The Effect of
N-terminal Changes on Arginyl-tRNA Synthetase from Escherichia coli
LIU Wen, LIU
Mo-Fang, XIA Xian, WANG En-Duo*, WANG Ying-Lai
( 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 )
Abstract An Asn2
deleted mutant of Escherichia coli arginyl-tRNA synthetase deleted Asn2
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 tRNAArg
at all. The mutant deleted Asn2
and Ile3 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 α-helix, more β-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.
Key words arginyl-tRNA synthetase; N-terminal; activity;
mutatation
*Corresponding author: Tel,
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