title: Acquisition of an insertion peptide for efficient aminoacylation by a halophile tRNA synthetase.
abstract: Enzymes of halophilic organisms contain unusual peptide motifs that are absent from their mesophilic counterparts. The functions of these halophile-specific peptides are largely unknown. Here we have identified an unusual peptide that is unique to several halophile archaeal cysteinyl-tRNA synthetases (CysRS), which catalyze attachment of cysteine to tRNA(Cys) to generate the essential cysteinyl-tRNA(Cys) required for protein synthesis. This peptide is located near the active site in the catalytic domain and is highly enriched with acidic residues. In the CysRS of the extreme halophile Halobacterium species NRC-1, deletion of the peptide reduces the catalytic efficiency of aminoacylation by a factor of 100 that largely results from a defect in kcat, rather than the Km for tRNA(Cys). In contrast, maintaining the peptide length but substituting acidic residues in the peptide with neutral or basic residues has no major deleterious effect, suggesting that the acidity of the peptide is not important for the kcat of tRNA aminoacylation. Analysis of general protein structure under physiological high salt concentrations, by circular dichroism and by fluorescence titration of tRNA binding, indicates little change due to deletion of the peptide. However, the presence of the peptide confers tolerance to lower salt levels, and fluorescence analysis in 30% sucrose reveals instability of the enzyme without the peptide. We suggest that the stability associated with the peptide can be used to promote proper enzyme conformation transitions in various stages of tRNA aminoacylation that are associated with catalysis. The acquisition of the peptide by the halophilic CysRS suggests an enzyme adaptation to high salinity.
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10 Street, Suite 220, Philadelphia, Pennsylvania 19107, USA.
authors.forename: Caryn
authors.initials: C
authors.lastname: Evilia
authors.forename: Ya-Ming
authors.initials: YM
authors.lastname: Hou
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000235:genetics; D049148:Aminoacylation; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D006217:Halobacterium / Q000201:enzymology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D011189:Potassium Chloride / Q000737:chemistry; D011487:Protein Conformation; D017384:Sequence Deletion
chemical_list: D010455:Peptides; D011189:Potassium Chloride; D000604:Amino Acyl-tRNA Synthetases; C019975:cysteinyl-tRNA synthetase