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 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 Modeling studies (P. C. E. Moody and A. G. W. Leslie, unpublished data) suggested that water 252 could play a part in transition state stabilization via a hydrogen bond to the oxyanion of the putative tetrahedral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

The functional significance of receptor phosphorylation in mediating the actions of glucocorticoids remains undefined. The identification of seven phosphorylation sites in the mouse glucocorticoid receptor (Bodwell, J. E., Orti, E., Coull, J. M., Pappin, D. J. C., Smith, L. I., and Swift, F. (1991) J. Biol. Chem. 266, 7549-7555) permits a direct examination of the potential regulatory role of glucocorticoid receptor phosphorylation in transactivation. Using oligonucleotide-directed mutagenesis of the mouse glucocorticoid receptor cDNA, we have substituted alanine or aspartate for the residues phosphorylated in this ligand-dependent transcription factor. COS-1 cells were cotransfected with mutant receptor cDNA expression vectors and a reporter plasmid containing the glucocorticoid-inducible mouse mammary tumor virus promoter linked to chloramphenicol acetyltransferase in order to characterize the effect of these substitutions on receptor-mediated gene expression. Substitution of alanine or aspartate at single phosphorylation sites does not prevent receptor transactivation. Receptors containing multiple substitutions of alanine or aspartate at the major phosphorylation sites in the acidic domain elicit levels of hormone-induced reporter gene expression that are comparable to wild-type receptors. Mutant receptors substituted with alanine at the five phosphorylation sites conserved among the rat, human, and mouse receptors exhibit a 22% decrease in transcriptional activity. Receptors mutated at all seven sites display a similar modest reduction. These results demonstrate that receptor phosphorylation at these seven identified residues is not a major determinant in glucocorticoid receptor transcriptional activity at the mouse mammary tumor virus promoter.
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PMID:Site-directed mutagenesis of the phosphorylation sites in the mouse glucocorticoid receptor. 840 99

A catalytically essential histidine residue (His-195) of chloramphenicol acetyltransferase (CAT) acts as a general base in catalysis, abstracting a proton from the primary hydroxy group of chloramphenicol. The pKa of His-195 has been determined from the pH-dependence of chemical modification. Both methyl 4-nitrobenzenesulphonate and iodoacetamide inactivate CAT by irreversible modification of His-195. The kinetics of inactivation by methyl 4-nitrobenzenesulphonate are pseudo-first-order, and the pH-dependence of inactivation yields a pKa value of 6.60. Iodoacetamide inactivation proceeds with second-order kinetics and a pKa value of 6.80. An alternative site of modification at the active site of CAT is the thiol group of Cys-31, a residue which has no catalytic role. On replacement of Cys-31 with alanine (Ala-31 CAT), the pH-dependence of iodoacetamide inactivation gives a pKa value of 6.66. The pKa values derived from chemical-modification experiments directed at His-195 are in agreement with the pKa values of 6.62 and 6.61 determined for wild-type and Ala-31 CAT respectively from the pH-dependence of kcat/Km.
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PMID:The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase. 843 83

The influenza A virus nucleoprotein (NP) is a phosphoprotein that encapsidates the viral genomic RNA. To map the in vivo phosphorylation site(s) of this protein, 32P-labeled NP was purified from cell cultures infected with influenza virus A/Victoria/3/75 by immunoaffinity chromatography. The purified protein was then subjected to chemical digestion with formic acid, which cleaves proteins at Asp-Pro bonds, and the resulting products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two of the phosphorylated products obtained were identified as fragments corresponding to the N-terminal 88 amino acids and to the C-terminal 196 residues of the NP. To identify the phosphate acceptor site(s) at the N-terminal phosphorylated region of NP, each of the seven serines within this region was individually changed to alanine by site-directed mutagenesis. The mutant proteins were then transiently expressed in mammalian cells and analyzed for their phosphorylation state. It was observed that the S-to-A mutation at position 3 drastically reduced the amount of 32P label incorporated into NP, whereas the other substitutions did not have a discernible effect on the phosphorylation level of the protein. In addition, all serine-altered proteins were tested for their functionality in an artificial system in which expression of a synthetic chloramphenicol acetyl-transferase RNA molecule is driven by influenza virus proteins synthesized from cloned genes. The results obtained demonstrate that all mutant proteins were competent to cooperate with the subunits of the viral polymerase for expression of the synthetic virus-like chloramphenicol acetyltransferase RNA in vivo. These data are discussed regarding the possible roles of NP phosphorylation for the viral replicative cycle.
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PMID:Serine 3 is critical for phosphorylation at the N-terminal end of the nucleoprotein of influenza virus A/Victoria/3/75. 864 69

