title: Fusion with anticodon binding domain of GluRS is not sufficient to alter the substrate specificity of a chimeric Glu-Q-RS.
abstract: Glutamyl-queuosine-tRNA(Asp) synthetase (Glu-Q-RS) is a paralog of glutamyl-tRNA synthetase (GluRS) and is found in more than forty species of proteobacteria, cyanobacteria, and actinobacteria. Glu-Q-RS shows striking structural similarity with N-terminal catalytic domain of GluRS (NGluRS) but it lacks the C-terminal anticodon binding domain (CGluRS). In spite of structural similarities, Glu-Q-RS and NGluRS differ in their functional properties. Glu-Q-RS glutamylates the Q34 nucleotide of the anticodon of tRNA(Asp) whereas NGluRS constitutes the catalytic domain of GluRS catalyzing the transfer of Glu on the acceptor end of tRNA(Glu). Since NGluRS is able to catalyze aminoacylation of only tRNA(Glu) the glutamylation capacity of tRNA(Asp) by Glu-Q-RS is surprising. To understand the substrate specificity of Glu-Q-RS we undertook a systemic approach by investigating the biophysical and biochemical properties of the NGluRS (1-301), CGluRS (314-471) and Glu-Q-RS-CGluRS, (1-298 of Glu-Q-RS fused to 314-471 from GluRS). Circular dichroism, fluorescence spectroscopy and differential scanning calorimetry analyses revealed absence of N-terminal domain (1-298 of Glu-Q-RS) and C-terminal domain (314-471 from GluRS) communication in chimera, in contrast to the native full length GluRS. The chimeric Glu-Q-RS is still able to aminoacylate tRNA(Asp) but has also the capacity to bind tRNA(Glu). However the chimeric protein is unable to aminoacylate tRNA(Glu) probably as a consequence of the lack of domain-domain communication.
authors.affiliation: Department of Biotechnology and Dr. B C Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700 019, India.
authors.forename: Sutapa
authors.initials: S
authors.lastname: Ray
authors.forename: Mickael
authors.initials: M
authors.lastname: Blaise
authors.forename: Bappaditya
authors.initials: B
authors.lastname: Roy
authors.forename: Saptaparni
authors.initials: S
authors.lastname: Ghosh
authors.forename: Daniel
authors.initials: D
authors.lastname: Kern
authors.forename: Rajat
authors.initials: R
authors.lastname: Banerjee
mesh_terms: D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000378:metabolism; D000926:Anticodon / Q000737:chemistry / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D002942:Circular Dichroism; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D000926:Anticodon; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; C487511:YadB protein, E coli; D000604:Amino Acyl-tRNA Synthetases