Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The deduced product of the Bacillus subtilis ytvP gene is similar to that of ORF13, a gene of unknown function in the Lactococcus lactis histidine biosynthesis operon. A B. subtilis ytvP mutant was auxotrophic for histidine. The only enzyme of the histidine biosynthesis pathway that remained uncharacterized in B. subtilis was histidinol phosphate phosphatase (HolPase), catalyzing the penultimate step of this pathway. HolPase activity could not be detected in crude extracts of the ytvP mutant, while purified glutathione S-transferase-YtvP fusion protein exhibited strong HolPase activity. These observations demonstrated that HolPase is encoded by ytvP in B. subtilis and led us to rename this gene hisJ. Together with the HolPase of Saccharomyces cerevisiae and the presumed HolPases of L. lactis and Schizosaccharomyces pombe, HisJ constitutes a family of related enzymes that are not homologous to the HolPases of Escherichia coli, Salmonella typhimurium, and Haemophilus influenzae.
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PMID:Histidinol phosphate phosphatase, catalyzing the penultimate step of the histidine biosynthesis pathway, is encoded by ytvP (hisJ) in Bacillus subtilis. 1032 33

The kinetic properties of bacterial and rat liver glutathione S-transferases (GST) active with dichloromethane (DCM) were compared. The theta class glutathione S-transferase (rGSTTI-1) from rat liver had an affinity for dihalomethanes lower by three orders of magnitude (K(app) > 50 mM) than the bacterial DCM dehalogenase/GST from Methylophilus sp. DM11. Unlike the bacterial DCM dehalogenase, the rat enzyme was unable to support growth of the dehalogenase minus Methylobacterium sp. DM4-2cr mutant with DCM. Moreover, the presence of DCM inhibited growth with methanol of the DM4-2cr transconjugant expressing the rat liver GSTT1-1. In Salmonella typhimurium TA1535, expression of rat and bacterial DCM-active GST from a plasmid in the presence of DCM yielded up to 5.3 times more reversions to histidine prototrophy in the transconjugant expressing the rat enzyme. Under the same conditions, however, GST-mediated conversion of DCM to formaldehyde was lower in cell-free extracts of the transconjugant expressing the rat GSTT1 than in the corresponding strain expressing the bacterial DCM dehalogenase. This provided new evidence that formaldehyde was not the main toxicant associated with GST-mediated DCM conversion, and indicated that an intermediate in the transformation of DCM by GST, presumably S-chloromethylglutathione, was responsible for the observed effects. The marked differences in substrate affinity of rat and bacterial DCM-active GST, as well as in the toxicity and genotoxicity associated with expression of these enzymes in bacteria, suggest that bacterial DCM dehalogenases/GST have evolved to minimise the toxic effects associated with glutathione-mediated catalysis of DCM conversion.
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PMID:Enzyme-mediated dichloromethane toxicity and mutagenicity of bacterial and mammalian dichloromethane-active glutathione S-transferases. 1035 Jan 86

The beta-toxin gene isolated from Clostridium perfringens type B was expressed as a glutathione S-transferase (GST) fusion gene in Escherichia coli. The purified GST-beta-toxin fusion protein from the E. coli transformant cells was not lethal. The N-terminal amino acid sequence of the recombinant beta-toxin (r toxin) isolated by thrombin cleavage of the fusion protein was G-S-N-D-I-G-K-T-T-T. Biological activities and molecular mass of r toxin were indistinguishable from those of native beta-toxin (n toxin) purified from C. perfringens type C. Replacement of Cys-265 with alanine or serine by site-directed mutagenesis resulted in little loss of the activity. Treatment of C265A with N-ethylmaleimide (NEM), which inactivated lethal activity of r toxin and n toxin, led to no loss of the activity. The substitution of tyrosine or histidine for Cys-265 significantly diminished lethal activity. In addition, treatment of C265H with ethoxyformic anhydride which specifically modifies histidyl residue resulted in significant decrease in lethal activity, but that of r toxin with the agent did not. These results showed that replacement of the cysteine residue at position 265 with amino acids with large size of side chain or introduction of functional groups in the position resulted in loss of lethal activity of the toxin. Replacement of Tyr-266, Leu-268 or Trp-275 resulted in complete loss of lethal activity. Simultaneous administration of r toxin and W275A led to a decrease in lethal activity of beta-toxin. These observations suggest that the site essential for the activity is close to the cysteine residue.
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PMID:Clostridium perfringens beta-toxin is sensitive to thiol-group modification but does not require a thiol group for lethal activity. 1035 19

