrolysis, with release of f[(3)H]methionine, upon addition of tRNA or CpCpA in the presence of acetone. This ribosomal catalyzed reaction has similar requirements, pH optimum, and antibiotic sensitivity to those of peptidyl transferase. Two antibiotics, lincomycin with E. coli ribosomes and anisomycin with reticulocyte ribosomes, inhibit peptide-bond formation and transesterification activities of peptidyl transferase, but stimulate hydrolysis of f[(3)H]Met-tRNA. Earlier studies have suggested peptidyl transferase activity is essential for R factor-dependent hydrolysis of f((3)H)Met-tRNA. These studies indicate that peptidyl transferase has the capacity for f((3)H)Met-tRNA hydrolysis and, therefore, may be responsible for peptidyl-tRNA cleavage during peptide chain termination.

Animals↗
PubMed1985

Identification of Yb-glutathione-S-transferase as a major rat liver protein labeled with dexamethasone 21-methanesulfonate.

Dexamethasone 21-methanesulfonate, an affinity label for glucocorticoid-binding proteins, was incubated with rat liver cytosol preparations. The predominant covalently labeled component was identified as Yb-glutathione-S-transferase on the basis of chromatographic properties, electrophoretic mobility, and specific retention by an anti-Yb-immunoadsorbent. Affinity labeling of this protein was blocked by excess dexamethasone. Preferential reactivity of dexamethasone 21-methanesulfonate with the Yb subclass of glutathione-S-transferase (glutathione transferase, EC 2.5.1.18) was also evident with mixtures containing the multiple forms of the enzyme. Yb-glutathione-S-transferase, the nonsaturable glucocorticoid-binding component of rat liver cytosol should, therefore, be reclassified; because of its high concentration and selective interaction with steroids, this enzyme may be an intracellular glucocorticoid-binding protein and, thereby, influence transport, metabolism, and action of the steroids.

Affinity Labels↗
PubMed1988

Amplification and increased expression of alpha class glutathione S-transferase-encoding genes associated with resistance to nitrogen mustards.

Glutathione-dependent enzymes play a central role in the protection of cells from cytotoxic chemicals and have been implicated in the intrinsic and acquired resistance of tumors to cytotoxic drugs. We have generated a Chinese hamster ovary line resistant to bifunctional nitrogen mustards and in this report have characterized and isolated the protein that represents the major observable phenotypic difference between the drug-sensitive and drug-resistant cell lines. This purified protein is shown to be an alpha class glutathione S-transferase comprising YcYc subunits and possessing a pI value of approximately 8.0. The intracellular level of the Yc subunit is elevated greater than 40-fold in the drug-resistant cell line, which could account for the increase in glutathione S-transferase (RX:glutathione R-transferase; EC 2.5.1.18) activity toward both 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Other glutathione S-transferase subunits within this gene family are also elevated. These changes are accompanied by a significant elevation in alpha class mRNA levels. Southern analysis indicates that the genes coding for these proteins are amplified 4- to 8-fold in the drug-resistant cell line. In addition, gamma-glutamyl transpeptidase [(5-glutamyl)-peptide:amino acid 5-glutamyltransferase; EC 2.3.2.2] activity is increased 3.6-fold in the drug-resistant Chinese hamster ovary cell line, which may explain the increase in cellular glutathione level. In this case no gene amplification was seen. These data indicate that gene amplification may be important in drug resistance toward alkylating agents and also that other enzymes in glutathione homeostasis are involved.

Animals↗
PubMed1989

Metabolism of ethanol and carcinogens by glutathione transferases.

