Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The moderate thermophile Bacillus stearothermophilus was used as a host in which to detect more thermostable variants of the B.pumilus chloramphenicol acetyltransferase (Cat-86) protein. Seventeen mutants were isolated and detected by their ability to grow in the presence of chloramphenicol at a previously restrictive temperature (58 degrees C). The genes encoding these proteins were sequenced; all 17 mutants carried the same C to T transition that conferred an amino acid substitution of alanine by valine at position 203 of the protein sequence. The wild-type and one mutant Cat-86 protein were purified to homogeneity using affinity chromatography, and kinetic and thermal stability studies were undertaken. Both enzymes had similar sp. act. in the region of 215 U/mg, with Km values for chloramphenicol in the range 13.8-15.4 microM and for acetyl CoA in the range 13.6-15.5 microM. The A203V mutant shows greater stability than the wild-type Cat-86 protein at temperatures above 50 degrees C and appears to pass through a transition state between 48 and 50 degrees C.
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PMID:Selection of a thermostable variant of chloramphenicol acetyltransferase (Cat-86). 143 64

Familial glucocorticoid resistance is a hypertensive, hyperandrogenic disorder characterized by increased serum cortisol concentrations in the absence of stigmata of Cushing's syndrome. Our previous studies of the first reported kindred showed a two- to threefold reduction in glucocorticoid receptor-ligand binding affinity in the propositus, and a lesser reduction in affinity in his mildly affected son and nephew. Glucocorticoid receptor cDNA from these three patients was amplified by polymerase chain reaction and sequenced. The cDNA nucleotide sequence was normal, except for nucleotide 2054, which substituted valine for aspartic acid at amino acid residue 641. The propositus was homozygous while the other relatives were heterozygous for the mutation. COS-7 monkey kidney cells were cotransfected with expression vectors for either wild type or Val 641-mutant receptors, together with the reporter plasmid pMMTV-CAT. Dexamethasone increased chloramphenicol acetyltransferase activity in cells expressing wild type receptor, but had no effect in cells expressing Val 641-mutant receptors, despite similar receptor concentrations, as indicated by Western blotting. The binding affinity for dexamethasone of the Val 641-mutant receptor was threefold lower than that of the wild type receptor. These results suggest that glucocorticoid resistance in this family is due to a point mutation in the steroid-binding domain of the glucocorticoid receptor.
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PMID:Point mutation causing a single amino acid substitution in the hormone binding domain of the glucocorticoid receptor in familial glucocorticoid resistance. 170 18

Stable transformants of the Jurkat T-cell line have been obtained that express either of two distinct forms of the type 1 human immunodeficiency virus nef gene: the nef-1-encoded protein (Nef-1) contains alanine, glycine, and valine at positions 15, 29, and 33, respectively; the protein specified by nef-2 (Nef-2) has threonine, arginine, and alanine at the corresponding positions. When Jurkat cells or their Nef-2-expressing transformants are treated with phorbol 12-myristate 13-acetate (PMA) plus either phytohemagglutinin (PHA) or antibodies against CD3 epsilon, T-cell receptor beta chain, or CD2, there is a prompt increase in interleukin 2 (IL-2) mRNA and intracellular calcium and in the IL-2 receptor alpha chain on the cell surface. Although cells expressing Nef-1 also induce calcium mobilization and the production of IL-2 receptor alpha chain, the formation of IL-2 mRNA is blocked in response to these stimuli. Moreover, Nef-1-expressing cells transfected with a plasmid in which the IL-2 promoter is fused to the chloramphenicol acetyltransferase (CAT) gene fail to induce CAT following treatment with PMA and PHA. By contrast, the parental and Nef-2-containing cells induce CAT normally. Nef-1-expressing cells can produce IL-2 mRNA in response to a combination of PMA and ionomycin, although much less efficiently than the parental Jurkat cells or Nef-2-expressing cells. These findings, and others described herein, suggest that the virally encoded Nef protein interferes with a signal emanating from the T-cell receptor complex that induces IL-2 gene transcription.
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PMID:Expression of the type 1 human immunodeficiency virus Nef protein in T cells prevents antigen receptor-mediated induction of interleukin 2 mRNA. 205 9

