enzyme YadB activates glutamate in the absence of tRNA and transfers the activated glutamate not on tRNAGlu but instead on tRNAAsp. tRNAAsp is able to accept two amino acids: aspartate charged by aspartyl-tRNA synthetase and glutamate charged byYadB. YadB transfers the activated glutamate on the cyclopenthene-diol ring of the modified nucleoside queuosine posttranscriptionally inserted at the wobble position of the anticodon-loop to form glutamylqueuosine
required, tetracoordinated to residues Cys101, Cys103, Cys119 and Tyr115. The zinc ion has a high impact on the enzyme structure and function. Enzyme Glu-Q-RS contain a single zinc-binding site in their putative tRNA acceptor stem-binding domain. Quantification by X-ray fluorescence spectrometry technique. Absence of saturating zinc in solution displays dramatic change in the solubility of the protein
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determination of strucure by molecular replacement, to 1.5 A resolution. Enzyme possesses a Zn2+ located in the putative tRNA acceptor stem-binding domain. The Zn2+ ion is coordinated by three cysteine and a tyrosine ligand
site-directed mutagenesis, in the mutant the zinc ion still remains coordinated but the variant becomes structurally labile and acquires aggregation capacity. The extent of aggregation of the protein is significantly decreased in presence of the small substrates and more particularly by adenosine triphosphate. Addition of zinc increases significantly the solubility of the variant. The aminoacylation assay reveals a decrease in activity of the mutant even after addition of zinc as compared to the wild-type, although the secondary structure of the protein is not altered. Unlike wild type Glu-Q-RS, the mutant shows a strong tendency to aggregate in solution at room temperature, addition of a saturating concentration of the small substrate ATP and/or L-Glu decreases the rate of aggregation of C101S/C103S Glu-Q-RS, ATP has the most efficient effect