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)

Transfection of several cell lines (HeLa, COS, PC-12, CA-77, and H4IIE C3) with pRSV-CAT by a variety of methods yielded rather low chloramphenicol acetyltransferase (CAT) activity in cell extracts. Extracts of these cells were found to interfere with the assay of added CAT. The extracts were capable of deacetylating acetylchloramphenicol and of accelerating the rate of hydrolysis of the acetyl-CoA present in the assay. Heating the cell extract to 60 degrees C for 10 min completely prevented the interference and slowed the hydrolysis of acetyl-CoA. Substantially higher CAT activities were observed when the extract was heat treated in the presence of EDTA prior to enzyme assay for most cell lines tested. This simple reliable method makes possible the accurate assessment of CAT activities in different cell lines. These observations are particularly pertinent to investigators studying tissue-specific gene expression.
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PMID:A method for increasing the sensitivity of chloramphenicol acetyltransferase assays in extracts of transfected cultured cells. 347 89

Naturally occurring chloramphenicol resistance in bacteria is normally due to the presence of the antibiotic inactivating enzyme chloramphenicol acetyltransferase (CAT) which catalyzes the acetyl-S-CoA-dependent acetylation of chloramphenicol at the 3-hydroxyl group. The product 3-acetoxy chloramphenicol does not bind to bacterial ribosomes and is not an inhibitor of peptidyltransferase. The synthesis of CAT is constitutive in E. coli and other Gram-negative bacteria which harbor plasmids bearing the structural gene for the enzyme, whereas Gram-positive bacteria such as staphylococci and streptococci synthesize CAT only in the presence of chloramphenicol and related compounds, especially those with the same stereochemistry of the parent compound and which lack antibiotic activity and a site of acetylation (3-deoxychloramphenicol). Studies of the primary structures of CAT variants suggest a marked degree of heterogeneity but conservation of amino acid sequence at and near the putative active site. All CAT variants are tetramers composed in each case of identical polypeptide subunits consisting of approximately 220 amino acids. The catalytic mechanism does not appear to involve an acyl-enzyme intermediate although one or more cysteine residues are protected from thiol reeagents by substrates. A highly reactive histidine residue has been implicated in the catalytic mechanism.
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PMID:Chloramphenicol acetyltransferase: enzymology and molecular biology. 634 Sep 55

The Escherichia coli Tn9 derived chloramphenicol resistance gene (camr) is functionally expressed in the yeast Saccharomyces cerevisiae. This gene was introduced into yeast cells as part of a hybrid yeast/E. coli shuttle plasmid. A number of plasmid associated yeast mutants overproducing the camr gene product, chloramphenicol acetyltransferase (acetyl-CoA: chloramphenicol 3-0-acetyltransferase, E.C. 2.3.1.28) were isolated. One of the plasmid mutants was analyzed in some detail. Even though this mutant showed a 1,000 fold overproduction of chloramphenicol acetyltransferase in the yeast host the level of RNA complementary to the camr gene was not increased. A deletion of 127 base pairs in the region immediately upstream from the 5' end of the camr gene appeared to be responsible for the "up" phenotype of this mutant. This mutation affected the expression of the camr gene in E. coli in a "down" fashion, in contrast to its effect in yeast.
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PMID:Expression of a prokaryotic gene in yeast: isolation and characterization of mutants with increased expression. 635 67

The mechanism of the enzymic reaction responsible for chloramphenicol resistance in bacteria was examined by steady-state kinetic methods. The forward reaction catalysed by chloramphenicol acetyltransferase leads to inactivation of the antibiotic. Use of alternative acyl donors and acceptors, as well as the natural substrates, has yielded data that favour the view that the reaction proceeds to the formation of a ternary complex by a rapid-equilibrium mechanism wherein the addition of substrates may be random but a preference for acetyl-CoA as the leading substrate can be detected. Chloramphenicol and acetyl-CoA bind independently, but the correlation between directly determined and kinetically derived dissociation constants is imperfect because of an unreliable slope term in the rate equation. The reverse reaction, yielding acetyl-CoA and chloramphenicol, was studied in a coupled assay involving citrate synthase and malate dehydrogenase, and is best described by a rapid-equilibrium mechanism with random addition of substrates. The directly determined dissociation constant for CoA is in agreement with that derived from kinetic measurements under the assumption of an independent-sites model.
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PMID:Analysis of the mechanism of chloramphenicol acetyltransferase by steady-state kinetics. Evidence for a ternary-complex mechanism. 659 36