A 25-residue peptide representing the membrane targeting N-terminal splice region of the cyclic AMP phosphodiesterase RD1 (RNPDE4A1) was synthesized, and its structure was determined by 1H NMR. Two independently folding helical regions were identified, separated by a highly mobile "hinge" region. The first helical region was formed by an N-terminal amphipathic alpha-helix, and the second consisted of multiple overlapping turns and contained a distinct compact, hydrophobic, tryptophan-rich domain (residues 14-20). Chimeric molecules, formed between the N-terminal region of RD1 and the soluble bacterial protein chloramphenicol acetyltransferase, were used in an in vitro system to determine the features within the splice region that were required for membrane association. The ability of RD1-chloramphenicol acetyltransferase chimera to become membrane-associated was not affected by deletion of any of the following regions: the apolar section (residues 2-7) of the first helical region, the polar part of this region together with the hinge region (residues 8-13), or the polar end of the C-terminal helical region (residues 21-25). In marked contrast, deletion of the compact, hydrophobic tryptophan-rich domain (residues 14-20) found in the second helical region obliterated membrane association. Replacement of this domain with a hydrophobic cassette of seven alanine residues also abolished membrane association, indicating that membrane-association occurred by virtue of specific hydrophobic interactions with residues within the compact, tryptophan-rich domain. The structure of this domain is well defined in the peptide, and although the region is helical, both the backbone and the distribution of side chains are somewhat distorted as compared with an ideal alpha-helix. Hydrophobic interactions, such as the "stacked" rings of residues Pro14 and Trp15, stabilize this domain with the side chain of residue Leu16 adopting a central position, interacting with the side chains of all three tryptophan residues 15, 19, and 20. These bulky side chains thus form a hydrophobic cluster. In contrast, the side chain of residue Val17 is relatively exposed, pointing out from the opposite "face" of the peptide. Although it appears that this compact, tryptophan-rich domain is responsible for membrane association, at present the target site and hence the specific interactions involved in membrane targeting by the RD1 splice region remain unidentified.
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PMID:Determination of the structure of the N-terminal splice region of the cyclic AMP-specific phosphodiesterase RD1 (RNPDE4A1) by 1H NMR and identification of the membrane association domain using chimeric constructs. 866 81

A cationic peptide amphiphile comprising an L-alanine residue interposed between a charged head group and a double-chain segment, N,N-dihexadecyl-N alpha-[6-(trimethylammonio)- hexanoyl]-L-alaninamide bromide (NC5Ala2C16), was synthesized and used to prepare sonicated liposomes. We examined the efficiency of this liposome in gene transfer according to the transient expression of chloramphenicol acetyltransferase (CAT). This cationic liposome reagent facilitates efficient DNA transfection in COS-7 cells. We determined the optimum conditions for NC5Ala2C16 liposome-mediated transfection. The optimal amounts of the amphiphile and plasmid DNA were determined to be about 100 micrograms and 10 micrograms per 35-mm dish, respectively. The activity of this liposome was greater than that of commercial reagents, lipofectin, and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate (DOTAP), and it was less toxic than lipofectin and DOTAP in COS-7 cells.
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PMID:Synthetic cationic amphiphile for liposome-mediated DNA transfection with less cytotoxicity. 879 87

The molecular signal for targeting catalases to peroxisomes has not been defined. In this study, a plant in vivo import system (tobacco BY-2 suspension culture cells) was used to test the current postulate that the peroxisome targeting signal (PTS) for mammalian catalases is the internal Ser-Lys-Leu (SKL) motif found approximately eight amino acid residues from the C-terminus. Elucidation of the catalase PTS has been hampered previously by the ubiquitous presence of catalase in peroxisomes. The current study was possible because antibodies to mammalian catalases did not recognize endogenous, tobacco peroxisome catalase. Rat and mouse liver catalases (Rcat and Mcat), with an internal Ser-His-Ile (SHI) and Ser-His-Met (SHM), respectively, and both with a C-terminal Ala-Asn-Leu (ANL), were expressed transiently in BY-2 cells and targeted to the peroxisomes. Sorting was demonstrated by double-label immunofluorescence colocalization of these catalases with tobacco catalase. Peroxisome targeting of Rcat was abolished as expected when the internal SHI residues were removed by deletion of three C-terminal portions (28, 16, or 11 residues). Surprisingly, peroxisome targeting was still abolished when SHI (or SHL produced by site-directed mutagenesis) were at the extreme C-terminus as a consequence of deleting eight residues. However, when SHL was at the C-terminus in full-sized Rcat via a mutation of ANL-COOH, the enzyme sorted to peroxisomes indicating that the position of the PTS is significant in Rcat. The importance of the internal context of the SHI (or SHL) was examined further by changing ANL-COOH to a non-SKL motif, AGS-COOH. This Rcat did not sort to the peroxisomes, nor did Rcat with its ANL-COOH deleted; these data indicated the necessity of the C-terminal tripeptide. Sufficiency of ANL was demonstrated when chloramphenicol acetyltransferase with an appended ANL-COOH was redirected from the cytosol to peroxisomes. Collectively, these results do not support the internal PTS hypothesis, but indicate that a type 1 PTS slightly divergent from the typical SKL motif serves as the necessary and sufficient PTS for rat liver and probably other eukaryotic catalases.
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PMID:Rat liver catalase is sorted to peroxisomes by its C-terminal tripeptide Ala-Asn-Leu, not by the internal Ser-Lys-Leu motif. 892 63