The role of conserved Asp-199 in chloramphenicol acetyltransferase (CAT) has been investigated by site-directed mutagenesis. Substitution of Asp-199 by alanine results in a thermolabile mutant enzyme (Ala-199 CAT) with reduced kcat(13-fold) but similar Km values to wild type CAT. Replacement by asparagine gives rise to a thermostable mutant enzyme (Asn-199 CAT) with much reduced kcat(1500-fold). Furthermore, Asn-199 CAT shows anomalous inactivation kinetics with the affinity reagent 3-(bromo-acetyl)chloramphenicol. These results favor a structural role for Asp-199 rather than a catalytic one, in keeping with crystallographic evidence for involvement of Asp-199 in a tight salt bridge with Arg-18. Replacement of Arg-18 by valine results in a mutant enzyme (Val-18 CAT) with similar properties to Ala-199 CAT. The catalytic imidazole of His-19 appears to be conformationally constrained by hydrogen bonding between N1-H and the carbonyl oxygen of the same residue and by ring stacking with Tyr-25.
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PMID:Substitutions in the active site of chloramphenicol acetyltransferase: role of a conserved aspartate. 306 55

Mutations at the cpxA locus of Escherichia coli K-12 affect cellular processes that are not otherwise related. We have now determined the physical and genetic structure of the E. coli chromosome in the region of cpxA (87.5 min). Our results indicate that cpxA is a single gene. Previous studies showed cpxA to be linked to tpiA. We therefore isolated two tpiA+ recombinant plasmids, pRA200 and pRA300, from EcoRI and BamHI digests of F'133, respectively. By genetic complementation or enzyme overproduction, the 9.5 kb EcoRI fragment in pRA200 was shown to include glpK, tpiA and cdh. The 13.6 kb BamHI fragment of pRA300 lacks glpK, but includes tpiA, pfkA and cpxA. Neither fragment complemented a deletion of the rha operon. These data indicate the chromosomal gene order: 87 min-rha-cpxA-pfkA-cdh-tpiA-glpK-88 min. The EcoRI and BamHI fragments overlap in an interval corresponding to about 8.2 kb of DNA. The total region of the E. coli K12 chromosome covered by the two fragments is about 15 kb. A terminal 2 kb EcoRI-BamHI fragment from pRA300 complemented the chromosomal cpxA2[Ts] allele with respect to isoleucine and valine synthesis, RNA bacteriophage sensitivity and surface exclusion in Hfr strains, and envelope protein composition. Complementation occurred when the fragment was subcloned in pBR325 but not when it was subcloned in pBR322, suggesting that the 2 kb fragment lacks expression sequences that are supplied by cat (chloramphenicol acetyltransferase gene) expression sequences of pBR325. The cpxA locus on the E. coli chromosome was established with respect to two chromosomal Tn10 insertions by a combination of genetic and physical analyses. The locus established by those analyses was consistent with the location of the 2 kb EcoRI-BamHI fragment in the physical map of the region. Physical analyses of (rha-pfkA) and (rha-tpiA) deletion strains showed that they lack cpxA and surrounding genes. Since these strains were viable, cpxA is not essential under all growth conditions.
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PMID:Physical and genetic structure of the glpK-cpxA interval of the Escherichia coli K-12 chromosome. 609 95

Mutations of the human androgen receptor gene were identified in five subjects from four families with androgen insensitivity syndrome. Individual exons of the androgen receptor gene were amplified by the polymerase chain reaction from genomic DNA and screened for sequence-dependent differences in their melting characteristics by denaturing gradient gel electrophoresis. DNA fragments from exons with altered mobility were sequenced. Four different single nucleotide base substitutions were found within exons 5, 6, and 7 encoding the steroid-binding domain of the androgen receptor. In one subject with ambiguous genitalia, amino acid residue 763 was changed from tyrosine to cysteine (TAC-->TGC; Y763C). Four subjects, including two siblings, had complete androgen insensitivity. In one subject, residue 779 was changed from arginine to tryptophan (CGC-->TGG; R779W), another subject (M807V) had a substitution of valine (GTG) for methionine (ATG) residue at position 807, and the two siblings (R855C) had a mutation in residue 855 changing arginine (CGC) to cysteine (TGC). Binding of the synthetic androgen ligand, methyltrienolone (R1881), by the mutant receptor Y763C was decreased by 54% compared to the normal receptor. Transcriptional activation of a mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter gene by AR mutant Y763C was negligible at 0.1 nM R1881 and only 55% at 10 nM R1881 when compared to the maximal response with the normal AR, as assessed by CAT activity. Mutant M807V retained only 22% of normal R1881 binding and mutant R855C was unable to bind the steroid. In accordance with the steroid binding, transcriptional activation of MMTV-CAT by M807V rose to only 26% of control in the presence of 10 nM R1881, a concentration at which R855C remained functionally inactive. In summary, missense mutations within the exons of the androgen receptor gene encoding the steroid-binding domain of the receptor are common causes of both partial and complete forms of androgen insensitivity syndrome.
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PMID:Human androgen insensitivity due to point mutations encoding amino acid substitutions in the androgen receptor steroid-binding domain. 758 99

