Natural variation of tyrosyl-tRNA synthetase and comparison with engineered mutants.
We report the cloning and sequence analysis of the gene for the tyrosyl-tRNA synthetase from Bacillus caldotenax and properties of the gene product. The amino acid sequence of the tyrosyl-tRNA synthetase was found to be 99% homologous with the corresponding enzyme from B. stearothermophilus, with only four amino acid differences. Two of these natural variations were found to involve active site residues of the enzyme and correspond to mutations that have been engineered previously in vitro. One, Thr-51----Ala-51, produced a more active enzyme, possessing a higher value of kcat/KM for ATP. Position 51 is a "hot spot" in the tyrosyl-tRNA synthetase, differing in enzymes derived from Escherichia coli, B. stearothermophilus, and B. caldotenax. The other, His-48----Asn-48, is found to be a neutral mutation but is in one of the rare regions that are conserved with other aminoacyl-tRNA synthetases. The equivalence of histidine and asparagine at position 48 extends the homology in this region to more enzymes. These residues, His-Ile-Gly-His, and now His-Ile-Gly-Asn, form part of the binding site for ATP in the transition state of the reaction. Although B. caldotenax is an obligate thermophile with an optimal growth temperature of 80 degrees C, as much as 20 degrees C above the growth optima of strains of Bacillus stearothermophilus, its tyrosyl-tRNA synthetase has an identical thermal stability in vitro to that from B. stearothermophilus.
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D004262:DNA Restriction Enzymes; D005796:Genes; D005798:Genes, Bacterial; D005818:Genetic Engineering; D014644:Genetic Variation; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics*; D009154:Mutation; D012689:Sequence Homology, Nucleic Acid; D014448:Tyrosine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism
D004262:DNA Restriction Enzymes; D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase