The lipoyl domain and its role in thiamin diphosphate-dependent oxidative decarboxylation.
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Binding Sites↗
Biomedical subjects
F Dardel
Publications and source records attributed to F Dardel.
Sequence-specific 1H-NMR assignments and secondary structure of the lipoyl domain of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex.
The lipoyl domain (residues 1-85) of the lipoate-acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been subjected to detailed structural analysis by means of two-dimensional (2D) 1H-NMR spectroscopy at 400 MHz. Sequence-specific proton resonance assignments were made, but at this field strength not all of the side-chain protons could be assigned, especially from complex spin systems like those of leucine, proline and lysine residues. Measurement of short-range interproton distances identified two extensive regions of beta-sheet, each containing four anti-parallel peptide strands. The lipoyl-lysine residue (Lys42) is located in a tight turn at a corner of one sheet, the N-terminal and C-terminal residues of the domain are close together in two adjacent beta-strands in the other. The lipoylated and unlipoylated forms of the domain have almost identical spectra, indicating that there is little, if any, conformational change in the protein as a result of the post-translational modification.
Acetyltransferases↗
Lysine 335, part of the KMSKS signature sequence, plays a crucial role in the amino acid activation catalysed by the methionyl-tRNA synthetase from Escherichia coli.
The KMSKS pattern, conserved among several aminoacyl-tRNA synthetase sequences, was first recognized in the Escherichia coli methionyl-tRNA synthetase through affinity labelling with an oxidized reactive derivative of tRNA(Met)f. Upon complex formation, two lysine residues of the methionyl-tRNA synthetase (Lys61 and 335, the latter being part of the KMSKS sequence) could be crosslinked by the 3'-acceptor end of the oxidized tRNA. Identification of an equivalent reactive lysine residue at the active centre of tyrosyl-tRNA synthetase designated the KMSKS sequence as a putative component of the active site of methionyl-tRNA synthetase. To probe the functional role of the labelled lysine residue within the KMSKS pattern, two variants of methionyl-tRNA synthetase containing a glutamine residue at either position 61 or 335 were constructed by using site-directed mutagenesis. Substitution of Lys61 slightly affected the enzyme activity. In contrast, the enzyme activities were very sensitive to the substitution of Lys335 by Gln. Pre-steady-state analysis of methionyladenylate synthesis demonstrated that this substitution rendered the enzyme unable to stabilize the transition state complex in the methionine activation reaction. A similar effect was obtained upon substituting Lys335 by an alanine instead of a glutamine residue, thereby excluding an effect specific for the glutamine side-chain. Furthermore, the importance of the basic character of Lys335 was investigated by studying mutants with a glutamate or an arginine residue at this position. It is concluded that the N-6-amino group of Lys335 plays a crucial role in the activation of methionine, mainly by stabilizing the transient complex on the way to methionyladenylate, through interaction with the pyrophosphate moiety of bound ATP-Mg2+. We propose, therefore, that the KMSKS pattern in the structure of an aminoacyl-tRNA synthetase sequence represents a signature sequence characteristic of both the pyrophosphate subsite and the catalytic centre.
Adenosine Triphosphate↗
Locations of the metG and mrp genes on the physical map of Escherichia coli.
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Chromosome Mapping↗
Expression in Escherichia coli of a sub-gene encoding the lipoyl domain of the pyruvate dehydrogenase complex of Bacillus stearothermophilus.
A sub-gene encoding the lipoyl domain (residues 1-85) of the lipoate acetyltransferase chain of the pyruvate dehydrogenase complex of Bacillus stearothermophilus was over-expressed in Escherichia coli. Approx. 80% of the domain was unlipoylated but most of the remainder was correctly lipoylated on Lys-42 and could be reductively acetylated by the B stearothermophilus enzyme complex. A small proportion (approx. 4%) of the domain carried an aberrant substituent, possibly an octanoyl group, on Lys-42. The 400 MHz 1H NMR spectra of the lipoylated and unlipoylated domains were essentially identical and closely resembled that of the native lipoyl domain.
Acetyltransferases↗
Transcription and regulation of expression of the Escherichia coli methionyl-tRNA synthetase gene.
The DNA sequence and transcriptional organization around the Escherichia coli methionyl-tRNA synthetase gene, metG, were resolved. This gene can be transcribed in vivo and in vitro from two distinct promoters separated by 510 nucleotides. The upstream promoter is located within the coding sequence of a divergent gene expressing a protein of Mr 39 kDa of unknown function. Transcription originating from this upstream promoter is attenuated by a Rho-independent terminator before entering the structural gene. This leader RNA contains several potentially stable secondary structures, one of which shows striking similarity to tRNA(Met), but no methionine-rich coding sequence. The regulation of metG expression was investigated by means of fusions to the lacZ gene. Transcription of a metG::lacZ fusion is induced in a metG mutant and, reciprocally, repression is observed in a methionyl-tRNA synthetase overproducing strain. A model of metG expression control is proposed.
