SERYL-TRANSFER-RNA 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 an d stability tests in KCl, NaCl and MgCl2 solutions. Although the natur al external environment of this archaebacterium is rich in sodium ions and poor in potassium ions, the converse being the case in the bacter ial cytosol, there is no large significant difference in activity and stability in vitro of the enzyme between solutions of NaCl and KCl. Lo w, but not high, concentrations of MgCl2 stabilize the enzyme. The enz yme 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 anti sera against the catalytic domain of the homologous E. coli enzyme. Th e gene coding for the H. marismortui enzyme has been isolated and sequ enced. The derived amino acid sequence is the first of a hyperhalophil ic aminoacyl-tRNA synthetase. The wild-type gene and a mutant gene wit h a deletion of the halophile-specific insertion were expressed in E. coli using the T7 RNA polymerase and the Thiofusion(TM) expression sys tems. None of the expressed proteins were enzymically active. A struct ural model has been produced by comparison with other seryl-tRNA synth etases which illustrates the high negative-charge density of the surfa ce of the hyperhalophilic enzyme.