Threonyl-tRNA synthetase from Thermus thermophilus: purification and some structural and kinetic properties.
Threonyl-tRNA synthetase (ThrRS) has been isolated from an extreme thermophile Thermus thermophilus strain HB8. The enzyme was purified to electrophoretic homogeneity by combinations of column chromatographies on DEAE-Sepharose, S-Sepharose, ACA-44 Ultrogel and HA-Ultrogel. Seventeen mg of purified enzyme were obtained from 1 kg of biomass. In parallel, purified aspartyl- and phenylalanyl-tRNA synthetases were obtained. The purified ThrRS is composed of two identical subunits with a molecular mass of about 77,000 (virtually the same as E coli ThrRS). The N-terminal sequence has been determined. The homology between the first 45 amino acid residues of ThrRS from T thermophilus and E coli is about 29%. A comparative study of tRNA(Thr) charging by ThrRS from E coli and T thermophilus reveals a similar efficiency of the reaction in both homologous systems. This efficiency remains unchanged for aminoacylation of tRNA(Thr) from T thermophilus by the heterologous ThrRS from E coli, but decreases 700 times for aminoacylation of E coli tRNA(Thr) by ThrRS from T thermophilus.
Department of Structure and Function of Ribosomes, Russian Academy of Sciences, Pushchino, Moscow region.
J
J
Zheltonosova
E
E
Melnikova
M
M
Garber
J
J
Reinbolt
D
D
Kern
C
C
Ehresmann
B
B
Ehresmann
D000595:Amino Acid Sequence; D001226:Aspartate-tRNA Ligase / Q000737:chemistry / Q000302:isolation & purification; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010652:Phenylalanine-tRNA Ligase / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000201:enzymology*; D013914:Threonine-tRNA Ligase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D001226:Aspartate-tRNA Ligase; D010652:Phenylalanine-tRNA Ligase; D013914:Threonine-tRNA Ligase