Nonoxidative alcohol metabolism to form fatty acid ethyl esters contributes to alcohol-related end-organ damage, and these products are formed by two synthase enzymes. We recently purified the major (pI 4.9) synthase from human myocardium. The N-terminal sequence (A P Y T V V Y F P V R G R X K A L R M L X A D) is greater than 73% identical with that of a neutral (pI 6.7) detoxification enzyme, glutathione transferase P from rat hepatocellular carcinoma (P P Y T I V Y F P V R G R C E A T R M L L A D). Moreover, both the major human fatty acid ethyl ester synthase and bovine liver glutathione transferase catalyze the formation of fatty acid ethyl esters (Vmax 105 and 98 nmol per hr per mg, respectively). In addition, both enzymes catalyze the formation of glutathione-xenobiotic conjugates (Vmax 67 and 335 mol per hr per mol of enzyme, respectively). Physiological concentrations of glutathione increase the rate of formation of fatty acid ethyl esters up to 5-fold, and the glutathione transferase substrate 1-chloro-2,4-dinitrobenzene is a potent inhibitor of human myocardial fatty acid ethyl ester synthase. Thus, the identification of the major form of human myocardial fatty acid ethyl ester synthase as an acidic glutathione transferase links alcohol and xenobiotic metabolism and may relate the enhancement of tumorigenesis by alcohol abuse with carcinogen-conjugation reactions.

Acyltransferases↗
PubMed1994

Construction, expression, and immunogenicity of the Schistosoma mansoni P28 glutathione S-transferase as a genetic fusion to tetanus toxin fragment C in a live Aro attenuated vaccine strain of Salmonella.

A vector has been constructed to allow genetic fusions of guest antigens via a hinge domain to the C terminus of the highly immunogenic C fragment of tetanus toxin. A fusion has been constructed with the gene encoding the protective 28-kDa glutathione S-transferase (EC 2.5.1.18) from Schistosoma mansoni. The recombinant vector has been electroporated into the nonvirulent Salmonella typhimurium aroA live vaccine strain SL3261. The corresponding chimeric protein is stably expressed in a soluble form in Salmonella as evaluated by Western blotting with fragment C and glutathione S-transferase antisera. Mice immunized intravenously with a single dose of the live recombinant bacteria elicit antibodies to both fragment C and glutathione S-transferase as detected by enzyme-linked immunosorbent assays. Furthermore, all of the mice were solidly protected when challenged with lethal doses of either tetanus toxin or the virulent Salmonella typhimurium strain C5. Mice have also elicited antibodies to fragment C and glutathione S-transferase after oral immunization. It may be that a live trivalent vaccine against typhoid, tetanus, and schistosomiasis is feasible.

Animals↗
PubMed2002

Transmutation of human glutathione transferase A2-2 with peroxidase activity into an efficient steroid isomerase.

A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

Binding Sites↗
PubMed2005

Nitration of tyrosine 92 mediates the activation of rat microsomal glutathione s-transferase by peroxynitrite.

There is increasing evidence that protein function can be modified by nitration of tyrosine residue(s), a reaction catalyzed by proteins with peroxidase activity, or that occurs by interaction with peroxynitrite, a highly reactive oxidant formed by the reaction of nitric oxide with superoxide. Although there are numerous reports describing loss of function after treatment of proteins with peroxynitrite, we recently demonstrated that the microsomal glutathione S-transferase 1 is activated rather than inactivated by peroxynitrite and suggested that this could be attributed to nitration of tyrosine residues rather than to other effects of peroxynitrite. In this report, the nitrated tyrosine residues of peroxynitrite-treated microsomal glutathione S-transferase 1 were characterized by mass spectrometry and their functional significance determined. Of the seven tyrosine residues present in the protein, only those at positions 92 and 153 were nitrated after treatment with peroxynitrite. Three mutants (Y92F, Y153F, and Y92F, Y153F) were created using site-directed mutagenesis and expressed in LLC-PK1 cells. Treatment of the microsomal fractions of these cells with peroxynitrite resulted in an approximately 2-fold increase in enzyme activity in cells expressing the wild type microsomal glutathione S-transferase 1 or the Y153F mutant, whereas the enzyme activity of Y92F and double site mutant was unaffected. These results indicate that activation of microsomal glutathione S-transferase 1 by peroxynitrite is mediated by nitration of tyrosine residue 92 and represents one of the few examples in which a gain in function has been associated with nitration of a specific tyrosine residue.

Amino Acid Sequence↗
PubMed1994

Consumption of Brussels sprouts results in elevated alpha-class glutathione S-transferase levels in human blood plasma.

