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M Härtlein

Publications and source records attributed to M Härtlein.

29 records · Page 2Linked to original sources
PubMed1991

Sequence, structural and evolutionary relationships between class 2 aminoacyl-tRNA synthetases.

Class 2 aminoacyl-tRNA synthetases, which include the enzymes for alanine, aspartic acid, asparagine, glycine, histidine, lysine, phenylalanine, proline, serine and threonine, are characterised by three distinct sequence motifs 1,2 and 3 (reference 1). The structural and evolutionary relatedness of these ten enzymes are examined using alignments of primary sequences from prokaryotic and eukaryotic sources and the known three dimensional structure of seryl-tRNA synthetase from E. coli. It is shown that motif 1 forms part of the dimer interface of seryl-tRNA synthetase and motifs 2 and 3 part of the putative active site. It is further shown that the seven alpha 2 dimeric synthetases can be subdivided into class 2a (proline, threonine, histidine and serine) and class 2b (aspartic acid, asparagine and lysine), each subclass sharing several important characteristic sequence motifs in addition to those characteristic of class 2 enzymes in general. The alpha 2 beta 2 tetrameric enzymes (for glycine and phenylalanine) show certain special features in common as well as some of the class 2b motifs. In the alanyl-tRNA synthetase only motif 3 and possibly motif 2 can be identified. The sequence alignments suggest that the catalytic domain of other class 2 synthetases should resemble the antiparallel domain found in seryl-tRNA synthetase. Predictions are made about the sequence location of certain important helices and beta-strands in this domain as well as suggestions concerning which residues are important in ATP and amino acid binding. Strong homologies are found in the N-terminal extensions of class 2b synthetases and in the C-terminal extensions of class 2a synthetases suggesting that these putative tRNA binding domains have been added at a later stage in evolution to the catalytic domain.

Adenosine Triphosphate↗
PubMed1991

Tyr-426 of the Escherichia coli asparaginyl-tRNA synthetase, an amino acid in a C-terminal conserved motif, is involved in ATP binding.

Sequence comparisons of the E. coli asparaginyl-tRNA synthetase (NRSEC) with aminocyl-tRNA synthetase sequences of class II enzymes show significant homologies with aspartyl- and lysyl-tRNA synthetases. Three conserved regions were found, one of which is located in the C-terminal part of the NRSEC sequence. Site-directed mutagenesis was performed in this conserved region. A single point mutation Tyr-426----Ser results in a 15-fold increase in the Km for ATP, while all the other kinetic parameters remain unchanged. The replacement of this Tyr-426 by a Phe does not affect the kinetic behaviour of the enzyme. These data indicate that Tyr-426 is part of the ATP binding site.

Adenosine Triphosphate↗
PubMed1990

A second class of synthetase structure revealed by X-ray analysis of Escherichia coli seryl-tRNA synthetase at 2.5 A.

The three-dimensional crystal structure of seryl-transfer RNA synthetase from Escherichia coli, refined at 2.5 A resolution, is described. It has an N-terminal domain that forms an antiparallel alpha helical coiled-coil, stretching 60 A out into the solvent and stabilized by interhelical hydrophobic interactions and an active-site alpha-beta domain based around a seven-stranded antiparallel beta sheet. Unlike the three other known synthetase structures, the enzyme contains no classical nucleotide-binding fold, and is the first representative of a second class of aminoacyl-tRNA synthetase structures.

Amino Acid Sequence↗
PubMed1989

Asparaginyl-tRNA synthetase from Escherichia coli has significant sequence homologies with yeast aspartyl-tRNA synthetase.

The Escherichia coli asnS gene codes for asparaginyl-tRNA synthetase (NRSEC). We have sequenced the asnS region, including 382 bp of the 5'-untranslated region, 1398 bp of the coding region and 280 bp of the 3'-untranslated region. The DNA-derived NRSEC amino acid (aa) sequence was confirmed by direct aa sequencing of the N-terminal parts of the native protein and of a 28-kDa internal fragment generated by trypsin digestion. The asnS gene product has been purified to homogeneity using three chromatographic steps. Sequence comparison of the deduced NRSEC sequence with all aminoacyl-tRNA synthetase sequences showed significant homologies with the yeast aspartyl-tRNA synthetase and weaker relationships with other aminoacyl-tRNA synthetases for aa with an XAX codon.

Amino Acid Sequence↗
PubMed1987

Molecular cloning and nucleotide sequence of the gene for Escherichia coli leucyl-tRNA synthetase.

The gene for Escherichia coli leucyl-tRNA synthetase leuS has been cloned by complementation of a leuS temperature sensitive mutant KL231 with an E.coli gene bank DNA. The resulting clones overexpress leucyl-tRNA synthetase (LeuRS) by a factor greater than 50. The DNA sequence of the complete coding regions was determined. The derived N-terminal protein sequence of LeuRS was confirmed by independent protein sequencing of the first 8 aminoacids. Sequence comparison of the LeuRS sequence with all aminoacyl-tRNA synthetase sequences available reveal a significant homology with the valyl-, isoleucyl- and methionyl-enzyme indicating that the genes of these enzymes could have derived from a common ancestor. Sequence comparison with the gene product of the yeast nuclear NAM2-1 suppressor allele curing mitochondrial RNA maturation deficiency reveals about 30% homology.

Amino Acid Sequence↗
PubMed1987

Cloning and characterization of the gene for Escherichia coli seryl-tRNA synthetase.

