Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

N-myristoylated viral polypeptide mu 1 was produced in COS cells transfected with a transient expression vector containing a DNA copy of the reovirus M2 gene. The mu 1 product was specifically cleaved to polypeptide mu 1C in cells that were cotransfected with the reovirus S4 gene and that expressed polypeptide sigma 3. Studies with site-specific mutants of the M2 gene demonstrated that conversion of mu 1 to mu 1C was dependent on myristoylation and the presence of the proteolytic cleavage sequence asparagine 42-proline 43 in mu 1, as well as on the presence of polypeptide sigma 3. The mu 1C product and polypeptide sigma 3 formed complexes that were immunoprecipitated by sigma 3-directed antibody, and a myristoylation-negative M2 double mutant, G2A-N42T, yielded mu 1 that did not undergo cleavage to mu 1C or bind sigma 3. However, the N42T single mutant did form immunoprecipitable complexes with sigma 3, indicating that binding can occur in the absence of cleavage. Polypeptide sigma 3 alternatively can bind double-stranded RNA and in COS cells stimulates translation of reporter chloramphenicol acetyltransferase mRNA translation, presumably by blocking double-stranded RNA-mediated activation of the eukaryotic initiation factor 2 alpha subunit kinase which inhibits the initiation of protein synthesis. Consistent with these observations and with the formation of mu 1C-sigma 3 complexes, coexpression of M2 with S4 DNA prevented the translational stimulatory effect of polypeptide sigma 3.
 ...
PMID:Reovirus polypeptide sigma 3 and N-terminal myristoylation of polypeptide mu 1 are required for site-specific cleavage to mu 1C in transfected cells. 154 57

The function of conserved Ser-148 of chloramphenicol acetyltransferase (CAT) has been investigated by site-directed mutagenesis. Modeling studies (P. C. E. Moody and A. G. W. Leslie, unpublished results) suggested that the hydroxyl group of Ser-148 could be involved in transition-state stabilization via a hydrogen bond to the oxyanion of the putative tetrahedral intermediate. Replacement of serine by alanine results in a mutant enzyme (Ala-148 CAT) with kcat reduced 53-fold and only minor changes in Km values for chloramphenicol and acetyl-CoA. The Ser-148----Gly substitution gives rise to a mutant enzyme (Gly-148 CAT) with kcat reduced only 10-fold. A water molecule may partially replace the hydrogen-bonding potential of Ser-148 in Gly-148 CAT. The three-dimensional structure of Ala-148 CAT at 2.34-A resolution is isosteric with that of wild-type CAT with two exceptions: the absence of the Ser-148 hydroxyl group and the loss of one poorly ordered water molecule from the active site region. The results are consistent with a catalytic role for Ser-148 rather than a structural one and support the hypothesis that Ser-148 is involved in transition-state stabilization. Ser-148 has also been replaced with cysteine and asparagine; the Ser-148----Cys mutation results in a 705-fold decrease in kcat and the Ser-148----Asn substitution in a 214-fold reduction in kcat. Removing the hydrogen bond donor (Ser-148----Ala or Gly) is less deleterious than replacing Ser-148 with alternative possible hydrogen bond donors (Ser-148----Cys or Asn).
 ...
PMID:Evidence for transition-state stabilization by serine-148 in the catalytic mechanism of chloramphenicol acetyltransferase. 210 33

The crystal structure of the Asp-199----Asn mutant of chloramphenicol acetyltransferase (CAT) has been determined to 2.35-A resolution. In wild-type CAT Asp-199 is involved in a fully buried intrasubunit salt bridge with Arg-18, an interaction that is adjacent to the active site. Replacement of aspartate with asparagine by site-directed mutagenesis disrupts this salt bridge and causes extensive conformational changes within the active site. The imidazole group of the catalytically essential His-195 is reoriented, with the loss of interactions thought to stabilize the preferred tautomer of this residue. Arg-18 and Asn-199 form three new intersubunit interactions as a result of large side-chain torsion angle changes which cause the movement of two polypeptide loops, some residues of which are up to 20 A away from the site of the mutation. The new interactions of Arg-18 and Asn-199 compensate for the loss of the buried salt bridge and afford near-wild-type thermostability to Asn-199 CAT, albeit with a greatly reduced activity.
 ...
PMID:Crystal structure of the aspartic acid-199----asparagine mutant of chloramphenicol acetyltransferase to 2.35-A resolution: structural consequences of disruption of a buried salt bridge. 227 9

The human ts11 gene was isolated on the basis of its ability to complement the mutation of the BHK cell cycle ts11 mutant, which is blocked in G1 at the nonpermissive temperature. This gene has now been identified as the structural gene for asparagine synthetase (AS) on the bases of sequence homology and the ability of exogenous asparagine to bypass the ts11 block. The ts11 (AS) mRNA has a size of about 2 kilobases and is induced in mid-G1 phase in human, mouse, and hamster cell lines. We have studied the organization and regulation of expression of the ts11 gene. The human ts11 gene consists of 13 exons (the first two noncoding) interspersed in a region of about 21 kilobases of DNA. Transient expression assays using the bacterial chloramphenicol acetyltransferase reporter gene identified two separate promoters: one (ts11 P1) contained in a 280-base-pair region upstream of the first exon and the other (ts11 P2) contained in the first intron. ts11 P1 produced about sixfold more chloramphenicol acetyltransferase activity than did ts11 P2 and had features of the promoters of housekeeping genes: high G + C content, multiple transcription start sites, absence of a TATA box, and presence of putative Sp1 binding sites. ts11 P2 contained a TATA sequence and other elements characteristic of a promoter, but so far we have no evidence of its physiological utilization. The ts11 gene was overexpressed in ts11 cells exposed to the nonpermissive temperature. Addition of asparagine to the culture medium led to a drastic decrease in mRNA levels and prevented G1 induction in serum-stimulated cells, which indicated that expression of the AS gene is regulated by a mechanism of end product inhibition.
 ...
PMID:Organization and expression of the cell cycle gene, ts11, that encodes asparagine synthetase. 256 68

