title: Structures of bacterial polynucleotide kinase in a michaelis complex with nucleoside triphosphate (NTP)-Mg2+ and 5'-OH RNA and a mixed substrate-product complex with NTP-Mg2+ and a 5'-phosphorylated oligonucleotide.
abstract: Clostridium thermocellum polynucleotide kinase (CthPnk), the 5'-end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from a nucleoside triphosphate (NTP) donor to a 5'-OH polynucleotide acceptor, either DNA or RNA. Here we report the 1.5-Å crystal structure of CthPnk-D38N in a Michaelis complex with GTP-Mg(2+) and a 5'-OH RNA oligonucleotide. The RNA-binding mode of CthPnk is different from that of the metazoan RNA kinase Clp1. CthPnk makes hydrogen bonds to the ribose 2'-hydroxyls of the 5' terminal nucleoside, via Gln51, and the penultimate nucleoside, via Gln83. The 5'-terminal nucleobase is sandwiched by Gln51 and Val129. Mutating Gln51 or Val129 to alanine reduced kinase specific activity 3-fold. Ser37 and Thr80 donate functionally redundant hydrogen bonds to the terminal phosphodiester; a S37A-T80A double mutation reduced kinase activity 50-fold. Crystallization of catalytically active CthPnk with GTP-Mg(2+) and a 5'-OH DNA yielded a mixed substrate-product complex with GTP-Mg(2+) and 5'-PO4 DNA, wherein the product 5' phosphate group is displaced by the NTP γ phosphate and the local architecture of the acceptor site is perturbed.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York, USA.
authors.forename: Ushati
authors.initials: U
authors.lastname: Das
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York, USA.
authors.forename: Li Kai
authors.initials: LK
authors.lastname: Wang
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Smith
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York, USA.
authors.forename: Annum
authors.initials: A
authors.lastname: Munir
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York, USA s-shuman@ski.mskcc.org.
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D048013:Acetivibrio thermocellus / Q000737:chemistry / Q000201:enzymology*; D018360:Crystallography, X-Ray; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry* / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D012313:RNA; D006160:Guanosine Triphosphate; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D008274:Magnesium