title: Mapping the stability determinants of bacterial tyrosyl transfer RNA synthetases by an experimental evolutionary approach.
abstract: The tyrosyl-tRNA synthetases from Bacillus stearothermophilus (Bst-TyrTS) and Escherichia coli (Eco-TyrTS) are 56% identical in amino acid sequence. To map and characterize the set of interactions that makes Bst-TyrTS more stable than Eco-TyrTS, a family of nine hybrid proteins was constructed between the two enzymes. The N-terminal part of each hybrid came from Eco-TyrTS and the C-terminal part from Bst-TyrTS. The stability and activity of these hybrids were estimated by experiments of thermal inactivation and tRNA charging. For all the hybrids, the temperature of half-inactivation in 30 minutes was above 44 degrees C and the rate of charging was at least 40% that of Bst-TyrTS. In general, the temperature of half-inactivation increased and the rate of charging decreased monotonically when the number of residues coming from the more stable and less active Bst-TyrTS increased. As a result, the rate of charging decreased when the temperature of half-inactivation increased. These results show that the sequences and structures of the two enzymes can replace each other locally and still give a stable and active TyrTS, and that the greater stability of Bst-TyrTS is due to cumulative changes of residues scattered along the sequence. They suggest that Bst-TyrTS is more rigid than Eco-TyrTS at low temperature. The existence of a few exceptional hybrids, having stabilities or activities lower than those of the neighbouring hybrids, shows that compensatory changes of residues have occurred between the two sequences during evolution. These exceptions could be explained by the systematic identification of the couples of residues that are in contact in the Bst-TyrTS structure and become heterologous in some hybrids.
authors.affiliation: Unité de Biochimie Cellulaire, Institut Pasteur, Paris, France.
authors.forename: V
authors.initials: V
authors.lastname: Guez-Ivanier
authors.forename: M
authors.initials: M
authors.lastname: Hermann
authors.forename: D
authors.initials: D
authors.lastname: Baldwin
authors.forename: H
authors.initials: H
authors.lastname: Bedouelle
mesh_terms: D001665:Binding Sites; D005075:Biological Evolution; D004926:Escherichia coli / Q000201:enzymology; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D014448:Tyrosine-tRNA Ligase