Seryl-tRNA synthetase from the extreme halophile Haloarcula marismortui--isolation, characterization and sequencing of the gene and its expression in Escherichia coli.
The seryl-tRNA synthetase from the extreme halophilic archaebacterium Haloarcula marismortui, belonging to the group Euryarchaeota, has been purified and its hyperhalophilic behavior demonstrated by activity and stability tests in KCl, NaCl and MgCl2 solutions. Although the natural external environment of this archaebacterium is rich in sodium ions and poor in potassium ions, the converse being the case in the bacterial cytosol. there is no large significant difference in activity and stability in vitro of the enzyme between solutions of NaCl and KCl. Low, but not high, concentrations of MgCl2 stabilize the enzyme. The enzyme aminoacylates tRNA from Escherichia coli even under the high salt conditions of the assay. A fluorescence study indicated that low salt denaturation of the hyperhalophilic enzyme is a biphasic process. The hyperhalophilic enzyme demonstrated immunological reactivity with antisera against the catalytic domain of the homologous E. coli enzyme. The gene coding for the H. marismortui enzyme has been isolated and sequenced. The derived amino acid sequence is the first of a hyperhalophilic aminoacyl-tRNA synthetase. The wild-type gene and a mutant gene with a deletion of the halophile-specific insertion were expressed in E. coli using the T7 RNA polymerase and the Thiofusion expression systems. None of the expressed proteins were enzymically active. A structural model has been produced by comparison with other seryl-tRNA synthetases which illustrates the high negative-charge density of the surface of the hyperhalophilic enzyme.
Taupin
C M
CM
EMBL Grenoble Outstation, France.
Härtlein
M
M
Leberman
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D005798:Genes, Bacterial; D016931:Halobacteriales / Q000201:enzymology / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D011489:Protein Denaturation; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012714:Serine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013499:Surface Properties
D011994:Recombinant Proteins; D012714:Serine-tRNA Ligase