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 noncollagenous proteins (NCPs) that predominate the bone matrix have recently been the focus of intense investigation because of their potential influence on cell attachment, Ca2+ and hydroxyapatite binding, and the mineralization of bone tissue. With the advent of molecular biology, all of the major NCPs of bone have been cloned and their amino acid sequences completely determined. While each of the proteins has distinct structural properties, some proteins appear to be part of gene families. Examples include the small proteoglycans, decorin and biglycan, as well as the gamma carboxyglutamic acid proteins, such as matrix gla protein and osteocalcin (bone gla protein). Some of the NCPs that are clearly not members of any known gene family still share several common characteristics. One such example of this "convergent evolution" is bone sialoprotein and osteopontin. Both are highly posttranslationally modified glycoproteins that share the cell attachment amino acid sequence RGD (arginine-glycine-aspartic acid), which facilitates the attachment of bone cells in vitro, yet they are clearly not related genetically. Using cDNAs and antisera as probes, the precise temporal localization of NCP expression has been determined, and it has been shown that NCPs are produced in skeletal, and in most cases, nonskeletal tissue as well. This observation implies that the functions of the NCPs are not necessarily limited to bone tissue. Many of the promoters for these genes have been isolated and functional domains determined by a combination of chloramphenicol acetyltransferase assay, gel shift, and footprint analyses. The most extensively studied promoter in the NCP category is osteocalcin, whose sensitivity to 1,25-dihydroxycholecalciferol has been delineated in detail. Future studies on the individual and cooperative activities of the NCPs in bone are likely to involve site-directed mutagenesis of cloned DNA and a combination of in vitro and in vivo functional analyses.
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PMID:Structure, expression, and regulation of the major noncollagenous matrix proteins of bone. 149 20

The X protein of hepatitis B virus (HBV) consists of 154 amino acids and trans-activates various cellular and viral promoters and enhancers. To investigate the essential amino acid sequences of X protein for trans-activation function, various mutations were introduced into the X open reading frame and analysed for trans-activation activity by chloramphenicol acetyltransferase assay. The amino acid sequences 46-52 (especially Pro-46, His-49 and His-52), 61-69 (especially Cys-61, Gly-67 to Pro-68 and Cys-69) and 132-139 (especially Phe-132, Cys-137 and His-139) of HBV X protein were found to be essential for the trans-activation function. These three sequences are included in the conserved amino acid sequences among hepadna virus X proteins. The first one could form a domain-like structure characteristic of histidine/aspartic acid requirement. The second and the third are homologous to the Kunitz domain of Kunitz-type serine protease inhibitors. The amino acids 5-27 region was found to make no positive contribution to the trans-activation function like the last 12 amino acids in the carboxy-terminal region [Takada, S. & Koike, K. (1990). Proc. Natl. Acad. Sci. USA, 87, 5628-5632]. From these findings, the trans-activation function of X protein appears to be dependent on at least two types of domain-like structures.
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PMID:Identification of three essential regions of hepatitis B virus X protein for trans-activation function. 154 57

Familial glucocorticoid resistance is a hypertensive, hyperandrogenic disorder characterized by increased serum cortisol concentrations in the absence of stigmata of Cushing's syndrome. Our previous studies of the first reported kindred showed a two- to threefold reduction in glucocorticoid receptor-ligand binding affinity in the propositus, and a lesser reduction in affinity in his mildly affected son and nephew. Glucocorticoid receptor cDNA from these three patients was amplified by polymerase chain reaction and sequenced. The cDNA nucleotide sequence was normal, except for nucleotide 2054, which substituted valine for aspartic acid at amino acid residue 641. The propositus was homozygous while the other relatives were heterozygous for the mutation. COS-7 monkey kidney cells were cotransfected with expression vectors for either wild type or Val 641-mutant receptors, together with the reporter plasmid pMMTV-CAT. Dexamethasone increased chloramphenicol acetyltransferase activity in cells expressing wild type receptor, but had no effect in cells expressing Val 641-mutant receptors, despite similar receptor concentrations, as indicated by Western blotting. The binding affinity for dexamethasone of the Val 641-mutant receptor was threefold lower than that of the wild type receptor. These results suggest that glucocorticoid resistance in this family is due to a point mutation in the steroid-binding domain of the glucocorticoid receptor.
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PMID:Point mutation causing a single amino acid substitution in the hormone binding domain of the glucocorticoid receptor in familial glucocorticoid resistance. 170 18

The inner core domain (residues approximately 221-454) of the dihydrolipoamide acetyltransferase component (E2P) of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae has been overexpressed in Escherichia coli strain JM105 via the expression vector pKK233-2. The truncated E2p was purified to apparent homogeneity. It exhibited catalytic activity (acetyl transfer from [1-14C]acetyl-CoA to dihydrolipoamide) very similar to that of wild-type E2p. The appearance of the truncated and wild-type E2p was also very similar, as observed by negative-stain electron microscopy, namely, a pentagonal dodecahedron. These findings demonstrate that the active site of E2p from S. cerevisiae resides in the inner core domain, i.e., catalytic domain, and that this domain alone can undergo self-assembly. The purified truncated E2p showed a tendency to aggregate. Aggregation was prevented by genetically engineered attachment of the interdomain linker segment (residues approximately 181-220) to the catalytic domain. All dihydrolipoamide acyltransferases contain the sequence His-Xaa-Xaa-Xaa-Asp-Gly near their carboxyl termini. By analogy with chloramphenicol acetyltransferase, the highly conserved His and Asp residues were postulated to be involved in the catalytic mechanism [Guest, J. R. (1987) FEMS Microbiol. Lett. 44, 417-422]. Substitution of the sole His residue in the S. cerevisiae truncated E2p, His-427, by Asn or Ala by site-directed mutagenesis did not have a significant effect on the kcat or Km values of the truncated E2p. However, the Asp-431----Asn, Ala, or Glu substitutions resulted in a 16-, 24-, and 3.7-fold reduction, respectively, in kcat, with little change in Km values.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Overexpression and mutagenesis of the catalytic domain of dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae. 227 45