Histidine protein kinases have been explored as potential antibacterial drug targets. The recent identification of two-component histidine kinases in fungi has led us to investigate the antifungal properties of three bacterial histidine kinase inhibitors (RWJ-49815, RWJ-49968, and RWJ-61907). All three compounds were found to inhibit growth of the Saccharomyces cerevisiae and Candida albicans strains, with MICs ranging from 1 to 20 microg/ml. However, deletion of SLN1, the only histidine kinase in S. cerevisiae, did not alter drug efficacy. In vitro kinase assays were performed by using the Sln1 histidine kinase purified from bacteria as a fusion protein to glutathione S-transferase. RWJ-49815 and RWJ-49968 inhibited kinase a 50% inhibitory concentration of 10 microM, whereas RWJ-61907 failed to inhibit at concentrations up to 100 microM. Based on these results, we conclude that these compounds have antifungal properties; however, their mode of action appears to be independent of histidine kinase inhibition.
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PMID:Antifungal properties and target evaluation of three putative bacterial histidine kinase inhibitors. 1039 Feb 25

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis was highly purified from the thermophilic bacterium Thermus thermophilus. The enzyme preparation showed a single band on SDS-polyacrylamide gel electrophoresis, a pH optimum of 7.5 and a temperature optimum at 60 degrees C. The native enzyme which is phosphorylated could, upon treatment with alkaline phosphatase, lose all activity. The inactive form could be reversibly activated by nucleotides in the order of NTP>NDP>NMP. When physiological polyamines were added to the purified enzyme in vitro, spermine or spermidine activated ODC by 140 or 40%, respectively, while putrescine caused a small inhibition. The basic amino acids lysine and arginine were competitive inhibitors of ODC, while histidine did not affect the enzyme activity. Among the phosphoamino acids tested, phosphoserine was the most effective activator of purified ODC. Polyamines added at high concentration to the medium resulted in a delay or in a complete inhibition of the growth of T. thermophilus, and in a decrease of the specific activity of ornithine decarboxylase. The decrease of ODC activity resulted from the appearance of a non-competitive inhibitor of ODC, the antizyme (Az). The T. thermophilus antizyme was purified by an ODC-Sepharose affinity column chromatography, as well as by immunoprecipitation using antibodies raised against the E. coli antizyme. The antizyme of E. coli inhibited the ODC of T. thermophilus, and vice versa. The fragment of amino acids 56-292 of the E. coli antizyme, produced as a fusion protein of glutathione S-transferase, did not inhibit the ODC of E. coli or T. thermophilus.
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PMID:Characterization of ornithine decarboxylase and regulation by its antizyme in Thermus thermophilus. 1039 69

