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)

The use of lactobacilli as starter organisms in food fermentation processes requires thorough knowledge of their reaction to the multitude of ecological factors including their response to stress. We have characterised the dnaK gene region of Lactobacillus sakei LTH681. Two chromosomal EcoRI fragments of 2.5 and 4.0 kb were identified using a homologous dnaK probe generated by PCR. The sequence analysis of the cloned fragments showed that the dnaK gene region consists of four heat shock genes with the organisation hrcA-grpE-dnaK-dnaJ. Comparison of the deduced amino acid sequences revealed high similarity to the corresponding heat shock proteins of Gram-positive bacteria. An upstream located orfY was found which exhibited substantial similarity (41.5%) to the chloramphenicol acetyltransferase of Enterobacter aerogenes. Northern hybridisation analysis revealed that the transcription of the genes is induced by heat shock (42 degrees C) as well as salt (6%) or ethanol (10%) stress. Several transcripts were detected including a polycistronic mRNA of 4.9 kb which represents the transcript of the complete dnaK gene region indicating a tetracistronic organisation of the dnaK operon. The other RNA fragments were identified as shorter transcripts (3.7 and 1.3 kb) or cleavage products of the polycistronic mRNAs. The transcription start sites of the dnaK operon were determined under inducing and non-inducing conditions. The site varied with the applied stress condition. A regulatory CIRCE element was identified located between the transcription and translation start site. The promoter region including CIRCE was transcriptionally fused to the beta-glucuronidase reporter gene gusA and expressed in L. sakei LTH681. The kinetics of transcriptional induction of gusA by heat shocking were identical to those of the dnaK operon confirming the involvement of the CIRCE element in regulation of gene expression.
 ...
PMID:Molecular characterisation of the dnaK operon of Lactobacillus sakei LTH681. 1055 84

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

It has been suggested that the common (betaalpha)(8)-barrel enzyme fold has evolved by the duplication and fusion of identical (betaalpha)(4)-half barrels, followed by the optimisation of their interface. In our attempts to reconstruct these events in vitro we have previously linked in tandem two copies of the C-terminal half barrel HisF-C of imidazole glycerol phosphate synthase from Thermotoga maritima and subsequently reconstituted in the fusion construct HisF-CC a salt bridge cluster present in wild-type HisF. The resulting recombinant protein HisF-C*C, which was produced in an insoluble form and unfolded with low cooperativity at moderate urea concentrations has now been stabilised and solubilised by a combination of random mutagenesis and selection in vivo. For this purpose, Escherichia coli cells were transformed with a plasmid-based gene library encoding HisF-C*C variants fused to chloramphenicol acetyltransferase (CAT). Stable and soluble variants were identified by the survival of host cells on solid medium containing high concentrations of the antibiotic. The selected HisF-C*C proteins, which were characterised in vitro in the absence of CAT, contained eight different amino acid substitutions. One of the exchanges (Y143C) stabilised HisF-C*C by the formation of an intermolecular disulfide bond. Three of the substitutions (G245R, V248M, L250Q) were located in the long loop connecting the two HisF-C copies, whose subsequent truncation from 13 to 5 residues yielded the stabilised variant HisF-C*C Delta. From the remaining substitutions, Y143H and V234M were most beneficial, and molecular dynamics simulations suggest that they strengthen the interactions between the half barrels by establishing a hydrogen-bonding network and an extensive hydrophobic cluster, respectively. By combining the loop deletion of HisF-C*C Delta with the Y143H and V234M substitutions, the variant HisF-C**C was generated. Recombinant HisF-C**C is produced in soluble form, forms a pure monomer with its tryptophan residues shielded from solvent and unfolds with similar cooperativity as HisF. Our results show that, starting from two identical and fused half barrels, few amino acid exchanges are sufficient to generate a highly stable and compact (betaalpha)(8)-barrel protein with wild-type like structural properties.
 ...
PMID:Stabilisation of a (betaalpha)8-barrel protein designed from identical half barrels. 1763 94


<< Previous 1 2