Changing the amino acid specificity of yeast tyrosyl-tRNA synthetase by genetic engineering

In an attempt to generate mutant aminoacyl-tRNA synthetases capable of char ging noncanonical amino acids, a series of yeast tyrosyl-tRNA synthetase (T yrRS) mutants was constructed by site-specific mutagenesis of putative acti ve site residues, which were deduced by analogy with those of Bacillus stea rothermophilus TyrRS. Among these mutants, one with the replacement of tyro sine at position 43 by glycine, "Y43G," was found to be able to utilize sev eral 3-substituted tyrosine analogues as substrates for aminoacylation. The catalytic efficiency (k(cat)/K-m) of mutant Y43G for aminoacylation with L -tyrosine was about 400-fold decreased as compared to that of the wild-type TyrRS. On the other hand, the ability to utilize 3-iodo-L-tyrosine was new ly generated in this mutant TyrRS, since the wild-type TyrRS could not acce pt 3-iodo-L-tyrosine at all under physiological conditions. This mutant Tyr RS should serve as a new tool for site-specific incorporation of non-canoni cal amino acids, such as those in 3-substituted tyrosine analogues, into pr oteins in an appropriate translation system in vivo or in vitro.