We have identified a novel gene, USP15, encoding a human ubiquitin-specific protease (USP). The USP15 protein consists of 952 amino acids with a predicted molecular mass of 109.2 kDa and contains the highly conserved Cys and His boxes present in all members of the UBP family of deubiquitinating enzymes. USP15 shares 60.5% sequence identity and 76% sequence similarity with the human homolog (UNP/Unph/USP4) of the mouse Unp proto-oncogene. Recombinant USP15 demonstrated ubiquitin-specific protease activity against engineered linear fusions of ubiquitin to beta-galactosidase and glutathione S-transferase. USP15 can also cleave the ubiquitin-proline bond, a property previously unique to Unp/UNP. Chromosomal mapping by fluorescence in situ hybridization and radiation hybrid analyses localized the USP15 gene to chromosome band 12q14, a different location than that of UNP (3p21.3). Analysis of expressed sequence tag databases reveals evidence of alternate polyadenylation sites in the USP15 gene and also indicates that the gene may possess an exon/intron structure similar to that of the Unp gene, suggesting they have descended from a common ancestor. A systematic nomenclature for the human USPs is proposed.
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PMID:Identification, functional characterization, and chromosomal localization of USP15, a novel human ubiquitin-specific protease related to the UNP oncoprotein, and a systematic nomenclature for human ubiquitin-specific proteases. 1044 27

The ram2 and cal1 genes encode the alpha and beta subunits of yeast geranylgeranyl protein transferase type I (GGPT-I), respectively. Arginine 166 of the beta subunit was changed to isoleucine (betaR166I), histidine 216 to aspartic acid (betaH216D), and asparagine 282 to alanine (betaN282A) by sequential PCR using mutagenic primers. The mutants were expressed under the same conditions as the wild-type and were assayed for GGPT-I activity. Wild-type yeast GGPT-I, alphaH145D, alphaD140N, betaR166I, betaH216D and betaN282A mutant GGPT-Is were partially purified by ammonium sulfate fractionation followed by a Q-Sepharose column. Characterization studies were performed using the active fraction of the Q-Sepharose column. In the chemical modification reactions, the catalytic activity of purified enzyme decreased in proportion to the concentration of modifying reagents, such as phenylglyoxal and diethyl pyrocarbonate (DEPC). Geranylgeranyl pyrophosphate (GGPP) protected the enzyme activity from the modification with phenylglyoxal. The measurement of GGPP binding to wild-type and five mutant GGPT-Is was performed by a gel-filtration assay. The binding of GGPP to the betaR166I mutant was low and the Km value for GGPP in the betaR166I mutant increased about 29-fold. Therefore, the results suggest a role for this arginine residue that directly influences the GGPP binding. The activity of the DEPC-modified GGPT-I was inhibited by 80% at 5 mM DEPC. The differential absorption at 242 nm may suggest that at this concentration the modified histidine residues were 1.5 mol per GGPT-I. The protein substrate, glutathione S-transferase fused undecapeptide (GST-CAIL) protected the enzyme from inactivation by DEPC, and the Km value for GST-CAIL in the betaH216D mutant increased about 12-fold. The trypsin digestion of [14C]DEPC-modified enzyme yielded a single radioactive peptide. As a result of the sequence of this radioactive peptide, the histidine 216 residue was assumed to be an essential part of binding of peptide substrate.
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PMID:Active site determination of yeast geranylgeranyl protein transferase type I expressed in Escherichia coli. 1049 Nov 63

We discovered a nuclear import pathway mediated by the product of the previously identified Saccharomyces cerevisiae gene PDR6 (pleiotropic drug resistance). This gene product functions as a karyopherin (Kap) for nuclear import. Consistent with previously proposed nomenclature, we have renamed this gene KAP122. Kap122p was localized both to the cytoplasm and the nucleus. As a prominent import substrate of Kap122p, we identified the complex of the large and small subunit (Toa1p and Toa2p, respectively) of the general transcription factor IIA (TFIIA). Recombinant GST-Kap122p formed a complex with recombinant His(6)-Toa1p/Toa2p. In wild-type cells, Toa1p and Toa2p were localized to the nucleus. Consistent with Kap122p being the principal Kap for import of the Toa1p-Toa2p complex, we found that deletion of KAP122 results in increased cytoplasmic localization of both Toa1p and Toa2p. Deletion of KAP122 is not lethal, although deletion of TOA1 and TOA2 is. Together these data suggest that Kap122p is the major Kap for the import of Toa1p-Toa2p into the nucleus. Like other substrate-Kap complexes, the Toa1p/Toa2p/Kap122p complex isolated from yeast cytosol or reconstituted from recombinant proteins, was dissociated by RanGTP but not RanGDP. Kap122p bound to nucleoporins, specifically, to the peptide repeat-containing fragments of Nup1p and Nup2p.
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PMID:The karyopherin Kap122p/Pdr6p imports both subunits of the transcription factor IIA into the nucleus. 1052 31

