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 aceEF-lpd operon of Escherichia coli encodes the pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3) subunits of the pyruvate dehydrogenase multienzyme complex (PDH complex). An isopropyl beta-D-thiogalactopyranoside-inducible expression system was developed for amplifying fully lipoylated wild-type and mutant PDH complexes to over 30% of soluble protein. The extent of lipoylation was related to the degree of aeration during amplification. The specific activities of the isolated PDH complexes and the E1p component were 50-75% of the values normally observed for the unamplified complex. This could be due to altered stoichiometries of the overproduced complexes (higher E3 and lower E1p contents) or inactivation of E1p. The chaperonin, GroEL, was identified as a contaminant which copurifies with the complex. Site-directed substitutions of an invariant glycine residue (G231A, G231S and G231M) in the putative thiamine pyrophosphate-binding fold of the E1p component had no effect on the production of high-molecular-mass PDH complexes but their E1p and PDH complex activities were very low or undetectable, indicating that G231 is essential for the structural or catalytic integrity of E1p. A minor correction to the nucleotide sequence, which leads to the insertion of an isoleucine residue immediately after residue 273, was made. Substitution of the conserved histidine and arginine residues (H602 and R603) in the putative active-site motif of the E2p subunit confirmed that H602 of the E. coli E2p is essential, whereas R603 could be replaced without inactivating E2p. Deletions affecting putative secondary structural elements at the boundary of the E2p catalytic domain inhibited catalytic activity without affecting the assembly of the E2p core or its ability to bind E1p, indicating that the latter functions are determined elsewhere in the domain. The results further consolidate the view that chloramphenicol acetyltransferase serves as a useful structural and functional model for the catalytic domain of the lipoate acyltransferases.
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PMID:Overproduction of the pyruvate dehydrogenase multienzyme complex of Escherichia coli and site-directed substitutions in the E1p and E2p subunits. 144 21

Hybrid promoter constructs were used to determine the DNA sequence requirements for stringent and growth rate control within a promoter region. The promoters were obtained by fusing complementing sequence regions located upstream and downstream from the GCGC discriminator motif of the growth rate regulated rRNA P1 promoter and a non-regulated tac promoter variant. The activities and the regulatory response of the hybrid promoters were determined in vivo using a promoter test vector system with the chloramphenicol acetyltransferase (CAT) reporter gene. Measurements were made at different growth rates and after starvation for isoleucine to induce the stringent response. Neither the upstream nor the downstream sequence of P1 relative to the GCGC discriminator motif conferred comparable regulatory features when fused to the complementing sequences of the non-regulated mutant tac promoter. A minor response to amino acid deprivation or changes in the growth rate was noted for the hybrid promoter with the rrnB P1 upstream segment and the tac downstream element, pointing to a slightly different importance of the two sequence elements for regulation. The parallel effects for stringent as well as growth rate regulation of the hybrid promoters supports the view of a common mechanism for both types of control. However, none of the promoter sequence elements on its own was able to restore the complete regulatory behaviour of their 'parent' promoters.
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PMID:The signal for growth rate control and stringent sensitivity in E. coli is not restricted to a particular sequence motif within the promoter region. 224 74

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

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 control of the expression of the pilin gene (pilE) in Neisseria gonorrhoeae under a wide variety of growth conditions has been studied. The expression of pilE was measured using transcriptional fusions between pilE and the gene encoding chloramphenicol acetyltransferase (CAT), and the level of pilin production was measured by Western blot analysis. Many of the conditions tested affected both growth rate and pilin gene expression (e.g. isoleucine, high osmolarity, high temperature, anaerobic growth, pH 6, urea and iron depletion). Changes in the level of many other proteins were also observed, depending on the conditions, indicating that gonococci undergo an adaptive response to environmental variations. Moreover, environment-induced changes in the level of many proteins, including pilin, seem to involve the pilA/pilB regulatory system, which has been previously proposed to modulate the expression of the gonococcal pilin gene.
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PMID:Control of Neisseria gonorrhoeae pilin gene expression by environmental factors: involvement of the pilA/pilB regulatory genes. 916 25