The effect of consumption of glucosinolate-containing Brussels sprouts on alpha-class glutathione S-transferase levels in human blood plasma was investigated in 10 healthy, male, non-smoking volunteers. Following a 3-week run-in period, five volunteers continued on a glucosinolate-free diet during a subsequent 3-week intervention period (control group), while the other five (sprouts group) consumed 300 g of cooked Brussels sprouts per day, at the expense of 300 g of a glucosinolate-free vegetable. alpha-Class glutathione S-transferases were measured by radioimmunoassay. In the control group, similar alpha-class glutathione S-transferase levels were observed in both periods (P = 0.814), while in the sprouts group the alpha-class glutathione S-transferase levels were elevated by a factor of 1.4 (P = 0.002). We hypothesize that the elevated alpha-class GST levels in plasma reflect GST-alpha induction in tissues such as liver and small intestine under non-toxic conditions. The present findings indicate that alpha-class GST levels in plasma may be used as a biomarker for alpha-class GST levels in tissues. In addition, they support the results of epidemiologic studies that consumption of cruciferous vegetables may result in a decreased cancer risk.

Adult↗
PubMed1984

Glutathione-mediated binding of dibromoalkanes to DNA: specificity of rat glutathione-S-transferases and dibromoalkane structure.

1,2-Dibromo-[1,2-14C]ethane was bound irreversibly to DNA when glutathione S-transferase or rat liver cytosolic components were added to incubations of calf thymus DNA and glutathione at 37 degrees C. There was no DNA binding of 1,2-dibromoethane when glutathione was absent or in incubations of DNA with microsomal proteins with or without NADPH, thus supporting the proposal that the major route of DNA binding by 1,2-dibromoethane occurs via conjugation to glutathione. In vitro binding of 1,2-dibromoethane occurred most effectively when the YaYc (or 'B') isozyme of glutathione S-transferase was included in incubations of DNA with 1,2-dibromoethane and glutathione. Other dihaloalkanes were incubated with DNA in the presence of glutathione S-transferase and [35S]glutathione. Of these, only 1,2-dibromo-3-chloropropane and tris-(2,3-dibromopropyl)-phosphate led to significant DNA binding of [35S]glutathione. 1,2-Dibromo-3-chloro-[1,3-14C]propane was bound to DNA when glutathione and glutathione S-transferase were present. However, even higher 1,2-dibromo-3-chloropropane binding to DNA occurred when cytosol or microsomes were included in incubations without glutathione. When glutathione was added to incubations containing cytosol and 1,2-dibromo-3-chloropropane, total DNA binding was decreased. Thus, the actual amount of DNA binding by dihaloethanes in vivo may be the result of a complicated balance among the opposing roles of glutathione conjugation in detoxicating and activating processes.

Animals↗
PubMed1985

Studies on the detoxication of microsomally-activated aflatoxin B1 by glutathione and glutathione transferases in vitro.

Aflatoxin B1 (AFB1)-8,9-oxide, the proposed ultimate carcinogen is conjugated enzymically with glutathione (GSH) to give 8-(S-glutathionyl)-9-hydroxy-8,9-dihydro AFB1 (AFB1-SG). The GSH conjugate isolated from rat bile was shown, on the basis of 1H n.m.r. to be identical to AFB1-SG. Of the seven soluble rat liver GSH transferases tested, namely GSH transferases 1-1, 1-2, 2-2, 3-3, 3-4, 4-4 and 5-5 (see reference 1 for the new system of nomenclature), only the first three were active with microsomally generated AFB1-8,9-oxide, their rates of conjugation being 1.1, 0.61 and 0.64 nmol/min/mg enzyme, respectively. AFB1-SG is a thioacetal, but it was not formed from the incubation of the hemiacetal, AFB1-8,9-dihydrodiol, with GSH or GSH plus GSH transferase 1-1 plus 1-2. The covalent binding of in vitro microsomally activated AFB1 to DNA and the formation of AFB1-SG were linearly related to AFB1 concentration in the range of 0.2-2 micrograms/ml. DNA binding was decreased by 38% by the competing formation of AFB1-SG throughout this range of concentrations. These results are in accord with the observation of Scott Appleton et al. (Cancer Res., 42, 3659-3662) that, in the rat in vivo, there is no evident threshold for the binding of AFB1 to DNA. These findings are also consistent with the further observation, reported in this paper that GSH and GSH transferases have no effect on the mutagenicity of microsomally activated AFB1 to Salmonella typhimurium TA 100.

