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Biomedical subjects

R Leberman

Publications and source records attributed to R Leberman.

At least 19 recordsLinked to original sources
PubMed1999

Archaeabacterial seryl-tRNA synthetases: adaptation to extreme environments and evolutionary analysis.

The aminoacyl-tRNA synthetases are ubiquitous enzymes which catalyze a crucial step of the cell life, the specific attachment of amino acids to their cognate tRNA. The amino acid sequences of three archaeal seryl-tRNA synthetases (SerRS) from Haloarcula marismortui and Methanococcus jannaschii, both belonging to the group of Euryarchaeota, and from Sulfolobus solfataricus, of the group of Crenarchaeota, were aligned with other eubacterial and eukaryal available SerRS sequences. In an attempt to identify some features of adaptation to extreme environments of these organisms, amino acid composition and amino acid substitutions between mesophilic and thermophilic SerRS were analyzed. In addition, universal phylogenetic trees of SerRS including the three known archaeal sequences, rooted by the threonyl-tRNA synthetases were inferred. Amino acid analyses of the SerRS revealed two ways of adaptation to thermophilic environments between the Eubacteria and the Archaea; most of the usually described amino acid substitutions were nonsignificant in the case of archaeal thermophilic SerRS and most amino acid composition biases seemed to be linked to the genome G+C content pressure. The phylogenetic analysis of the SerRS showed the Archaea to be paraphyletic, H. marismortui emerging with the Gram-positive Bacteria, M. jannaschii being near the root of the tree, and S. solfataricus branching with Eucarya.

Adaptation, Physiological↗
PubMed1998

The crystal structure of asparaginyl-tRNA synthetase from Thermus thermophilus and its complexes with ATP and asparaginyl-adenylate: the mechanism of discrimination between asparagine and aspartic acid.

The crystal structure of Thermus thermophilus asparaginyl-tRNA synthetase has been solved by multiple isomorphous replacement and refined at 2.6 A resolution. This is the last of the three class IIb aminoacyl-tRNA synthetase structures to be determined. As expected from primary sequence comparisons, there are remarkable similarities between the tertiary structures of asparaginyl-tRNA synthetase and aspartyl-tRNA synthetase, and most of the active site residues are identical except for three key differences. The structure at 2.65 A of asparaginyl-tRNA synthetase complexed with a non-hydrolysable analogue of asparaginyl-adenylate permits a detailed explanation of how these three differences allow each enzyme to discriminate between their respective and very similar amino acid substrates, asparagine and aspartic acid. In addition, a structure of the complex of asparaginyl-tRNA synthetase with ATP shows exactly the same configuration of three divalent cations as previously observed in the seryl-tRNA synthetase-ATP complex, showing that this a general feature of class II synthetases. The structural similarity of asparaginyl- and aspartyl-tRNA synthetases as well as that of both enzymes to the ammonia-dependent asparagine synthetase suggests that these three enzymes have evolved relatively recently from a common ancestor.

Adenosine Triphosphate↗
PubMed1998

Identification of YHR019 in Saccharomyces cerevisiae chromosome VIII as the gene for the cytosolic asparaginyl-tRNA synthetase.

Exploiting the asparagine auxotrophy of the Saccharomyces cerevisiae mutant strain 8556a, we have isolated the gene for the cytosolic asparaginyl-tRNA synthetase (AsnRS) of S. cerevisiae, by functional complementation of the mutation affecting this strain. The isolated gene could be identified to the open reading frame YHR019, called DED81, located on chromosome VIII. The mutant gene from the 8556a strain, asnrs-1, was amplified from genomic DNA by PCR. This gene contains a point mutation, leading to the replacement of a glycine residue by a serine in a region of the protein probably important for the asparaginyl-adenylate recognition. The protein encoded by YHR019 is very similar to cytosolic AsnRS from other eukaryotic sources. In a phylogenetic analysis based on AsnRS sequences from various organisms, the eukaryotic sequences were clustered. Expression of YHR019 in Escherichia coli demonstrated that a yeast AsnRS activity was produced. The recombinant enzyme was purified to homogeneity in three chromatography steps. We showed that the recombinant S. cerevisiae AsnRS was able to charge unfractionated yeast tRNA, but not E. coli tRNA, with asparagine.

Amino Acyl-tRNA Synthetases↗
PubMed1998

Analysis and overexpression in Escherichia coli of a staphylococcal gene encoding seryl-tRNA synthetase.

