engleski

The role of zinc ion in the active site of atypical archael seryl-tRNA synthetase: Structure and biochemical analysis

Aminoacyl-tRNA synthetases are the key enzymes in ensuring the fidelity of translation. They are responsible for correct attachment of the appropriate amino acids to their specific tRNA. Seryl-tRNA synthetases (SerRSs), specific for amino acid serine, can be divided into two quite divergent groups, bacterial-type and methanogenic-type enzymes. The standard or bacterial-type SerRS has been found in the majority of organisms (prokaryotes, eukaryotes and archaea) and the methanogenic-type enzyme in methanogenic archaea, with the exception of Methanosarcina mazei and Methanosarcina acetivorans. Therefore, we decided to crystallize and solve the structure of the methanogenic-type seryl-tRNA synthetase. Recombinant enzyme from Methanosarcina barkeri has been purified from Eschericia coli bacterial cells and the conditions for crystallization of apo-protein have been determined. In addition, the structure of M. barkeri seryl-tRNA synthetase has been solved also in complex with: serine, ATP and seryl-adenylat analogue, 5’-O-(N-(L-seryl)sulfamoyl)adenozine. In contrast to bacterial type, methanogenic-type seryl-tRNA synthetase relies on zinc ion for amino acid selection. Additionally, tRNA binding domain of the methanogenic enzyme is very different both in sequence and structure. The results of this study also showed that M. barkeri seryl-tRNA synthetase has the unique insertion in catalytic core. The role of this insertion in positioning of the tRNA-binding domain with respect to the catalytic core has been determined by gel-shift and aminoacylation experiments. Finally, we performed directed mutagenesis in the active site to determine the enzyme specificity for serine recognition.

protein synthesis; aminoacyl-tRNA; seryl-tRNA synthetase; zinc

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