Amino Acid Sequence↗
Methionyl-tRNA synthetase from E. coli--a review.
Methionyl-tRNA synthetase (MetRS) from E coli is a dimer composed of 2 identical subunits of Mr 76 kDa. A fully active monomeric fragment (64 kDa) could be obtained by mild proteolysis of the native dimer. Earlier studies reviewed in Blanquet et al (1979) have compared the catalytic mechanisms of native and trypsin-modified MetRS. Moreover, the truncated form of the enzyme was crystallized and its 3-D structure solved at low resolution. In the last few years, the availability of the corresponding metG gene has facilitated the development of studies using affinity labelling and site-directed mutagenesis techniques. In parallel, the 3-D structure has been solved at a resolution of 2.5 A. These convergent approaches have allowed significant progress in the understanding of the structure-function relationships of this enzyme, and, in particular, of the rules governing the recognition of tRNA.
Amino Acid Sequence↗
Computer simulation of DNA ligation: determination of initial DNA concentrations favouring the formation of recombinant molecules.
A computer program was used to simulate the dynamic process of a ligation of DNA fragments. More specifically, the influence of the initial DNA fragments lengths and concentrations on the relative abundance of the various end-products was systematically investigated. Depending on the nature of the DNA extremities (asymmetric or symmetric, dephosphorylated or not), sets of initial conditions could be found that optimized the yield of active recombinant molecules. These results can be directly used to increase the efficiency of the ligation step, in particular for the construction of cDNA or genomic libraries.
Algorithms↗
Genetic engineering of methionyl-tRNA synthetase: in vitro regeneration of an active synthetase by proteolytic cleavage of a methionyl-tRNA synthetase--beta-galactosidase chimeric protein.
The construction of a family of plasmids carrying derivatives of metG, the gene for E. coli methionyl-tRNA synthetase, is described. These plasmids allow expression of native or truncated forms of the enzyme and easy purification of the products. To facilitate the characterization of modified enzymes with very low catalytic activity, a specialized vector was constructed, in which metG was fused in frame with lacZ, the gene for beta-galactosidase. This plasmid expresses a methionyl-tRNA synthetase-beta-galactosidase chimeric protein, which is shown to carry the activities of both enzymes. This hybrid can be purified in a single step of affinity chromatography for beta-galactosidase. The methionyl-tRNA synthetase moiety can be regenerated by mild proteolysis, thus providing a simple method for purifying and studying mutated proteins.
Amino Acyl-tRNA Synthetases↗
DNAid: a Macintosh full screen editor featuring a built-in regular expression interpreter for the search of specific patterns in biological sequences using finite state automata.
DNAid is a full screen and multi-window sequence editor designed as an aid for the easy manipulation of DNA and protein sequences on Macintosh computers. In addition to the classical editing capabilities, powerful analysis and search functions are available from within the editor. Restriction mapping, translation and alignment of homologous sequences are supported by DNAid, furthermore a pattern matching language is included which allows searches for user-defined strict or fuzzy signals within biological sequences. Patterns are translated into finite state automata which allow very efficient searches.
Amino Acid Sequence↗
Open reading frames in the control regions of the phenylalanyl-tRNA synthetase operon of E. coli.
The pheST operon codes for the two subunits of phenylalanyl-tRNA synthetase and it expression is controlled by attenuation in a way similar to many amino acid biosynthetic operons. The nucleotide sequence of the control regions of the operon indicates the presence of several open reading frames besides that of the leader peptide. One of these open reading frames, called the alternative leader peptide, starts at about the same place as the leader peptide and ends after the terminator of the attenuator. Another open reading frame, called the terminator peptide, starts after the terminator and covers about half the distance to pheS, the first structural gene of the operon. The present report shows that, in fact, the only open reading frame to be translated efficiently is the leader peptide itself. The alternative leader peptide and the terminator peptide are both translated at a negligible rate.
Amino Acyl-tRNA Synthetases↗
PEGASE: a machine language program for DNA sequence analysis on Apple II microcomputer using a binary coding of nucleotides.
The core of a 6502 machine language program for DNA sequence analysis on Apple II microcomputer is described. Use of a binary coding of nucleotides allows interactive data manipulation on a low-cost configuration with execution times similar to those of larger computers. The PEGASE system should prove useful and easy to use in routine sequence handling and experiment design.
Algorithms↗
A microcomputer program for comparison and alignment of DNA sequence gel readings.
During the course of determining the sequence of a large DNA fragment, it is necessary to cross-check numerous gel readings from different DNA fragments, in order to track and eliminate mistakes. An algorithm is presented that takes advantage of the high degree of homology between such sequences to construct an alignment of the matching regions. It does not require knowledge of a starting homology zone, neither large memory areas, even for sequences of several kilobases, and it can overcome large gaps or mismatch zones that correspond for instance to misinterpretation of compressions on sequence gels. This algorithm has been implemented in 6502 assembly language on an Apple II computer as an extension to the PEGASE sequence handling system.
Algorithms↗