We have sequenced and expressed in Escherichia coli the gene encoding the seryl-tRNA synthetase from the pathogenic bacterium Staphylococcus aureus. The overexpressed and purified recombinant enzyme was able to aminoacylate unfractionated tRNA from E. coli. Its activity was not affected by antibodies raised against and inhibiting the E. coli seryl-tRNA synthetase.

Amino Acid Sequence↗
PubMed1997

Preliminary X-ray diffraction studies on asparaginyl-tRNA synthetase from Thermus thermophilus.

The recombinant asparginyl-tRNA synthetase from the thermophilic bacterium Thermus thermophilus expressed in Escherichia coli has been crystallized from PEG 6000 solutions. Depending on the PEG concentrations the crystals were in either tetragonal or hexagonal space groups. Although generally smaller, the latter (space group P6(4)22) diffracted better, to a resolution of 2.8 A. Using the coordinates of the yeast aspartyl-tRNA synthetase structure molecular replacement methods were applied to both tetragonal and hexagonal crystals; a solution was found which gave excellent crystal packing in both space groups.

Journal Article↗
PubMed1997

Seryl-tRNA synthetase from the extreme halophile Haloarcula marismortui--isolation, characterization and sequencing of the gene and its expression in Escherichia coli.

The seryl-tRNA synthetase from the extreme halophilic archaebacterium Haloarcula marismortui, belonging to the group Euryarchaeota, has been purified and its hyperhalophilic behavior demonstrated by activity and stability tests in KCl, NaCl and MgCl2 solutions. Although the natural external environment of this archaebacterium is rich in sodium ions and poor in potassium ions, the converse being the case in the bacterial cytosol. there is no large significant difference in activity and stability in vitro of the enzyme between solutions of NaCl and KCl. Low, but not high, concentrations of MgCl2 stabilize the enzyme. The enzyme aminoacylates tRNA from Escherichia coli even under the high salt conditions of the assay. A fluorescence study indicated that low salt denaturation of the hyperhalophilic enzyme is a biphasic process. The hyperhalophilic enzyme demonstrated immunological reactivity with antisera against the catalytic domain of the homologous E. coli enzyme. The gene coding for the H. marismortui enzyme has been isolated and sequenced. The derived amino acid sequence is the first of a hyperhalophilic aminoacyl-tRNA synthetase. The wild-type gene and a mutant gene with a deletion of the halophile-specific insertion were expressed in E. coli using the T7 RNA polymerase and the Thiofusion expression systems. None of the expressed proteins were enzymically active. A structural model has been produced by comparison with other seryl-tRNA synthetases which illustrates the high negative-charge density of the surface of the hyperhalophilic enzyme.

Amino Acid Sequence↗
PubMed1997

Characterization of a temperature-sensitive Escherichia coli mutant and revertants with altered seryl-tRNA synthetase activity.

A mutation in the structural gene coding for seryl-tRNA synthetase in temperature-sensitive Escherichia coli K28 has been reported to alter the level of enzyme expression at high temperature (R. J. Hill and W. Konigsberg, J. Bacteriol. 141:1163-1169, 1980). We identified this mutation as a C-->T transition in the first base of codon 386, resulting in a replacement of histidine by tyrosine. The steady-state levels of serS mRNA in K28 and in the wild-type strains are very similar. Pulse-chase labeling experiments show a difference in protein stability, but not one important enough to account for the temperature sensitivity of K28. The main reason for the temperature sensitivity of K28 appears to be the low level of specific activity of the mutant synthetase at nonpermissive temperature, not a decreased expression level. Spontaneous temperature-resistant revertants were selected which were found to have about a fivefold-higher level of SerRS than the K28 strain. We identified the mutation responsible for the reversion as being upstream from the -10 sequence in the promoter region. The steady-state levels of serS mRNA in the revertants are significantly higher than that in the parental strain.

Cloning, Molecular↗
PubMed1996

Asparaginyl-tRNA synthetase from Thermus thermophilus HB8. Sequence of the gene and crystallization of the enzyme expressed in Escherichia coli.