A fusion gene construct, in which the coding sequence for bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA: chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) was placed under the control of the regulatory region of the Drosophila gene encoding the 70-kilodalton heat shock protein [Di Nocera, P.P. & Dawid, I.B. (1983) Proc. Natl. Acad. Sci. USA 80, 7095-7098], was microinjected into the cytoplasm of unfertilized sea urchin eggs. Pluteus-stage embryos developing from the injected eggs were exposed to high temperature conditions that we found would elicit an endogenous sea urchin heat shock response. These embryos express the gene for CAT and, after heat treatment, display 8-10 times more CAT enzyme activity than do extracts from control embryos cultured at normal temperatures. The injected DNA is present in high molecular weight concatenates and, during development, is amplified about 100-fold. Amplified sequences are responsible for all or most of the induced CAT enzyme activity.
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PMID:Inducible expression of a cloned heat shock fusion gene in sea urchin embryos. 659 99

The Escherichia coli R factor-derived chloramphenicol resistance (camr) gene is functionally expressed in the yeast Saccharomyces cerevisiae. the gene was introduced by transformation into yeast cells as part of a chimeric plasmid, pYT11-LEU2, constructed in vitro. The plasmide vector consists of the E. coli plasmid pBR325 (carrying the camr gene), the yeast 2-micron DNA plasmid, and the yeast LEU2 structural gene. Yeast cells harboring pYT11-LEU2 acquire resistance to chloramphenicol and cell-free extracts prepared from such cells contain chloramphenicol acetyltransferase (acetyl-CoA: chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28), the enzyme specified by the camr gene in E. coli. Resistance to chloramphenicol and the presence of chloramphenicol acetyltransferase activity segregate with the yeast marker LEU2, carried by the transforming plasmid, during both mitotic growth and meiotic division.
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PMID:Functional expression in yeast of the Escherichia coli plasmid gene coding for chloramphenicol acetyltransferase. 698 64

The antibiotic fusidic acid and certain closely related steroidal compounds are potent competitive inhibitors of the type I variant of chloramphenicol acetyltransferase (CATI). In the absence of crystallographic data for CATI, the structural determinants of steroid binding were identified by (1) construction in vitro of genes encoding chimaeric enzymes containing segments of CATI and the related type III variant (CATIII) and (2) site-directed mutagenesis of the gene encoding CATIII, followed by kinetic characterisation of the substituted variants. Replacement of four residues of CATIII (Gln92, Asn146, Tyr168 and Ile172) by their equivalents from CATI yields an enzyme variant that is susceptible to competitive inhibition by fusidate with respect to chloramphenicol (Ki = 5.4 microM). The structure of the complex of fusidate and the Q92C/N146F/Y168F/I172V variant, determined at 2.2 A resolution by X-ray crystallography, reveals the inhibitor bound deep within the chloramphenicol binding site and in close proximity to the side-chain of His195, an essential catalytic residue. The aromatic side-chain of Phe146 provides a critical hydrophobic surface which interacts with non-polar substituents of the steroid. The remaining three substitutions act in concert both to maintain the appropriate orientation of Phe 146 and via additional interactions with the bound inhibitor. The substitution of Gln92 by Cys eliminates a critical hydrogen bond interaction which constrains a surface loop (residues 137 to 142) of wild-type CATIII which must move in order for fusidate to bind to the enzyme. Only two hydrogen bonds are observed in the CAT-fusidate complex, involving the 3-alpha-hydroxyl of the A-ring and both hydroxyl of Tyr25 and NE2 of His195, both of which are also involved in hydrogen bonds with substrate in the CATIII-chloramphenicol complex. In the acetyl transfer reaction catalysed by CAT, NE2, of His195 serves as a general base in the abstraction of a proton from the 3-hydroxyl of chloramphenicol as the first chemical step in catalysis. The structure of the CAT-inhibitor complex suggests that deprotonation of the 3-alpha-hydroxyl of bound fusidate by this mechanism could produce an oxyanion nucleophile analogous to that seen with chloramphenicol, but one which is incorrectly positioned to attack the thioester carbonyl of acetyl-CoA, accounting for the observed failure of CAT to acetylate fusidate.
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PMID:Steroid recognition by chloramphenicol acetyltransferase: engineering and structural analysis of a high affinity fusidic acid binding site. 750 Mar 66

