The effect of N-terminal changes on arginyl-tRNA synthetase from Escherichia coli.
An Asn(2) deleted mutant of Escherichia coli arginyl-tRNA synthetase deleted Asn(2) and a chimera mutant, in which the N-terminal 23 amino acid residues of yeast arginyl-tRNA synthetase were appended to the N-terminus of Escherichia coli synthetase, were synthesized and studied. The expression of the deletion and chimera mutants in Escherichia coli formed inclusion bodies, presumably due to improper folding of the proteins. Relative to the native enzyme, the deletion mutant showed full amino acid activation activity and a 26% reduction in aminoacylation activity, while the chimera mutant lost 93% and 96% activities in aminoacid activation and aminoacylation, respectively, and did not aminoacylate yeast tRNA(Arg) at all. The mutant deleted Asn(2) and Ile(3) was able to be expressed in Escherichia coli but not stable to be purified. The emission maximum wavelength in the fluorescence spectra of the chimera mutants shifted to longer one and the corresponding intensities decreased, when compared with those of the native enzyme. The data show that the conformation of the mutants are different and the tryptophan residues in the mutants are more exposed than those in the native enzyme. An estimate of the secondary structure of the mutant enzymes from their far ultraviolet CD spectra showed that the chimera mutant contained less alpha-helix, more beta-sheet and slightly higher fraction of random coil, as compared with the native enzyme. The results indicate that an intact N-terminal domain of E.coli arginyl-tRNA synthetase is important to its activity and correct folding.
Liu
Wen
W
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China. wed@server.shcnc.ac.cn
Liu
Mo-Fang
MF
Xia
Xia
X
Wang
En-Duo
ED
Wang
Ying-Lai
YL
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism; D001126:Arginine-tRNA Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D009154:Mutation; D012347:RNA, Transfer, Arg / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D020539:Sequence Analysis, Protein; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence
D012347:RNA, Transfer, Arg; D011993:Recombinant Fusion Proteins; D001120:Arginine; D001126:Arginine-tRNA Ligase