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)

We have constructed a small, transposition-defective derivative of the transposon Tn10 that carries the chloramphenicol acetyltransferase gene of pACYC184. This new genetic element, Tn10d-Cam, transposes when Tn10 transposase is provided from a multi-copy plasmid. Transposon insertion mutagenesis of Salmonella typhimurium was performed by using a strain carrying a Tn10d-Cam insertion in an Escherichia coli F' episome as the donor in transductional crosses into recipients that carried a plasmid expressing Tn10 transposase. Tn10d-Cam insertion mutations were also generated by complementation in cis of Tn10d-Cam by a cotransducible Tn10 element that overproduces transposase. Here, transposase was provided only transiently, and the Tn10d-Cam insertion mutations were recovered in a transposase-free strain. Cis complementation was used for mutagenesis of a plasmid target. The site specificity of insertion and the effect of insertions on expression of a downstream gene were investigated, using Tn10d-Cam insertions in a plasmid carrying a segment of the histidine operon.
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PMID:Characterization of Tn10d-Cam: a transposition-defective Tn10 specifying chloramphenicol resistance. 284 6

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

Chloramphenicol acetyltransferase [acetyl-CoA:chloramphenicol O3-acetyltransferase; EC 2.3.1.28] is the enzyme responsible for high-level bacterial resistance to the antibiotic chloramphenicol. It catalyzes the transfer of an acetyl group from acetyl CoA to the primary hydroxyl of chloramphenicol. The x-ray crystallographic structure of the type III variant enzyme from Escherichia coli has been determined and refined at 1.75-A resolution. The enzyme is a trimer of identical subunits with a distinctive protein fold. Structure of the trimer is stabilized by a beta-pleated sheet that extends from one subunit to the next. The active site is located at the subunit interface, and the binding sites for both chloramphenicol and CoA have been characterized. Substrate binding is unusual in that the two substrates approach the active site via clefts on opposite molecular "sides." A histidine residue previously implicated in catalysis is appropriately positioned to act as a general base catalyst in the reaction.
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PMID:Structure of chloramphenicol acetyltransferase at 1.75-A resolution. 328 84

Bacterial resistance to the antibiotic chloramphenicol is normally mediated by chloramphenicol acetyltransferase (CAT), which utilizes acetyl coenzyme A as the acyl donor in the inactivation reaction. 3-(Bromoacetyl)chloramphenicol, an analogue of the acetylated product of the forward reaction catalyzed by CAT, was synthesized as a probe for accessible and reactive nucleophilic groups within the active site. Extremely potent covalent inhibition was observed. Affinity labeling was demonstrated by the protection afforded by chloramphenicol at concentrations approaching Km for the substrate. Inactivation was stoichiometric, 1 mol of the inhibitor covalently bound per mole of enzyme monomer, with complete loss of both the acetylation and hydrolytic activities associated with CAT. N3-(Carboxymethyl)histidine was identified as the only alkylated amino acid, implicating the presence of a unique tautomeric form of a reactive imidazole group at the catalytic center. The proteolytic digestion of CAT modified with 3-(bromo[14C]-acetyl)chloramphenicol yielded three labeled peptide fractions separable by reverse-phase high-pressure liquid chromatography. Each peptide fraction was sequenced by fast atom bombardment mass spectrometry; the labeled peptide in each case was found to span the highly conserved region in the primary structure of CAT, which had been tentatively assigned as the active site. The rapid, stoichiometric, and specific alkylation of His-189, taken together with the high degree of conservation of the adjacent amino acid residues, strongly suggests a central role for His-189 in the catalytic mechanism of CAT.
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PMID:3-(Bromoacetyl)chloramphenicol, an active site directed inhibitor for chloramphenicol acetyltransferase. 386 88

