Escherichia coli glutaminyl-tRNA synthetase. II. Characterization of the glnS gene product

Glutaminyl-tRNA synthetase has been purified by a simple, two-column procedure from an Escherichia coli K12 strain carrying the glnS structural gene on plasmid pBR322. The primary sequence of this enzyme as derived from the DNA sequence (see accompanying paper) has been confirmed. Manual Edman degradation was used to identify the NH2-terminal sequence of the protein. Oligopeptides scattered throughout the primary sequence of glutaminyl-tRNA synthetase were sequenced by the gas chromatographic-mass spectrometric method and matched to the theoretical peptides derived from the translated DNA sequence. The expected carboxyl terminus at position 550 was verified by carboxypeptidase B digestion. The primary sequence of glutaminyl-tRNA synthetase contains no extensive sequence repeats. A search was made for sequence homologies between this enzyme and the few other aminoacyl-tRNA synthetases for which primary sequences are available. A single homologous region is shared by at least three of the synthetases examined here.     

Escherichia coli glutaminyl-tRNA synthetase. I. Isolation and DNA sequence of the glnS gene

We have isolated a lambda-transducing phage carrying the gene (glnS) for Escherichia coli glutaminyl-tRNA synthetase. The location of the glnS gene within the 13.5-kilobase E. coli DNA transducing fragment was determined by genetic means. The glnS gene was recloned into plasmid pBR322 and its nucleotide sequence was established. The DNA sequence translates to a protein of 550 amino acids.     

Phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase by the gene products of dnaK and dnaJ in Escherichia coli K-12 cells

In Escherichia coli K-12, the heat shock protein DnaK and DnaJ participate in phosphorylation of both glutaminyl-tRNA synthetase and threonyl-tRNA synthetase since when cellular proteins extracted from the dnaK7(Ts), dnaK756(Ts) and dnaJ259(Ts) mutant cells labeled with 32Pi at 42 degrees C were analyzed by two-dimensional gel electrophoresis, no phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase was observed while phosphorylation of both aminoacyl-tRNA synthetases was detected in the samples extracted from wild-type cells.     

Participation of the dnaK and dnaJ gene products in phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase of Escherichia coli K-12.

The heat shock proteins DnaK and DnaJ of Escherichia coli participate in phosphorylation of both glutaminyl-tRNA synthetase and threonyl-tRNA synthetase. When cellular proteins extracted from the dnaK7(Ts) and dnaJ259(Ts) mutant cells labeled with 32Pi at 42 degrees C were analyzed by two-dimensional gel electrophoresis, no phosphorylation of these proteins was observed when they were compared with those from wild-type cells.     

Escherichia coli glutaminyl-tRNA synthetase is electrostatically optimized for binding of its cognate substrates

Natural evolution has resulted in protein molecules displaying a wide range of binding properties that include extremes of affinity and specificity. A detailed understanding of the principles underlying protein structure-function relationships, particularly with respect to binding properties, would greatly enhance molecular engineering and ligand design studies. Here, we have analyzed the interactions of an aminoacyl-tRNA synthetase for which strong evolutionary pressure has enforced high specificity for substrate binding and catalysis. Electrostatic interactions have been identified as one efficient mechanism for enhancing binding specificity; as such, the effects of charged and polar groups were the focus of this study. The binding of glutaminyl-tRNA synthetase from Escherichia coli to several ligands, including the natural substrates, was analyzed. The electrostatic complementarity of the enzyme to its ligands was assessed using measures derived from affinity optimization theory. The results were independent of the details of the calculational parameters, including the value used for the protein dielectric constant. Glutamine and ATP, two of the natural ligands, were found to be extremely complementary to their binding sites, particularly in regions seen to make electrostatic interactions in the structure. These data suggest that the optimization of electrostatic interactions has played an important role in guiding the evolution of this enzyme. The results also show that the enzyme is able to effectively select for high affinity and specificity for the same chemical moieties both in the context of smaller substrates, and in that of a larger reactive intermediate. The regions of greatest non-complementarity between the enzyme and ligands are the portions of the ligand that make few polar contacts with the binding site, as well as the sites of chemical reaction, where overly strong electrostatic binding interactions with the substrate could hinder catalysis. The results also suggest that the negative charge on the phosphorus center of glutaminyl-adenylate plays an important role in the tight binding of this intermediate, and thus that adenylate analogs that preserve the negative charge in this region may bind substantially tighter than analogs where this group is replaced with a neutral group, such as the sulfamoyl family, which can make similar hydrogen bonds but is uncharged.     