Aflatoxin B1↗
PubMed2000

Dissection of the two transferase activities of the Pasteurella multocida hyaluronan synthase: two active sites exist in one polypeptide.

Type A Pasteurella multocida, an animal pathogen, employs a hyaluronan [HA] capsule to avoid host defenses. PmHAS, the 972-residue membrane-associated hyaluronan synthase, catalyzes the transfer of both GlcNAc and GlcUA to form the HA polymer. To define the catalytic and membrane-associated domains, pmHAS mutants were analyzed. PmHAS1-703 is a soluble, active HA synthase suggesting that the carboxyl-terminus is involved in membrane association of the native enzyme. PmHAS1-650 is inactive as a HA synthase, but retains GlcNAc-transferase activity. Within the pmHAS sequence, there is a duplicated domain containing a short motif, Asp-Gly-Ser, that is conserved among many beta-glycosyltransferases. Changing this aspartate in either domain to asparagine, glutamate, or lysine reduced the HA synthase activity to low levels. The mutants substituted at residue 196 possessed GlcUA-transferase activity while those substituted at residue 477 possessed GlcNAc-transferase activity. The Michaelis constants of the functional transferase activity of the various mutants, a measure of the apparent affinity of the enzymes for the precursors, were similar to wild-type values. Furthermore, mixing D196N and D477K mutant proteins in the same reaction allowed HA polymerization at levels similar to the wild-type enzyme. These results provide the first direct evidence that the synthase polypeptide utilizes two separate glycosyltransferase sites.

Amino Acid Motifs↗
PubMed1977

Peptidyl transferase activity in rat skeletal muscle ribosomes after protein restriction.

Rats were fed for 6 consecutive days a diet containing casein supplemented with 3 g/kg methionine. The protein level was either 20% or 3%. Skeletal muscle ribosomes were isolated and the activity of the ribosomal enzyme peptidyl transferase was determined. [3H]puromycin acted as acceptor for tRNA bound peptide provided it was located at the ribosomal peptidyl site. The results showed that the rate of peptidyl transferase activity was 13% lower after protein restriction. The differences amounted to 6% when diphtheria toxin and NAD were added as an inhibitor of elongation factor2. This factor catalyzes translocation of peptidyl-tRNA from ribosomal acceptor to peptidyl site. Between 0.11 and 0.15 pmoles peptidyl-tRNA per pmole ribosome were originally accessable as a substrate for peptidyl transferase. The results indicate that peptidyl transferase contributes to a decrease in protein synthesis after protein restriction; but that other components also contribute to the decrease observed.

Acyltransferases↗
PubMed1995

Role for the highly conserved region of domain IV of 23S-like rRNA in subunit-subunit interactions at the peptidyl transferase centre.

The function of the highly conserved and accessible region of domain IV of 23S rRNA (positions 1900-1981 in Escherichia coli 23S rRNA) was investigated by subjecting it to a random mutagenesis procedure that produced single-site mutations efficiently. Nine single-site mutants were selected that were recessive lethal. High levels of mutated 23S rRNA were expressed in E. coli and extracted ribosomes were investigated for their content of mutated rRNA. The peptidyl transferase activity of the ribosomes was also estimated using a newly developed method involving selective inhibition of chromosome-encoded ribosomes by clindamycin. Two of the mutants, U1940A and U1955G, yielded 50S subunits that were defective in subunit-subunit association but active in peptidyl transferase activity and five, U1926C, U1946C, U1979C, U1982A and G1984A, produced 50S subunits that were defective in both subunit-subunit interactions and peptidyl transferase activity. We infer that the large conserved rRNA region generates a complex structure that plays an essential role in maintaining and modulating subunit-subunit interactions and argue that its involvement in the peptidyl transferase centre is secondary, possibly involving the correct alignment of protein L2.

