title: Identification of the catalytic residues of sequence-specific and histidine-free ribonuclease colicin E5.
abstract: Colicin E5 cleaves tRNAs for Tyr, His, Asn and Asp in their anticodons to abolish protein synthesis in Escherichia coli. We previously showed how its C-terminal RNase domain, E5-CRD, recognizes the anticodon bases but the catalytic mechanism remained to be elucidated. Although the reaction products with 5'-OH and 2',3'-cyclic phosphate ends suggested a similar mechanism to those of RNases A and T1, E5-CRD does not have the His residues necessary as a catalyst in usual RNases. To identify residues important for the catalytic reaction, mutants as to all residues within 5 Å from the central phosphorus of the scissile phosphodiester bond were prepared. Evaluation of the killing activities of the mutant colicins and the RNase activities of the mutant E5-CRDs suggested direct involvement of Arg33, Lys25, Gln29 and Lys60 in the reaction. Particularly, Arg33 plays a critical role and Ile94 provides a structural support of Arg33. Crystal structure of the complex of E5-CRD(R33Q)/dGpdUp showed structural and binding functional integrity of this mutant protein, suggesting involvement of Arg33 in the catalytic reaction. The structure of the free E5-CRD, we also determined, showed great flexibility of a flap region, which facilitates the access of Lys60 to the substrate in an induced-fit manner.
authors: Department of Biotechnology, The University of Tokyo, Yayoi 1-1-1, Tokyo 113-8654, Japan.
authors: Sakura
authors: S
authors: Inoue-Ito
authors: Shunsuke
authors: S
authors: Yajima
authors: Shinya
authors: S
authors: Fushinobu
authors: Shugo
authors: S
authors: Nakamura
authors: Tetsuhiro
authors: T
authors: Ogawa
authors: Makoto
authors: M
authors: Hidaka
authors: Haruhiko
authors: H
authors: Masaki
mesh_terms: D055162:Biocatalysis; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006639:Histidine; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012343:RNA, Transfer / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D006639:Histidine; D012343:RNA, Transfer; D004722:Endoribonucleases; D012260:Ribonucleases; C515828:colicin E5 protein, E coli