Maize mitochondrial seryl-tRNA synthetase recognizes Escherichia coli tRNASer in vivo and in vitro

In our studies to analyze the structure/function relationships among cytoplasmic and organellar seryl-tRNA synthetases (SerRS), we have characterized a Zea mays cDNA (SerZMm) encoding a protein with significant similarity to prokaryotic SerRS enzymes. To demonstrate the functional identity of SerZMm, the gene sequence encoding the putative mature protein was cloned. This construct complemented in vivo a temperature-sensitive Escherichia coli serS mutant strain. The mature SerZMm protein overexpressed in Escherichia coli efficiently aminoacylated bacterial tRNA^Ser in vitro, while yeast tRNA was a poor substrate. These data identify SerZMm as an organellar maize seryl-tRNA synthetase, the first plant organellar SerRS to be cloned. The analysis of its N-terminal targeting signal suggests a mitochondrial function for the SerZMm protein in maize.     

The core region of human glutaminyl-tRNA synthetase homologies with the Escherichia coli and yeast enzymes.

We have isolated from a Lambda-gt 11 library a human cDNA clone with one open reading frame of about 2400 bases. A stretch of about 350 amino acids in the deduced amino acid sequence is up to 40 percent identical with parts of the known amino acid sequences of E. coli and yeast glutaminyl (Gln)-tRNA synthetase. The isolated cDNA sequence corresponds to an internal section of a 5500 bases long mRNA that codes for a 170 kDa polypeptide associated with Gln-tRNA synthetase. Thus, the human enzyme is about three times larger than the E. coli and two times larger than the yeast Gln-tRNA synthetase. The three enzymes share an evolutionarily conserved core but differ in amino acid sequences linked to the N-terminal and C-terminal side of the core.     

A fluorescence spectroscopic study of glutaminyl-tRNA synthetase from Escherichia coli and its implications for the enzyme mechanism

Interaction between Escherichia coli glutaminyl-tRNA synthetase (GlnRS) and its substrates have been studied by fluorescence quenching. In the absence of other substrates, glutamine, tRNA(Gln) and ATP bind with dissociation constants of 460, 0.22 and 180 microM, respectively. The presence of other substrates has either no effect or, at best a weak effect, on binding of ligands. Attempts to isolate enzyme-bound aminoacyl adenylate did not succeed. Binding of the phosphodiester, 5'-(methyl)adenosine monophosphate (MeAMP), to GlnRS was studied by fluorescence quenching and radioactive-ligand binding. tRNA also only has a weak effect on phosphodiester binding. Selectively pyrene-labeled GlnRS was used to obtain shape and size information for free GlnRS. A comparison with the GlnRS shape in the GlnRS/tRNA(Gln) crystal structure indicates that no major change in shape and size occurs upon tRNA(Gln) binding to GlnRS. 5,5'-Bis(8-anilino-1-naphthalene sulfonate) (bis-ANS), a non-covalent fluorescent probe, was also used to probe for conformational changes in GlnRS. This probe also indicated that no major conformational change occurs upon tRNA(Gln) binding. We conclude that lack of tRNA-independent pyrophosphate-exchange activity in this enzyme is not a result of either lack of glutamine or ATP binding in the absence of tRNA, or formation of aminoacyl adenylate and slow release of pyrophosphate. A conformational change is implied upon tRNA binding, which promotes pyrophosphate exchange. Fluorescence studies indicate that this conformational change must be limited and local in nature.     

In vivo and in vitro function of the Escherichia coli periplasmic cysteine oxidoreductase DsbG

We have characterized in vivo and in vitro the recently identified DsbG from Escherichia coli. In addition to sharing sequence homology with the thiol disulfide exchange protein DsbC, DsbG likewise was shown to form a stable periplasmic dimer, and it displays an equilibrium constant with glutathione comparable with DsbA and DsbC. DsbG was found to be expressed at approximately 25% the level of DsbC. In contrast to earlier results (Andersen, C. L., Matthey-Dupraz, A., Missiakas, D., and Raina, S. (1997) Mol. Microbiol. 26, 121-132), we showed that dsbG is not essential for growth and that dsbG null mutants display no defect in folding of multiple disulfide-containing heterologous proteins. Overexpression of DsbG, however, was able to restore the ability of dsbC mutants to express heterologous multidisulfide proteins, namely bovine pancreatic trypsin inhibitor, a protein with three disulfides, and to a lesser extent, mouse urokinase (12 disulfides). As in DsbC, the putative active site thiols in DsbG are completely reduced in vivo in a dsbD-dependent fashion, as would be expected if DsbG is acting as a disulfide isomerase or reductase. However, the latter is not likely because DsbG could not catalyze insulin reduction in vitro. Overall, our results indicate that DsbG functions primarily as a periplasmic disulfide isomerase with a narrower substrate specificity than DsbC.