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

Dihydrolipoamide acetyltransferase (E2p) is the structural and catalytic core of the pyruvate dehydrogenase multienzyme complex. In Azotobacter vinelandii E2p, residues Ser558, His610', and Asn614' are potentially involved in transition state stabilization, proton transfer, and activation of proton transfer, respectively. Three active site mutants, S558A, H610C, and N614D, of the catalytic domain of A. vinelandii E2p were prepared by site-directed mutagenesis and enzymatically characterized. The crystal structures of the three mutants have been determined at 2.7, 2.5, and 2.6 A resolution, respectively. The S558A and H610C mutants exhibit a strongly (200-fold and 500-fold, respectively) reduced enzymatic activity whereas the substitution of Asn614' by aspartate results in a moderate (9-fold) reduced activity. The decrease in enzymatic activity of the S558A and H610C mutants is solely due to the absence of the hydroxyl and imidazole side chains, respectively, and not due to major conformational rearrangements of the protein. Furthermore the sulfhydryl group of Cys610' is reoriented, resulting in a completely buried side chain which is quite different from the solvent-exposed imidazole group of His610' in the wild-type enzyme. The presence of Asn614' in A. vinelandii E2p is exceptional since all other 18 known dihydrolipoamide acyltransferase sequences contain an aspartate in this position. We observe no difference in conformation of Asp614' in the N614D mutant structure compared with the conformation of Asn614' in the wild-type enzyme. Detailed analysis of all available structures and sequences suggests two classes of acetyltransferases: one class with a catalytically essential His-Asn pair and one with a His-Asp-Arg triad as present in chloramphenicol acetyltransferase [Leslie, A. G. W. (1990) J. Mol. Biol. 213, 167-186] and in the proposed active site models of Escherichia coli and yeast E2p.
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PMID:Crystallographic and enzymatic investigations on the role of Ser558, His610, and Asn614 in the catalytic mechanism of Azotobacter vinelandii dihydrolipoamide acetyltransferase (E2p). 770 42

Alteration of the charge of surface lysyl residues of chloramphenicol acetyltransferase (CAT) by site-directed mutagenesis was used to increase the charge difference between the subunits of two naturally occurring enzyme variants (CATI and CATIII). The introduced charge change greatly facilitates the purification of CATI/CATIII and CATIII/CATIII hybrid trimers by ion-exchange chromatography. Hybrids containing only one functional active site per trimer were generated in vitro by reversible denaturation of mixtures of "active" subunits (retention of a catalytic histidine at position 195) and "inactive" subunits (with alanine replacing histidine 195). Such hybrids were used (1) to demonstrate that the previously observed novel binding of a steroidal antibiotic (fusidic acid) by CATI involves amino acid residues at each subunit interface and (2) to identify specific residues contributing to such interactions. A pre-steady-state kinetic characterization of homotrimers containing the H195A substitution also revealed that fusidate binding to CATI may, like chloramphenicol binding, involve a hydrogen bond with the catalytic histidine residue. In addition, confirmation of the fact that His-195 interacts with chloramphenicol in CATI as well as in CATIII makes it likely that it is essential for the catalytic mechanism of all naturally occurring variants of CAT, as first suggested by structural evidence for the type III enzyme (Leslie, 1990).
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PMID:Properties of hybrid active sites in oligomeric proteins: kinetic and ligand binding studies with chloramphenicol acetyltransferase trimers. 775 72

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

Site-directed mutagenesis was employed to make two single amino acid substitutions for highly conserved amino acid residues near the C-terminus of the 783-amino acid mouse glucocorticoid receptor. Substitution of leucine for histidine-781 caused little or no change in the concentration of dexamethasone required for half-maximal activation of a chloramphenicol acetyltransferase reporter gene expressed from a mouse mammary tumor virus promoter. However, when phenylalanine-780 was changed to alanine, the half-maximal concentrations of various agonists were increased as follows, compared with the wild-type glucocorticoid receptor: triamcinolone acetonide by 7-fold, dexamethasone by 25-fold, and hydrocortisone and deoxycorticosterone by more than 150-fold. Binding of labeled steroids by the mutant receptor in vitro and in vivo was also decreased. In contrast, this mutation caused a small decrease in the concentration of RU486 required for antagonist or partial agonist activity. Thus, the phenyl group of phenylalanine-780 of the mouse glucocorticoid receptor is an important determinant of ligand binding affinity and specificity.
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PMID:Phenylalanine-780 near the C-terminus of the mouse glucocorticoid receptor is important for ligand binding affinity and specificity. 805 63


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