NDLI: A Mutant Escherichia coli Tyrosyl-tRNA Synthetase Utilizes the Unnatural Amino Acid Azatyrosine More Efficiently than Tyrosine

Content Provider	Journal of Biological Chemistry (JBC)
Author	Hamano-Takaku, Fumie Iwama, Toshiharu Saito-Yano, Shio Takaku, Kazuaki Monden, Yoshiaki Kitabatake, Makoto Söll, Dieter Nishimura, Susumu
Abstract	Alloproteins, proteins that contain unnatural amino acids, have immense potential in biotechnology and medicine. Although various approaches for alloprotein production exist, there is no satisfactory method to produce large quantities of alloproteins containing unnatural amino acids in specific positions. The tyrosine analogue azatyrosine,l-β-(5-hydroxy-2-pyridyl)-alanine, can convert theras-transformed phenotype to normal phenotype, presumably by its incorporation into cellular proteins. This provided the stimulus for isolation of a mutant tyrosyl-tRNA synthetase (TyrRS) capable of charging azatyrosine to tRNA. A plasmid library of randomly mutatedEscherichia coli tyrS (encoding TyrRS) was made by polymerase chain reaction techniques. The desired TyrRS mutants were selected by screening for in vivo azatyrosine incorporation of E. coli cells transformed with the mutanttyrS plasmids. One of the clones thus isolated, R-6-A-7, showed a 17-fold higher in vivo activity for azatyrosine incorporation than wild-type TyrRS. The mutant tyrS gene contained a single point mutation resulting in replacement of phenylalanine by serine at position 130 in the protein. Structural modeling revealed that position 130 is located close to Asp182, which directly interacts with tyrosyladenylate. Kinetic analysis of aminoacyl-tRNA formation by the wild-type and mutated F130S TyrRS enzymes showed that the specificity for azatyrosine, measured by the ratios ofk cat/K m for tyrosine and the analogue, increased from 17 to 36 as a result of the F130S mutation. Thus, the high discrimination against azatyrosine is significantly reduced in the mutant enzyme. These results suggest that utilization of F130S TyrRS for in vivo protein biosynthesis may lead to efficient production of azatyrosine-containing alloproteins.
Related Links	http://www.jbc.org/content/275/51/40324.abstract
Ending Page	40328
Starting Page	40324
Page Count	5
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	51
Volume Number	275
DOI	10.1074/jbc.M003696200
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2000-12-22
Access Restriction	Open
Subject Keyword	Tyrosyl-tRNA synthetase (TyrRS) Wild-type, TCA, trichloroacetic acid (wt) ENZYME CATALYSIS AND REGULATION
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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