title: Preliminary X-ray crystallographic analysis of an engineered glutamyl-tRNA synthetase from Escherichia coli.
abstract: The nature of interaction between glutamyl-tRNA synthetase (GluRS) and its tRNA substrate is unique in bacteria in that many bacterial GluRS are capable of recognizing two tRNA substrates: tRNAGlu and tRNAGln. To properly understand this distinctive GluRS-tRNA interaction it is important to pursue detailed structure-function studies; however, because of the fact that tRNA-GluRS interaction in bacteria is also associated with phylum-specific idiosyncrasies, the structure-function correlation studies must also be phylum-specific. GluRS from Thermus thermophilus and Escherichia coli, which belong to evolutionarily distant phyla, are the biochemically best characterized. Of these, only the structure of T. thermophilus GluRS is available. To fully unravel the subtleties of tRNAGlu-GluRS interaction in E. coli, a model bacterium that can also be pathogenic, determination of the E. coli GluRS structure is essential. However, previous attempts have failed to crystallize E. coli GluRS. By mapping crystal contacts of a homologous GluRS onto the E. coli GluRS sequence, two surface residues were identified that might have been hindering crystallization attempts. Accordingly, these two residues were mutated and crystallization of the double mutant was attempted. Here, the design, expression, purification and crystallization of an engineered E. coli GluRS in which two surface residues were mutated to optimize crystal contacts are reported.
authors.affiliation: Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII M, Kolkata 700 054, India.
authors.forename: Nipa
authors.initials: N
authors.lastname: Chongdar
authors.affiliation: Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII M, Kolkata 700 054, India.
authors.forename: Saumya
authors.initials: S
authors.lastname: Dasgupta
authors.affiliation: Department of Biochemistry, Bose Institute, P-1/12 CIT Scheme VII M, Kolkata 700 054, India.
authors.forename: Ajit Bikram
authors.initials: AB
authors.lastname: Datta
authors.affiliation: Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII M, Kolkata 700 054, India.
authors.forename: Gautam
authors.initials: G
authors.lastname: Basu
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005975:Glutamate-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D005975:Glutamate-tRNA Ligase; D008239:Lysine; D000409:Alanine
keywords: Escherichia coli; glutamyl-tRNA synthetase