Heat shock transcription factor 1 (HSF1) is constitutively expressed in mammalian cells and negatively regulated for DNA binding and transcriptional activity. Upon exposure to heat shock and other forms of chemical and physiological stress, these activities of HSF1 are rapidly induced. In this report, we demonstrate that constitutive phosphorylation of HSF1 at serine residues distal to the transcriptional activation domain functions to repress transactivation. Tryptic phosphopeptide analysis of a collection of chimeric GAL4-HSF1 deletion and point mutants identified a region of constitutive phosphorylation encompassing serine residues 303 and 307. The significance of phosphorylation at serines 303 and 307 in the regulation of HSF1 transcriptional activity was demonstrated by transient transfection and assay of a chloramphenicol acetyltransferase reporter construct. Whereas the transfected wild-type GAL4-HSF1 chimera is repressed for transcriptional activity and derepressed by heat shock, mutation of serines 303 and 307 to alanine results in derepression to a high level of constitutive activity. Similar results were obtained with mutation of these serine residues in the context of full-length HSF1. These data reveal that constitutive phosphorylation of serines 303 and 307 has an important role in the negative regulation of HSF1 transcriptional activity at control temperatures.
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PMID:Repression of the heat shock factor 1 transcriptional activation domain is modulated by constitutive phosphorylation. 912 59

Transcriptional regulation of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in hepatic gluconeogenesis, by insulin was investigated with the use of adenovirus vectors encoding various mutant signaling proteins. Insulin inhibited transcription induced by dexamethasone and cAMP of a chloramphenicol acetyltransferase (CAT) reporter gene fused with the PEPCK promoter sequence in HL1C cells stably transfected with this construct. A dominant negative mutant of phosphoinositide (PI) 3-kinase blocked insulin inhibition of transcription of the PEPCK-CAT fusion gene, whereas a constitutively active mutant of PI 3-kinase mimicked the effect of insulin. Although a constitutively active mutant of Akt (protein kinase B) inhibited PEPCK-CAT gene transcription induced by dexamethasone and cAMP, a mutant Akt (Akt-AA) in which the phosphorylation sites targeted by insulin are replaced by alanine did not affect the ability of insulin to inhibit transcription of the fusion gene. Akt-AA almost completely inhibited insulin-induced activation of both endogenous and recombinant Akt in HL1C cells. Furthermore, neither a kinase-defective mutant protein kinase Clambda (PKClambda), which blocked insulin-induced activation of endogenous PKClambda, nor a dominant negative mutant of the small GTPase Rac prevented inhibition of PEPCK-CAT gene transcription by insulin. These data suggest that phosphoinositide 3-kinase is important for insulin-induced inhibition of PEPCK gene transcription and that a downstream effector of phosphoinositide 3-kinase distinct from Akt, PKClambda, and Rac may exist for mediating the effect of insulin.
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PMID:Dominant negative forms of Akt (protein kinase B) and atypical protein kinase Clambda do not prevent insulin inhibition of phosphoenolpyruvate carboxykinase gene transcription. 1040 89

Viral expression systems allow for the rapid production of large amounts of recombinant protein in cell culture. In particular, Sindbis virus vectors now exist that make possible the expression of a variety of heterologous proteins in mammalian culture systems. Unfortunately, infection of cultured cells with Sindbis virus vectors typically results in apoptotic cell death, as demonstrated in the current study by DNA laddering and fluorescence microscopy. Fortunately, it has recently been demonstrated that apoptosis can be inhibited in vitro by certain chemical reagents that are capable of blocking specific steps during the cell death cascade. In this study, a rat prostate carcinomal cell line, AT3-neo, was infected with a Sindbis virus vector containing the gene for chloramphenicol acetyltransferase (dsSV-CAT) in the presence of several representative antiapoptotic chemicals and analyzed for cell viability as well as recombinant protein production. N-acetylcysteine (NAC), pyrrolidine dithiocarbamate (PDTC), bongkrekic acid (BA), and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) all exhibited the capacity to limit apoptosis in the infected cells. In fact, after just 1 day, percentage viabilities of the cells exposed to chemical reagents were between 72% and 91%, compared with 44% for the untreated controls. Furthermore, cells maintained on these agents were able to survive the infection from 1 to 3 days longer than the control samples. In addition to providing gains in cell viability, chemical treatment allowed for higher levels of recombinant protein production in most cases. Maximum chloramphenicol acetyltransferase (CAT) productivities in cells maintained on BA, NAC, and Z-VAD.fmk were 1.7-, 2.2-, and 3.9-fold higher than those obtained from the untreated cultures. Consequently, the addition of chemical reagents to culture media as a means of inhibiting apoptosis may be a valuable tool in the cell culture industry, where cell death severely limits productivity levels and adds significantly to production costs.
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PMID:Antiapoptosis chemicals prolong productive lifetimes of mammalian cells upon Sindbis virus vector infection. 1048 28


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