ATP-citrate lyase (CL) catalyzes the conversion of citrate and CoA to oxaloacetate (OA) and acetyl-CoA. As the coupled malic dehydrogenase (MDH) assay is not able either to study the effect of oxaloacetate (OA) on CL activity or to measure accurately CL activity in biological samples, a new assay was developed. The CL-citrate coupled CAT assay measures the amount of acetyl-CoA formed by transferring radiolabeled acetyl-CoA synthesized from [14C]citrate to chloramphenicol with chloramphenicol acetyltransferase (CAT). Employing this assay, the rate of increase in acetyl-CoA synthesis from citrate is linear with respect to added CL. Kinetic values for ATP, CoA and citrate are similar to those obtained using the MDH assay. The effect of CL phosphorylation on enzyme activity was determined. CL phosphorylated by cAMP-dependent protein kinase or by this kinase and glycogen synthase kinase-3 (GSK-3) decreases the apparent Vmax without changing the apparent Km. The effect of OA, a product of the enzyme reaction, on CL activity was also determined. Computational analysis of the data obtained without added OA and at three concentrations of OA indicate that the apparent Km for the substrate is not altered even though the apparent Vmax is decreased. The effect of OA on the activity of phosphorylated enzyme was also determined. OA decreases the apparent Vmax of the phosphorylated enzyme to the same extent as in control CL. This assay is able to measure CL activity in cytosol from 3T3-L1 adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of oxaloacetate and phosphorylation on ATP-citrate lyase activity. 766 53

The imidazole N epsilon 2 of His-195 plays an essential part in the proposed general base mechanism of chloramphenicol acetyltransferase (CAT), hydrogen bonding to and a abstracting a proton from the primary hydroxyl group of chloramphenicol. Replacement of His-195 by alanine or glutamine results in apparent decreases in kcat of (9 x 10(5)- and (3 x 10(5))-fold, respectively, whereas Km values for both substrates (chloramphenicol and acetyl-CoA) are similar to those of wild-type CAT. The structure of Gln-195 CAT has been solved at 2.5-A resolution and is largely isosteric with that of wild-type CAT. Substitution of His-195 by glutamate resulted in a (5 x 10(4))-fold decrease in kcat together with a 3-fold increase in the Km for chloramphenicol. Direct determination of binding constants for both substrates demonstrated that these substitutions result in only small decreases in the affinity of CAT for acetyl-CoA (Kd values increased 2- to 3-fold), whereas chloramphenicol Kd values are elevated 26-, 20-, and 53-fold for Ala-195 CAT, Gln-195 CAT and Glu-195 CAT, respectively. The pH dependence of kcat/Km yields apparent pKa values of 6.5 and 6.7 for Ala-195 CAT and Gln-195 CAT, respectively, which are very similar to that (6.6) determined for the ionization of His-195 in wild-type CAT. In contrast, the pH dependence of kcat/Km for Glu-195 CAT (pKa = 8.3) is very different from that of wild-type CAT.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Replacement of catalytic histidine-195 of chloramphenicol acetyltransferase: evidence for a general base role for glutamate. 790 44

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


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