The gene for the asparaginyl-tRNA synthetase, a class IIb enzyme, from the extreme thermophile Thermus thermophilus HB8 has been cloned and sequenced. Sequence analysis revealed an open reading frame that codes for a protein of 438 amino acid residues (50875 Da). Codon usage in the asparaginyl-tRNA synthetase gene (asnS) is similar to the characteristic usage in the genes for proteins from bacteria of the genus Thermus, and the G+C content in the third position of the codons is as high as 94%. The amino acid sequence of asparaginyl-tRNA synthetase from T. thermophilus shows high similarity with other bacterial asparaginyl-tRNA synthetase sequences (30-55% identity). By expression of the T. thermophilus asnS gene in Escherichia coli, the thermostable enzyme was overproduced and purified to homogeneity by heat treatment and two chromatography steps. The protein obtained is remarkably thermostable and retains 50% of its initial tRNA aminoacylation activity after 1 h of incubation at 90 degrees C or 21 h at 85 degrees C. Crystals of the enzyme were obtained from polyethylene glycol 6000 solutions by vapour diffusion techniques. The crystals diffract X-rays beyond 2.8 A.

Amino Acid Sequence↗
PubMed1996

Interconversion of crystals of the Escherichia coli EF-Tu.EF-Ts complex between high- and low-diffraction forms.

Crystals of the complex formed between the two bacterial polypeptide elongation factors, EF-Tu and EF-Ts, produced from solutions of PEG 6000 can be of two morphologically similar forms both of space group P2(1)2(1)2(1). One form diffracts to only about 3 A resolution, the other to better than 2.4 A resolution. These forms can be interconverted and the transformation of one into the other has been shown to be solely a result of dehydration/hydration processes. By designing a suitable soaking protocol and careful control of the experimental parameters for data collection at cryotemperatures, complete data sets for the high-resolution form could be obtained.

Journal Article↗
PubMed1996

The structure of the Escherichia coli EF-Tu.EF-Ts complex at 2.5 A resolution.

The crystal structure of the EF-Tu.EF-Ts complex from Escherichia coli has been determined to a resolution of 2.5 A. The complex contains two subunits of each of the elongation factors. The two EF-Ts molecules form a tight dimer, but there is little contact between the two EF-Tu molecules. The interaction of EF-Ts with EF-Tu results principally in the disruption of the Mg2+ ion binding site, thereby reducing the affinity of EF-Tu for guanine nucleotides.

Amino Acid Sequence↗
PubMed1995

An immunodominant antigen of Brugia malayi is an asparaginyl-tRNA synthetase.

Lymphatic filariasis is caused by infection with the filarial nematodes Brugia malayi, Brugia timori, Wuchereria bancrofti and Onchocerca volvulus which collectively infect about 200 million persons throughout the world. Protein sequence homology analysis of a major nematode antigen suggested that it was a class II aminoacyl-tRNA synthetase. The overproduction, purification and verification that the major B. malayi antigen is an asparaginyl-tRNA synthetase is described.

Amino Acid Sequence↗
PubMed1995

Seryl-tRNA synthetase from Escherichia coli: functional evidence for cross-dimer tRNA binding during aminoacylation.

Escherichia coli seryl-tRNA synthetase (SerRS) is a homo-dimeric class II aminoacyl-tRNA synthetase. Each subunit is composed of two distinct domains: the N-terminal domain is a 60 A long, arm-like coiled coil structure built up of two antiparallel alpha-helices, whereas the C-terminal domain, the catalytic core, is an alpha-beta structure overlying a seven-stranded antiparallel beta-sheet. Deletion of the arm-like domain (SerRS delta 35-97) does not affect the amino acid activation step of the reaction, but reduces aminoacylation activity by more than three orders of magnitude. In the present study, it was shown that the formation of heterodimers from two aminoacylation defective homodimers, the N-terminal deletion and an active site mutant (SerRS E355Q), restored charging activity. The aminoacylation activity in a mixture containing the heterodimers was compared to that of solutions containing the same concentrations of homodimer. The activity of the mixture was eight times higher than the activities of the homodimer solutions, and reached 50% of the theoretical value that would be expected if 50% of the mixture was in the heterodimer form and assuming that a heterodimer contains only one active site. These results are in full agreement with the structural analysis of E. coli SerRS complexed with its cognate tRNA and provide functional evidence for the cross-dimer binding of tRNA in solution.

Acylation↗
PubMed1995

Stoichiometry of the EF-Tu.GTP complex with aminoacyl-tRNA: ternary of quinternary?