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.
 ...
PMID:Substitutions in the active site of chloramphenicol acetyltransferase: role of a conserved aspartate. 306 55

Several genes homologous to the methyl-accepting chemotaxis proteins (MCPs) of Escherichia coli have been cloned and characterized from the Gram-positive bacterium, Bacillus subtilis. Sequence analysis reveals four large open reading frames, designated mcpA, mcpB, tlpA, and tlpB, each encoding a predicted 72-kDa protein. These proteins exhibit strong homology to chemoreceptors from several organisms, although similarity is limited to the C-terminal domain. These transducer genes were mapped to a chromosomal position of 279 degrees, which is distant from previously identified fla, mot, or che loci. Each gene was inactivated by insertion of a nonpolar chloramphenicol acetyltransferase cassette in the N-terminal region. In vivo methylation of the bacterial strain deficient in mcpA revealed the loss of several methylated bands in the range of the MCP previously designated as H1, and greatly reduced methylation of the MCP designated as H2. Furthermore, this bacterial strain exhibited a chemotaxis deficiency toward glucose and alpha-methyl-glucoside. Inactivation of mcpB caused a reduction in methylation of the MCP designated as H3, while chemotaxis toward asparagine, aspartate, glutamine, and histidine was significantly impaired in this strain. Despite strong homology, inactivation of tlpA and tlpB did not result in an observed deficiency in chemotaxis. Most unusually, these mutant strains exhibited a striking tendency to adhere together and resisted disaggregation.
 ...
PMID:Cloning and characterization of genes encoding methyl-accepting chemotaxis proteins in Bacillus subtilis. 818 84

Glucosidase I initiates the processing of asparagine (N-) linked glycoproteins by removing the distal alpha1,2-linked glucosyl residue of the tetradecasaccharide Glc(3)Man(9)GlcNAc(2). The gene encoding this enzyme was isolated and its structural organization and promoter activity determined. The major transcript for glucosidase I on northern blot appeared to be 3.1 kb; Southern blotting and DNA sequencing indicated the size of the gene to be 6.8 kb, comprising four exons separated by three introns. The first exon encodes the cytoplasmic tail and transmembrane domain; the fourth encodes the putative catalytic domain of the enzyme. Exon-intron junctions are flanked by consensus splice donor and acceptor sequences. Transcription initiation sites were mapped by primer extension, ribonuclease protection assay and RT-PCR analysis. Primer extension results showed multiple initiation sites at -150, -156, and -272 bp relative to the translation initiation codon ATG. Sequence analysis of 5' flanking region showed no canonical TATA box, a high GC content, Sp1 and ETF binding sites (typical of a housekeeping gene promoter). Also noteworthy, the promoter region contains several generic STAT factor binding sites, one nearly perfect, and two half GR binding elements. Other cis- acting elements recognized by transcription factors such as AP-2, NF-kappaB, estrogen receptor, and progesterone receptor (PR) were also present in the putative promoter region. To determine the promoter activity, a construct encompassing the region between -2114 to -5 bp of the putative promoter was ligated to the chloramphenicol acetyltransferase (CAT) reporter plasmid and transiently transfected into COS 7 cells. CAT assay results clearly show transcriptional activity of the promoter.
 ...
PMID:Genomic organization and promoter activity of glucosidase I gene. 1040 45

Nonribosomal peptide synthetases (NRPSs) make many natural products of clinical importance, but a deeper understanding of the protein domains that compose NRPS assembly lines is required before these megasynthetases can be effectively engineered to produce novel drugs. The N-terminal amide bond-forming condensation (C) domain of the enterobactin NRPS EntF was excised from the multidomain synthetase using endpoints determined from sequence alignments and secondary structure predictions. The isolated domain was well-folded when compared by circular dichroism to the vibriobactin NRPS VibH, a naturally free-standing C domain. The EntF domain was also fully functional in an assay based on a synthetic small-molecule substrate, seryl N-acetylcysteamine. Active site mutants of the EntF C domain were surprisingly inactive in vitro as compared to their VibH counterparts, yet maintained the overall domain structure. An in vivo assay was developed in the context of the full-length EntF protein to more sensitively probe the activity level of the C domain mutants, and this supported strong effects for the active site mutations. The crucial role of histidine-138 was confirmed by assay of the full-length protein in vitro. These results suggest a strong resemblance of catalysis by the EntF C domain to chloramphenicol acetyltransferase, including an active site organized by an arginine-aspartate salt bridge, a key histidine acting as a general base, and an asparagine instead of a serine stabilizing the proposed tetrahedral intermediate by hydrogen bonding. The precise definition of a functional C domain excised from a NRPS should aid efforts at swapping NRPS domains between assembly lines.
 ...
PMID:Dissection of the EntF condensation domain boundary and active site residues in nonribosomal peptide synthesis. 1256 37