The human immunodeficiency virus type 1 Vpr is a virion-associated protein that is incorporated in trans into viral particles, presumably via an interaction with the p6 domain of the Gag polyprotein precursor. Recently, several studies demonstrated that Vpr fusion proteins could be used as intravirion inactivating agents. In this study, we compared different Vpr-chloramphenicol acetyltransferase (CAT) fusion proteins for their virion incorporation ability and their effect on the infectivity of HIV viruses. Our deletion analysis indicates that both the N-terminal alpha-helical domain and the leucine/isoleucine-rich (LR) domain located in the middle region of Vpr are required for optimal virion incorporation of Vpr-CAT fusion proteins. The C-terminal basic region, associated with Vpr's ability to mediate cell cycle arrest in G2, was not required for virion incorporation, thus allowing the development of Vpr-based chimeric proteins devoid of any effect on cell growth. The fusion of Vpr at the N- or C-terminus of CAT targeted with equal efficiency the chimeric protein into virions. While the virion incorporation of most Vpr-CAT fusion proteins tested in this study was dependent on the presence of an intact p6 domain, fusion proteins containing only the N-terminal alpha-helical domain of Vpr (amino acid 1 to 42) were incorporated into virions in a p6-independent manner. Virion incorporation of Vpr-CAT fusion proteins was shown to decrease viral infectivity. Moreover, the insertion of HIV protease-cleavage sites between Vpr and CAT not only efficiently delivered and released the cleaved CAT product into HIV viral particles, but also greatly potentiated the inhibition of progeny virion infectivity. Overall, our study: (1) defines the Vpr sequence requirement and configuration necessary for the specific and optimal incorporation of Vpr fusion protein into HIV particles; (2) shows that Vpr fusion proteins have the ability to suppress HIV infectivity by targeting multiple steps of viral morphogenesis.
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PMID:HIV-1 Vpr-chloramphenicol acetyltransferase fusion proteins: sequence requirement for virion incorporation and analysis of antiviral effect. 1049 Jul 69

In Escherichia coli mRNA, the arginine codons AGA/AGG and the isoleucine codon AUA are rarely used with frequencies of about 0.35% and 0.41%, respectively. Six genes with a different number of these codons were expressed in an E. coli in vitro coupled transcription/translation system, which contained either tRNA prepared from E. coli cells carrying a plasmid with argU and ileX genes encoding rare tRNAs (tRNA(arg)(AGA/AGG) and tRNA(ile)(AUA)), designated codon-plus tRNA, or normal tRNA from cells lacking the plasmid. Genes having a low number of the rare codons, such as genes encoding chloramphenicol acetyltransferase and anti-gp120 single-chain Fv (artificially constructed to remove rare codons), were expressed at similar levels using with both tRNA preparations. On the other hand, the use of codon-plus tRNA increased the expression levels of genes having a relatively large number of the rare codons, including genes encoding archaeal proteins, green fluorescent protein of jelly fish origin, and a single-chain Fv of mouse origin, by about 20% higher than that using normal tRNA.
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PMID:Dosage effect of minor arginyl- and isoleucyl-tRNAs on protein synthesis in an Escherichia coli in vitro coupled transcription/translation system. 1623 46

A lysate-based thermostability and activity profile is described for chloramphenicol acetyltransferase (CAT) expressed in trifluoroleucine, T (CAT T). CAT and 13 single-isoleucine CAT mutants were expressed in medium supplemented with T and assayed for thermostability on cell lysates. Although fluorinated mutants, L82I T and L208I T, showed losses in thermostability, the L158I T fluorinated mutant demonstrated an enhanced thermostability relative to CAT T. Further characterization of L158I T suggested that T at position 158 contributed to a portion of the observed loss in thermostability upon global fluorination.
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PMID:Fluorinated chloramphenicol acetyltransferase thermostability and activity profile: improved thermostability by a single-isoleucine mutant. 1784 47

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