The stoichiometry of the complex formed between the Escherichia coli polypeptide elongation factor EF-Tu, GTP and valyl-tRNA(val) has been determined by non-enzymatic deacylation studies on mixtures of the components at well-defined concentrations. A titration end-point was found corresponding to a 1:1 complex of EF-Tu.GTP with the aminoacylated-tRNA i.e. formation of a ternary complex. The result conforms to the classical model of the elongation step and not to the revolutionary proposition of the formation of a 2:2:1 complex; quinternary complex (EF-Tu.GTP)2.aa-RNA.

Escherichia coli↗
PubMed1995

Isolation and characterization of an Escherichia coli seryl-tRNA synthetase mutant with a large increase in Km for serine.

A mutant of Escherichia coli resistant to serine hydroxamate which has a large increase in Km for serine of seryl-tRNA synthetase is described. The mutant serS gene was cloned and sequenced and was found to contain a single-base-pair mutation, resulting in the substitution of the residue alanine 262 by valine in motif 2. The methyl side chain of alanine 262 is not exposed at the active site, and molecular modeling indicated that replacement of alanine 262 by valine does not significantly affect the configuration of amino acids at the active site. This finding suggests that the residue at this position may be involved in a conformational change (possibly induced by ATP binding) which is necessary for optimal binding of the cognate amino acid.

Base Sequence↗
PubMed1994

A new additive for protein crystallization.

The potential usefulness of the new zwitterionic solubilizing agent, dimethyl ethylammonium propane sulfonate (NDSB195), in protein crystallization was shown using hen egg-white lysozyme. In the presence of this agent, highly diffracting crystals were obtained using ammonium sulphate as a precipitant, whereas in its absence only amorphous precipitates were obtained. The crystals possess a triclinic unit cell not previously described and diffract to a resolution of 2 A. To ascertain that the new reagent had not produced significant changes in the protein fold the structure was determined to a resolution of 2.6 A. Only minor differences were observed (notably in regions of crystal contacts) with the known tetragonal lysozyme structure (Brookhaven Protein Data Bank entry 1HEL).

Crystallization↗
PubMed1994

Seryl-tRNA synthetase from Escherichia coli: implication of its N-terminal domain in aminoacylation activity and specificity.

Escherichia coli seryl-tRNA synthetase (SerRS) a dimeric class II aminoacyl-tRNA synthetase with two structural domains charges specifically the five iso-acceptor tRNA(ser) as well as the tRNA(sec) (selC product) of E. coli. The N-terminal domain is a 60 A long arm-like coiled coil structure built of 2 long antiparallel a-h helices, whereas the C-terminal domain is a alpha-beta structure. A deletion of the N-terminal arm of the enzyme does not affect the amino acid activation step of the reaction, but reduces dramatically amino-acylation activity. The Kcat/Km value for the mutant enzyme is reduced by more than 4 orders of magnitude, with a nearly 30 fold increased Km value for tRNA(ser). An only slightly truncated mutant form (16 amino acids of the tip of the arm replaced by a glycine) has an intermediate aminoacylation activity. Both mutant synthetases have lost their specificity for tRNA(ser) and charge also non-cognate type 1 tRNA(s). Our results support the hypothesis that class II synthetases have evolved from an ancestral catalytic core enzyme by adding non-catalytic N-terminal or C-terminal tRNA binding (specificity) domains which act as determinants for cognate and anti-determinants for non-cognate tRNAs.

Acylation↗
PubMed1994

Crystal structures at 2.5 angstrom resolution of seryl-tRNA synthetase complexed with two analogs of seryl adenylate.

Crystal structures of seryl-tRNA synthetase from Thermus thermophilus complexed with two different analogs of seryl adenylate have been determined at 2.5 A resolution. The first complex is between the enzyme and seryl-hydroxamate-AMP (adenosine monophosphate), produced enzymatically in the crystal from adenosine triphosphate (ATP) and serine hydroxamate, and the second is with a synthetic analog of seryl adenylate (5'-O-[N-(L-seryl)-sulfamoyl]adenosine), which is a strong inhibitor of the enzyme. Both molecules are bound in a similar fashion by a network of hydrogen bond interactions in a deep hydrophilic cleft formed by the antiparallel beta sheet and surrounding loops of the synthetase catalytic domain. Four regions in the primary sequence are involved in the interactions, including the motif 2 and 3 regions of class 2 synthetases. Apart from the specific recognition of the serine side chain, the interactions are likely to be similar in all class 2 synthetases.

Adenosine↗