Seryl-tRNA synthetase is the gene product of the serS locus in Escherichia coli. Its gene has been cloned by complementation of a serS temperature sensitive mutant K28 with an E. coli gene bank DNA. The resulting clones overexpress seryl-tRNA synthetase by a factor greater than 50 and more than 6% of the total cellular protein corresponds to the enzyme. The DNA sequence of the complete coding region and the 5'- and 3' untranslated regions was determined. Protein sequence comparison of SerRS with all available aminoacyl-tRNA synthetase sequences revealed some regions of significant homology particularly with the isoleucyl- and phenylalanyl-tRNA synthetases from E. coli.

Amino Acid Sequence↗
PubMed1987

Crystals of seryl-tRNA synthetase from Escherichia coli. Preliminary crystallographic data.

Crystals of seryl-tRNA synthetase from Escherichia coli can be grown from ammonium sulphate/octyl glucoside solutions in two days. The crystals appear to be very suitable for X-ray analysis, diffracting to at least 2.8 A resolution and being resistant to radiation damage. The crystals are monoclinic (space group C2) with cell parameters a = 148.2 A, b = 90.6 A, c = 69.5 A and beta = 119.0 degrees. Depending on whether the asymmetric unit is the enzyme monomer (Mr 48,414) or dimer the Vm value would be either 4.12 or 2.10 A3/dalton. Although the former would indicate a rather high solvent content, other proteins crystallized in the presence of octyl glucoside have Vm values similar to this.

Amino Acyl-tRNA Synthetases↗
PubMed1984

Study of regulation and transport of hemolysin by using fusion of the beta-galactosidase gene (lacZ) to hemolysin genes.

Operon and gene fusions between lacZ and the hemolysin genes, hlyC and hlyA, were performed. These two genes are essential for the synthesis of active hemolysin and are transcribed from a common promoter (p1). Whereas the amount of hemolysin produced in Escherichia coli is not changed by altering the hly gene dose, beta-galactosidase activity follows the gene dosage in both types of fusions when lacZ comes under the control of p1. This indicates that hemolysin is not negatively regulated on the transcription or translation level. The products of the gene fusions hlyC::lacZ and hlyA::lacZ were identified in maxicells as stable proteins of 146,000 and 220,000 daltons, respectively. Both fusion proteins possess beta-galactosidase activity indicating that the performed fusions of lacZ to the hly genes do not destroy the reading frame of hlyC and hlyA. The fusion proteins HlyC-beta-gal and HlyA-beta-gal were predominantly detected in the cytoplasm, confirming previous data which suggested that the primary gene products of hlyC and hlyA are not transported across the cytoplasmic membrane.

Bacterial Proteins↗
PubMed1983

Transport of hemolysin by Escherichia coli.

The hemolytic phenotype in Escherichia coli is determined by four genes. Two (hlyC and hlyA) determine the synthesis of a hemolytically active protein which is transported across the cytoplasmic membrane. The other two genes (hlyBa and hlyBb) encode two proteins which are located in the outer membrane and seem to form a specific transport system for hemolysin across the outer membrane. The primary product of gene hlyA is a protein (protein A) of 106,000 daltons which is nonhemolytic and which is not transported. No signal peptide can be recognized at its N-terminus. In the presence of the hlyC gene product (protein C), the 106,000-dalton protein is processed to the major proteolytic product of 58,000 daltons, which is hemolytically active and is transported across the cytoplasmic membrane. Several other proteolytic fragments of the 106,000-dalton protein are also generated. During the transport of the 58,000-dalton fragment (and possible other proteolytic fragments of hlyA gene product), the C protein remains in the cytoplasm. In the absence of hlyBa and hlyBb the entire hemolytic activity (mainly associated with the 58,000-dalton protein) is located in the periplasm: Studies on the location of hemolysin in hlyBa and hlyBb mutants suggest that the gene product of hlyBa (protein Ba) binds hemolysin and leads it through the outer membrane whereas the gene product of hlyBb (protein Bb) releases hemolysin from the outer membrane. This transport system is specific for E coli hemolysin. Other periplasmic enzymes of E coli and heterologous hemolysin (cereolysin) are not transported.

Base Sequence↗
PubMed1983

Cloning and expression in Escherichia coli and Bacillus subtilis of the hemolysin (cereolysin) determinant from Bacillus cereus.

From a cosmid gene bank of Bacillus cereus GP4 in Escherichia coli we isolated clones which, after several days of incubation, formed hemolysis zones on erythrocyte agar plates. These clones contained recombinant cosmids with B. cereus DNA insertions of varying lengths which shared some common restriction fragments. The smallest insertion was recloned as a PstI fragment into pJKK3-1, a shuttle vector which replicates in Bacillus subtilis and E. coli. When this recombinant plasmid (pJKK3-1 hly-1) was transformed into E. coli, it caused hemolysis on erythrocyte agar plates, but in liquid assays no external or internal hemolytic activity could be detected with the E. coli transformants. B. subtilis carrying the same plasmid exhibited hemolytic activity at levels comparable to those of the B. cereus donor strain. The hemolysin produced in B. subtilis seemed to be indistinguishable from cereolysin in its sensitivity to cholesterol, activation by dithiothreitol, and inactivation by antibodies raised against cereolysin. When the recombinant DNA carrying the cereolysin gene was used as a probe in hybridization experiments with chromosomal DNA from a streptolysin O-producing strain of Streptococcus pyogenes or from listeriolysin-producing strains of Listeria monocytogenes, no positive hybridization signals were obtained. These data suggest that the genes for these three SH-activated cytolysins do not have extended sequence homology.

Bacillus cereus↗