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

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 dihydrolipoamide succinyltransferase (E2o) component of the 2-oxoglutarate dehydrogenase multienzyme complex is composed of 24 subunits arranged with 432 point group symmetry. The catalytic domain (CD) of the E2o component catalyzes the transfer of a succinyl group from the S-succinyldihydrolipoyl moiety to coenzyme A. The crystal structure of the Escherichia coli E2oCD has been solved to 3.0 A resolution using molecular replacement phases derived from the structure of the catalytic domain from the Azotobacter vinelandii dihydrolipoamide acetyltransferase (E2pCD). The refined model of the E. coli E2oCD consists of residues 172 to 404 and has an R-factor of 0.205 (Rfree=0.249) for 9696 reflections between 20.0 and 3.0 A resolution. Although both E2oCD and E2pCD form 24mers, subtle changes in the orientations of two helices in E2oCD increase the stability of the E2oCD 24mer in comparison to the less stable A. vinelandii E2pCD 24mer. Like E2pCD and chloramphenicol acetyltransferase (CAT), the active site of E2oCD is located in the middle of a channel formed at the interface between two 3-fold related subunits. Two of the active-site residues (His375 and Thr323) have a similar orientation to their counterparts in E2pCD and CAT. A third catalytic residue (Asp379) assumes a conformation similar to the corresponding residue in E2pCD (Asn614), but different from its counterpart in CAT (Asp199). Binding of the substrates to E2oCD is proposed to induce a change in the conformation of Asp379, allowing this residue to form a salt bridge with Arg184 that is analogous to that formed between Asp199 and Arg18 in CAT. Computer models of the active site of E2o complexed with dihydrolipoamide and with coenzyme A led to the identification of the probable succinyl-binding pocket. The residues which form this pocket (Ser330, Ser333, and His348) are probably responsible for E2o's substrate specificity.
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PMID:Crystal structure of the truncated cubic core component of the Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex. 967 95