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.
Highlights
A highly conserved protein motif characteristic of amino acids [8].No amino acid sequence similarity could be
Class I1 aminoacyl tRNA synthetaseswas found to detected between E. coli Asparagine synthetase (AS) A and E. coli AS B
The alignment was most striking for aspar- distantly diverged members of the same family comes from tyl tRNA synthetase, anenzyme with catalyticsimilaritiesto asparagine synthetase.To test whether this sequence reflects a conserved function, site-directed mutagenesis was used to replace the codon for ArgZgs of asparagine synthetase A, which aligns with an invariant arginine in the Class I1 aminoacyl tRNA synan alignment of E. coli AS B with human AS, which shows clear evidence for homology, with 37% identical amino acids [8].E. coli AS A is not significantly related to any sequence in the protein or DNA databases as determined by FASTA searches (Ref. 8).’
Summary
From the $Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, Florida 32610 and the §Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104. With the closest resemblance being to the subgroup of ArgPSse, ven to a lysine, resulted in a loss of asparagine aspartyl tRNA synthetases The hypothesis that this particsynthetase activity.Directed random mutagenesis was ular region of E. coli AS Ais involved in enzyme function was used to create a variety of codon changes which tested using site-directed mutagenesis. This is consistent enzymes with amino acid substitutions yielding soluble enzyme, 13 with changes within theconserved 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.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