The catalytic domain of dihydrolipoamide transacylase (E2c) of bovine branched-chain alpha-keto acid dehydrogenase complex (BCKAD) was overexpressed in Escherichia coli. The E2c catalyzes a reversible acyl transfer reaction between acyl-CoA and dihydrolipoamide, which also occurs spontaneously with a much slower rate. The benzene extracts of both the enzyme-catalyzed and the spontaneous reactions mixture have identical ultraviolet absorbance spectra with a maximum at 233-234 nm, which is characteristic of S-acyldihydrolipoamide. The spontaneous reaction rate of various acyl-CoA is in the order of acetoacetyl-CoA > acetyl-CoA > isobutyryl-CoA > isovaleryl-CoA. In other words, the spontaneous acyl transfer is faster when the substituent (R) of acyl-CoA (R-CO-S-CoA) is a more electron-withdrawing group. This result indicates that a negative charge occurs in the substrate during the acyl transfer process. The function of the active-site histidine (His391) and serine (Ser338) of bovine E2c was analyzed by site-directed mutagenesis. Substitution of His391 or Ser338 with alanine caused drastic decreases in catalytic efficiencies by 3-4 orders of magnitude. The residual activity of H391A increased as the pH of the reaction buffer was elevated. These data support the base-catalyzed mechanism inferred from that of chloramphenicol acetyltransferase (CAT). In this reaction, the active-site histidine acts as a general base, and the active-site serine provides a hydrogen bond to the putative negatively charged tetrahedral transition state. Moreover, when Ala348 was changed to valine, the catalytic efficiency for isovaleryl-CoA decreased about 10-fold, and that for acetyl-CoA increased about 3-fold.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Site-directed mutagenesis and functional analysis of the active-site residues of the E2 component of bovine branched-chain alpha-keto acid dehydrogenase complex. 794 94

The androgen receptor (AR) from a patient with Reifenstein syndrome (incomplete androgen insensitivity syndrome) was characterized. The patient's pubic skin fibroblasts had normal androgen binding. However, when incubated at 41 C, fibroblasts from the patient had a marked decrease in androgen binding as compared with normal fibroblasts. Analysis of coding sequences of the androgen receptor gene revealed a single nucleotide substitution in exon E, resulting in an amino acid change from glycine (GGG) to valine (GTG) at amino acid 743 within the steroid binding domain of AR. Reconstruction of this mutation by site-directed mutagenesis into a human AR complementary DNA followed by expression in COS1 cells led to production of a mutant AR with no significant difference in androgen binding when cells were incubated with androgen at room temperature. However, in contrast to wild type AR expressed in COS1 cells, the mutant AR had markedly lower androgen-binding affinity at 41 C. The mutant receptor could still stimulate a reporter gene at 37 C but this transcriptional stimulation was also decreased when compared with wild type AR receptor in a chloramphenicol acetyltransferase assay. These results suggest that partial androgen resistance in this patient with Reifenstein syndrome is due to a single point mutation in the steroid binding domain of the androgen receptor.
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PMID:A single amino acid substitution (gly743 --> val) in the steroid-binding domain of the human androgen receptor leads to Reifenstein syndrome. 832 32

The structure of the type III variant of chloramphenicol acetyltransferase reveals that Thr-174, a conserved residue, is hydrogen-bonded to a bound water molecule (water 252). Modeling studies (P. C. E. Moody and A. G. W. Leslie, unpublished data) suggested that water 252 could play a part in transition state stabilization via a hydrogen bond to the oxyanion of the putative tetrahedral intermediate. In addition, water 252 is one of three bound water molecules hydrogen-bonded to the 1-hydroxyl group of chloramphenicol in the chloramphenicol acetyltransferase-chloramphenicol binary complex. A combination of site-directed mutagenesis and the use of an alternative substrate has allowed the quantitation of the energetic contribution of each of the interactions made by water 252 to catalysis. Thr-174 was replaced by alanine, valine, and isoleucine, each substitution removing the hydroxyl group hydrogen-bonded to water 252. Steady-state kinetic analysis of the mutant enzymes was carried out using both chloramphenicol and 1-deoxy-chloramphenicol as acetyl acceptors. The substitutions at Thr-174 result in a fall in kcat and in decreased affinities for each acetyl acceptor in the binary complexes and also in the ternary complexes with acetyl-CoA. From the calculated free energies in the transition state, the hydrogen bond between water 252 and the oxyanion of the tetrahedral intermediate can be estimated to contribute 0.9 kcal mol-1 toward transition state stabilization, whereas the free energy of the hydrogen bonds between the 1-hydroxyl of chloramphenicol and three bound water molecules provides 1.6 kcal mol-1.
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PMID:Transition state stabilization by chloramphenicol acetyltransferase. Role of a water molecule bound to threonine 174. 840 36

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