Malonyl-CoA synthetase (MCS) catalyses the formation of malonyl-CoA in a two-step reaction consisting of the adenylation of malonate with ATP followed by malonyl transfer from malonyl-AMP to CoA. In order to identify amino acid residues essential for each step of the enzyme, catalysis based on chemical modification and database analysis, Arg-168, Lys-170, and His-206 were selected for site-directed mutagenesis. Glutathione-S-transferase-fused enzyme (GST-MCS) was constructed and mutagenized to make R168G, K170M, R168G/K170M and H206L mutants, respectively. The MCS activity of soluble form GST-MCS was the same as that of wild-type MCS. Circular dichroism spectra for the four mutant enzymes were nearly identical to that for the GST-MCS, indicating that Arg-168, Lys-170 and His-206 are not important for conformation but presumably for substrate binding and/or catalysis. HPLC analysis of products revealed that the intermediate malonyl-AMP is not accumulated during MCS catalysis and that none of the mutant enzymes accumulated it either. Kinetic analysis of the mutants revealed that Lys-170 and His-206 play a critical role for ATP binding and the formation of malonyl-AMP, whereas Arg-168 is critical for formation of malonyl-CoA and specificity for malonyl-AMP. Molecular modelling based on the crystal structures of luciferase and gramicidin S synthetase 1 provided MCS structure which could fully explain all these biochemical data even though the MCS model was generated by comparative modelling.
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PMID:Identification of residues essential for a two-step reaction by malonyl-CoA synthetase from Rhizobium trifolii. 1054 46

DnaJ proteins are located in various compartments of the eukaryotic cell. As previously shown, peroxisomes and glyoxysomes possess a membrane-anchored form of DnaJ protein located on the cytosolic face. Hints as to how the membrane-bound co-chaperone interacts with cytosolic soluble chaperones were obtained by examining the affinity between the DnaJ protein and various potential partners of the Hsp70 family. Two genes encoding cytosolic Hsp70 isoforms were isolated and characterized from cucumber cotyledons. In addition, cDNAs encoding Hsp70 forms attributed to the cytosol, plastids and the lumen of the endoplasmic reticulum were prepared. His-tagged DnaJ proteins and glutathione S-transferase-Hsp70 fusion proteins were constructed. Using these tools, it was demonstrated that the soluble His-tagged form of DnaJ protein exclusively binds the cytosolic isoform 1 of Hsp70. This interaction was further analyzed by characterizing the interaction between the glyoxysome-bound form of the DnaJ protein and various isoforms of Hsp70. Specific binding to the glyoxysomal surface was only observed in the case of cytosolic isoform 1 of Hsp70. This interaction was strictly dependent on the presence of ADP. Glyoxysomes did not bind other cytosolic or plastidic isoforms or the BiP-related form of Hsp70. Analyzing the enzymatic properties of cytosolic Hsp70s, we showed that the ATPase-modulating activity of DnaJ was highest when isoform 1 was assayed. Collectively, the data indicate that the partner of the DnaJ protein anchored at the glyoxysomal membrane is the cytosolic isoform 1 of Hsp70. In addition to the chaperones located at the surface of glyoxysomes, two isoforms of Hsp70 and one soluble form of DnaJ protein were detected in the glyoxysomal matrix.
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PMID:The membrane-bound DnaJ protein located at the cytosolic site of glyoxysomes specifically binds the cytosolic isoform 1 of Hsp70 but not other Hsp70 species. 1065 11


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