title: The zinc-binding site of Escherichia coli glutamyl-tRNA synthetase is located in the acceptor-binding domain. Studies by extended x-ray absorption fine structure, molecular modeling, and site-directed mutagenesis.
abstract: The zinc contents of fragments of Escherichia coli glutamyl-tRNA synthetase, as well as the conservation of the CYC sequence only in zinc-containing glutamyl-tRNA synthetases, suggested that the 98CYCX24-CRHSHEHHADDEPC138 includes some or all residues involved in binding its zinc atom (Liu, J., Lin, S.-X., Blochet, J.-E., Pézolet, M., and Lapointe, J. (1993) Biochemistry 32, 11390-11396). Extended x-ray absorption fine structure (EXAFS) shows that this zinc atom has a four-coordinate non-planar coordination environment with 3 sulfur and 1 nitrogen atoms with bond lengths, respectively, 2.37 +/- 0.02 A and 2.01 +/- 0.02 A, presumably belonging to 3 cysteine residues and 1 histidine residue. Conservative replacement of each histidine and cysteine residue of the 98C-138C segment, respectively, with glutamine (Q) and serine (S), yields variants H129Q, H131Q, H132Q, and C138S (which sustain the growth at 42 degrees C of E. coli JP1449, whose glutamyl-tRNA synthetase is thermosensitive) and C98S, C100S, C125S, and H127Q (which do not). The amount of this enzyme in these mutants is at least 1 order of magnitude larger than that in a wild type strain; however, no glutamyl-tRNA synthetase activity is detectable in extracts of the variants C100S and C125S, whereas its specific activity in those of C98S and H127Q is about 10-fold lower than in cells overproducing the wild type enzyme or the variants H129Q, H131Q, H132Q, and C138S. These results indicate that the zinc atom present in E. coli glutamyl-tRNA synthetase is bound by the 2 evolutionarily conserved cysteines at positions 98 and 100, and by Cys125 and His127. Molecular modeling of the N-terminal half of this enzyme, using the known structure of E. coli glutaminyl-tRNA synthetase, supports this conclusion and suggests that the 98C-127H segment does not have the characteristics of the classical zinc fingers.
authors.affiliation: Département de Biochimie, Faculté des Sciences et de Génie, Université Laval, Ste-Foy, Québec, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Liu
authors.forename: Y
authors.initials: Y
authors.lastname: Gagnon
authors.forename: J
authors.initials: J
authors.lastname: Gauthier
authors.forename: L
authors.initials: L
authors.lastname: Furenlid
authors.forename: P J
authors.initials: PJ
authors.lastname: L'Heureux
authors.forename: M
authors.initials: M
authors.lastname: Auger
authors.forename: O
authors.initials: O
authors.lastname: Nureki
authors.forename: S
authors.initials: S
authors.lastname: Yokoyama
authors.forename: J
authors.initials: J
authors.lastname: Lapointe
mesh_terms: D015502:Absorptiometry, Photon / Q000379:methods; D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology; D001665:Binding Sites; D004331:Drosophila melanogaster / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005975:Glutamate-tRNA Ligase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D005975:Glutamate-tRNA Ligase; D015032:Zinc