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)

Dihydrolipoamide dehydrogenase (E3; EC 1.8.1.4) is the common component of the three mammalian alpha-ketoacid dehydrogenase complexes and the glycine cleavage system. To study regulation of E3 gene expression, a 12-kilobase clone from a human leukocyte genomic library was isolated, and a 1.8-kilobase fragment containing part of the first intron, the first exon, and 1.5 kilobases of the 5' flanking region of the E3 gene was sequenced. The nucleotide sequence of the E3 promoter region revealed consensus sequences for several DNA binding proteins but no apparent TATA box or Sp1 sites. Although the 1.6-kilobase 5' flanking region has a low percentage of G+C (44%), the nucleotide sequence between +1 and -150 base pairs has a G+C content of 67%. Primer extension analysis showed a major transcriptional start site located 95 nucleotides upstream from the translation initiation codon. A series of 5' deletions from the E3 promoter-regulatory region were ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene, and the resulting constructs were transfected into HepG2 cells. The longest E3 promoter-CAT construct had a relatively high level of CAT enzyme activity, and deletion of a promoter element between -769 and -1223 base pairs resulted in a 3-fold increase in reporter gene expression. These results suggest that the human E3 promoter has characteristics of housekeeping and facultative promoters and that a negative regulatory element is present between 769 and 1223 base pairs upstream from the transcription start site.
 ...
PMID:Characterization of the transcriptional regulatory region of the human dihydrolipoamide dehydrogenase gene. 133 63

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.
 ...
PMID:Overproduction of the pyruvate dehydrogenase multienzyme complex of Escherichia coli and site-directed substitutions in the E1p and E2p subunits. 144 21

Site-directed mutagenesis was performed in the protease-sensitive region, between the lipoyl and catalytic domains and in the catalytic domain, of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii. The interaction of the mutated enzymes with the peripheral components pyruvate dehydrogenase (E1p) and lipoamide dehydrogenase (E3) was studied by gel filtration experiments, analytical ultracentrifugation and reconstitution of the pyruvate dehydrogenase complex. Upon binding of peripheral components, the 24-subunit core of A. vinelandii wild-type E2p dissociates into tetramers. Four E1p or E3 dimers can bind to a tetramer. Binding is mutually exclusive, resulting in an active complex containing one E3 and three E1p dimers. Large deletions of the protease-sensitive region of E2p resulted in a total loss of the E1p and E3 binding. A small deletion (delta P361-R362) or the point mutation K367Q in the protease-sensitive region did not influence E3 binding, but affected E1p binding strongly, although with excess E1p almost complete reconstitution was reached. For E2p with the point mutation R416D in the N-terminal region of the catalytic domain only 16% overall activity could be measured in reconstituted complexes. This is due to a very weak E1p/E2p interaction, whereas the E3 binding was not affected. The point mutation R416D did not influence the catalytic activity of E2p, although a function for this residue in the formation of the active site was predicted from amino acid similarities with chloramphenicol acetyltransferase type III from Escherichia coli. Deletion of the complete Ala + Pro-rich sequence between the protease-sensitive region and the catalytic domain did not affect the enzymological properties of E2p, nor the affinity for E1p or E3. A further deletion of 20 N-terminal residues from the catalytic domain destroyed the E2p activity. From gel filtration experiments it was concluded that the quaternary structure was unaffected, as was E3 binding. E1p binding was lost and, in contrast to the wild-type enzyme, no dissociation of the core upon addition of E3 was observed. This mutant enzyme possesses, like E. coli E2p, six E3 binding sites and clearly shows that interaction of E3 or E1p with the E1p sites and dissociation are linked processes. It is concluded that the binding site for E3 is located on the N-terminal part of the protease-sensitive region. In contrast, the binding site for E1p consists of two regions, one located on the protease-sensitive region and one of the catalytic domain. These regions are separated by a flexible sequence of about 20 amino acids.
 ...
PMID:Site-directed mutagenesis of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Binding of the peripheral components E1p and E3. 176 97

The 5'-flanking region of the murine dihydrolipoamide dehydrogenase (Dld) gene was characterized for its promoter activity. DNase I footprinting analysis of the promoter region (-545 bp to +41 bp) revealed six major protein-binding domains (termed P1 to P6) that were protected by NIH3T3 fibroblast nuclear extracts. Transient transfection assays, using a series of nested deletions of the 2.5 kb 5'-flanking region ligated to the chloramphenicol acetyltransferase reporter gene, identified that the -42-bp to +41-bp region, which harbours the P1, P2, and P3 domains, had minimal transcriptional activity. When the 5'-flanking region was extended from -42 bp to -82 bp, there was an approx. 5-fold increase in promoter activity. To identify further the cis elements involved in transcription of the Dld gene (-82 bp to +41 bp), a series of mutations were introduced into this region and evaluated for functional effects using transient transfection and electrophoretic mobility shift assays. Mutation or deletion of the CACGAC direct repeat, located from -61 bp to -46 bp, resulted in minimal promoter activity. Mutation of the Ets motif, located from -37 bp to -32 bp, reduced the minimal promoter activity by approx. 50%, whereas the deletion of this motif almost abolished the promoter activity. These results indicate that: (i) the Ets motif is required for the minimal promoter activity and (ii) the CACGAC direct repeat enhances promoter activity. Database searches failed to identify the direct repeat with the CACGAC motif and hence the CACGAC sequence may represent a novel motif. The requirement of both the Ets motif and the direct repeat element for optimal promoter activity represents a unique combination for gene transcription.
 ...
PMID:Roles of an Ets motif and a novel CACGAC direct repeat in transcription of the murine dihydrolipoamide dehydrogenase (Dld) gene. 1005 37

Dihydrolipoamide dehydrogenase is a common component of four multienzyme complexes which are involved in oxidation of carbohydrates, lipids and amino acids. To better understand the regulation of human DLD gene expression, we have analyzed the proximal promoter region of this gene. DNase I footprinting analysis of the promoter region (-322 to +47 bp) revealed four major protein-binding domains (termed P1-P4). Nested deletions and site-specific mutations of approximately 100 bp proximal promoter region identified two elements, TACGAC direct repeat sequence and cAMP-response element (CRE)-like site, which are localized in the P2 and P1 domains, respectively, and mediate basal transcription of the DLD gene. Electrophoretic mobility supershift assays showed that the CRE-like site is associated with CRE binding protein. Interestingly, when DLD promoter constructs (-1.8 kb to +47 bp and -78 to +47 bp) fused with the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into human HepG2 cells either in the presence or absence of 0.5 mM 8-Br-cAMP, the levels of CAT expression remained unaffected. In addition, endogenous DLD mRNA levels in HepG2 cells also remained unaffected by treatment with 0.5 mM 8-Br-cAMP. These results indicate that the CRE binding protein is essential for basal transcription of the human DLD promoter, but does not confer cAMP-dependent gene regulation.
 ...
PMID:Human dihydrolipoamide dehydrogenase gene transcription is mediated by cAMP-response element-like site and TACGAC direct repeat. 1146 32