PMID:1346128
| Citation |
Hinchman, SK, Henikoff, S and Schuster, SM (1992) A relationship between asparagine synthetase A and aspartyl tRNA synthetase. J. Biol. Chem. 267:144-9 |
|---|---|
| Abstract |
A highly conserved protein motif characteristic of Class II aminoacyl tRNA synthetases was found to align with a region of Escherichia coli asparagine synthetase A. The alignment was most striking for aspartyl tRNA synthetase, an enzyme with catalytic similarities to asparagine synthetase. To test whether this sequence reflects a conserved function, site-directed mutagenesis was used to replace the codon for Arg298 of asparagine synthetase A, which aligns with an invariant arginine in the Class II aminoacyl tRNA synthetases. The resulting genes were expressed in E. coli, and the gene products were assayed for asparagine synthetase activity in vitro. Every substitution of Arg298, even to a lysine, resulted in a loss of asparagine synthetase activity. Directed random mutagenesis was then used to create a variety of codon changes which resulted in amino acid substitutions within the conserved motif surrounding Arg298. Of the 15 mutant enzymes with amino acid substitutions yielding soluble enzyme, 13 with changes within the conserved region were found to have lost activity. These results are consistent with the possibility that asparagine synthetase A, one of the two unrelated asparagine synthetases in E. coli, evolved from an ancestral aminoacyl tRNA synthetase. |
| Links | |
| Keywords |
Amino Acid Sequence; Aspartate-Ammonia Ligase/genetics; Aspartate-Ammonia Ligase/metabolism; Aspartate-tRNA Ligase/genetics; Aspartate-tRNA Ligase/metabolism; Base Sequence; Catalysis; Codon; Escherichia coli/enzymology; Escherichia coli/metabolism; Gene Expression; Molecular Sequence Data; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Sequence Alignment |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
References
See Help:References for how to manage references in EcoliWiki.