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.
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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

Genes encoding chloramphenicol acetyltransferase in gram-positive bacteria are induced by chloramphenicol. Induction reflects an ability of the drug to stall a ribosome at a specific site in cat leader mRNA. Ribosome stalling at this site alters downstream RNA secondary structure, thereby unmasking the ribosome-binding site for the cat coding sequence. Here, we show that ribosome stalling in the cat-86 leader is a function of leader codons 2 through 5 and that stalling requires these codons to be presented in the correct reading frame. Codons 2 through 5 specify Val-Lys-Thr-Asp. Insertion of a second copy of the stall sequence 5' to the authentic stall sequence diminished cat-86 induction fivefold. Thus, the stall sequence can function in ribosome stalling when the stall sequence is displaced from the downstream RNA secondary structure. We suggest that the stall sequence may function in cat induction at two levels. First, the tetrapeptide specified by the stall sequence likely plays an active role in the induction strategy, on the basis of previously reported genetic suppression studies (W. W. Mulbry, N. P. Ambulos, Jr., and P.S. Lovett, J. Bacteriol. 171:5322-5324, 1989). Second, we show that embedded within the stall sequence of cat leaders is a region which is complementary to a sequence internal in 16S rRNA of Bacillus subtilis. This complementarity may guide a ribosome to the proper position on leader mRNA or potentiate the stalling event, or both. The region of complementarity is absent from Escherichia coli 16S rRNA, and cat genes induce poorly, or not at all, in E. coli.
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PMID:Four codons in the cat-86 leader define a chloramphenicol-sensitive ribosome stall sequence. 229 82

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

Previously, a cDNA was constructed so that transcription by T7 RNA polymerase yielded a approximately 1-kb negative-sense analog of genomic RNA of human respiratory syncytial virus (RSV) containing the gene for chloramphenicol acetyltransferase (CAT) under the control of putative RSV transcription motifs and flanked by the RSV genomic termini. When transfected into RSV-infected cells, this minigenome was "rescued," as evidenced by high levels of CAT expression and the production of transmissible particles which propagated and expressed high levels of CAT expression during serial passage (P.L. Collins, M. A. Mink, and D. S. Stec, Proc. Natl. Acad. Sci. USA, 88:9663-9667, 1991). Here, this cDNA, together with a second one designed to yield an exact-copy positive-sense RSV-CAT RNA antigenome, were each modified to contain a self-cleaving hammerhead ribozyme for the generation of a nearly exact 3' end. Each cDNA was transfected into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase, together with plasmids encoding the RSV N, P, and L proteins, each under the control of a T7 promoter. When the plasmid-supplied template was the mini-antigenome, the minigenome was produced. When the plasmid-supplied template was the minigenome, the products were mini-antigenome, subgenomic polyadenylated mRNA and progeny minigenome. Identification of progeny minigenome made from the plasmid-supplied minigenome template indicates that the full RSV RNA replication cycle occurred. RNA synthesis required all three RSV proteins, N, P, and L, and was ablated completely by the substitution of Asn for Asp at position 989 in the L protein. Thus, the N, P, and L proteins were sufficient for the synthesis of correct minigenome and antigenome, but this was not the case for subgenomic mRNA, indicating that the requirements for RNA replication and transcription are not identical. Complementation with N, P, and L alone yielded an mRNA pattern containing a large fraction of molecules of incomplete, heterogeneous size. In contrast, complementation with RSV (supplying all of the RSV gene products) yielded a single discrete mRNA band. Superinfection with RSV of cells staging N/P/L-based RNA synthesis yielded the single discrete mRNA species. Some additional factor supplied by RSV superinfection appeared to be involved in transcription, the most obvious possibility being one or more additional RSV gene products.
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PMID:RNA replication by respiratory syncytial virus (RSV) is directed by the N, P, and L proteins; transcription also occurs under these conditions but requires RSV superinfection for efficient synthesis of full-length mRNA. 763 14

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

Specific interaction between the nucleocapsid protein (N) and the phosphoprotein (P) of vesicular stomatitis virus (VSV), an important step in the life-cycle of the virus, was studied by using a two-hybrid system. Plasmids encoding P fused with the yeast GAL4 DNA-binding domain (pGALP) and N fused with the herpes simplex virus VP16 transactivating region (pVPN) were transfected into CHO cells along with a reporter plasmid encoding chloramphenicol acetyltransferase (CAT). The ability of N and P to associate in vivo was measured by activation of the CAT gene by the VP16 transactivating region. Transfection of plasmids pGALP and pVPN resulted in a high level of CAT activity, indicating that the N and P portions of the fusion proteins associated very strongly with each other. Progressive C-terminal deletions of the P protein revealed two regions that are important for association with the N protein: the N-terminal acidic domain and the C-terminal basic domain. Phosphorylation of P protein was not required for N-P association. Various deletions and mutations of the N protein revealed the C-terminal 5 amino acids (Val-Glu-Phe-Asp-Lys), in particular the amino acids Val-Glu-Phe, to be critical for N association with P. This two-hybrid system can be used in other viral systems to study the interaction between proteins involved in transcription and replication.
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PMID:Mapping of interacting domains between the nucleocapsid protein and the phosphoprotein of vesicular stomatitis virus by using a two-hybrid system. 823 1


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