Base Sequence↗
PubMed2000

Glutathione S-transferase in mucus of rat small intestine.

Glutathione S-transferases in the small intestine function in detoxification of electrophilic compounds ingested in foods, dietary supplements, and orally administered drug preparations. Although the required substrate glutathione (GSH) is synthesized in the intestinal enterocytes, the rate of synthesis is slow compared to both the maximal GST activity and the rate of uptake of luminal GSH. GSH is supplied to the intestinal lumen in the bile, and normal luminal concentrations in the rat are about 250 microM. The present study was designed to test the hypothesis that exogenous GSH is used for intestinal conjugation by glutathione S-transferase. The results show that 250 microM of extracellular GSH stimulated conjugation of 1-chloro-2,4-dinitrobenzene by approximately 300% in rat intestinal enterocyte preparations. However, an unexpected finding was that most of this stimulated activity did not depend upon uptake of GSH by the enterocytes but was due to glutathione S-transferase associated with mucus. Immunohistochemistry of glutathione S-transferase in the intact small intestine confirmed that a portion of the GST is present in the mucus layer. The presence of this detoxication enzyme in the extracellular mucus layer provides a novel mechanism for preventing direct contact of potentially toxic dietary electrophiles with the intestinal enterocytes.

Animals↗
PubMed2000

Perillyl alcohol, an inhibitor of geranylgeranyl transferase, induces apoptosis of immortalized human vascular smooth muscle cells in vitro.

The isoprenoids geranylgeraniol and farnesol are required for lipid modification of several important cellular proteins. In this study the effect of perillyl alcohol (PA), an inhibitor of geranylgeranyl transferase, was examined on proliferation and apoptosis of immortalized human vascular smooth muscle cells (HVSMCs) derived from saphenous vein. PA induced a time- and concentration-dependent inhibition of cell proliferation over 3 days and 48 h exposure to PA inhibited [methyl-3H-thymidine incorporation induced by 10% fetal calf serum in a concentration-dependent manner. Flow cytometry analysis indicated that PA reduced the proportion of cells in S phase and increased apoptosis. PA-induced apoptosis was confirmed by terminal deoxynucleotidyl transferase (TdT) reagent-based immunocytochemistry. In contrast, the selective inhibitors of farnesyl transferase B581 and BZA-5B were without effect. In view of the proposed role of proliferation and apoptosis in myointimal hyperplasia, inhibitors of geranylgeranyl transferases may have therapeutic potential in cardiovascular disease.

Alkyl and Aryl Transferases↗
PubMed2004

Genetic characterization of pcpS, encoding the multifunctional phosphopantetheinyl transferase of Pseudomonas aeruginosa.

Fatty acid synthases (primary metabolism), non-ribosomal peptide synthases and polyketide synthases (secondary metabolism) contain phosphopantetheinyl (Ppant)-dependent carrier proteins that must be made functionally active by transfer of the 4'-Ppant moiety from coenzyme A. These reactions are usually catalysed by dedicated Ppant transferases. Although rich in Ppant-dependent carrier proteins, it was previously shown that Pseudomonas aeruginosa possesses only one Ppant transferase, encoded by pcpS, which functions in both primary and secondary metabolism. Consistent with this notion are our findings that pcpS can genetically complement mutations in the Escherichia coli acpS and entD genes, encoding the apo-acyl carrier protein (ACP) synthase of fatty acid synthesis and a Ppant transferase of enterobactin synthesis, respectively. It also complements a Bacillus subtilis sfp mutation affecting a gene encoding a Ppant transferase essential for surfactin synthesis. A pcpS insertion mutant could only be constructed in a strain carrying the E. coli acpS gene on a chromosomally integrated element in trans, implying that the in vitro essentiality of pcpS is due to its requirement for activation of apo-ACP of fatty acid synthesis. The conditional pcpS mutant is non-fluorescent, does not produce pyoverdine and pyochelin, and does not grow in the presence of iron chelators. The data presented here for the first time confirm that PcpS plays an essential role in both fatty acid and siderophore metabolism.

Bacterial Proteins↗