title: Structural and biochemical analysis of the phosphate donor specificity of the polynucleotide kinase component of the bacterial pnkp•hen1 RNA repair system.
abstract: Clostridium thermocellum Pnkp is the end-healing and end-sealing subunit of a bacterial RNA repair system. CthPnkp is composed of three catalytic modules: an N-terminal 5'-OH polynucleotide kinase, a central 2',3' phosphatase, and a C-terminal ligase. The crystal structure of the kinase domain bound to ATP•Mg(2+) revealed a rich network of ionic and hydrogen-bonding contacts to the α, β, and γ phosphates. By contrast, there are no enzymic contacts to the ribose and none with the adenine base other than a π-cation interaction with Arg116. Here we report that the enzyme uses ATP, GTP, CTP, UTP, or dATP as a phosphate donor for the 5'-OH kinase reaction. The enzyme also catalyzes the reverse reaction, in which a polynucleotide 5'-PO4 group is transferred to ADP, GDP, CDP, UDP, or dADP to form the corresponding NTP. We report new crystal structures of the kinase in complexes with GTP, CTP, UTP, and dATP in which the respective nucleobases are stacked on Arg116 but make no other enzymic contacts. Mutating Arg116 to alanine elicits a 10-fold increase in Km for ATP but has little effect on kcat. These findings illuminate the basis for nonspecific donor nucleotide utilization by a P-loop phosphotransferase.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute , New York, New York 10065, United States.
authors.forename: Ushati
authors.initials: U
authors.lastname: Das
authors.forename: Li Kai
authors.initials: LK
authors.lastname: Wang
authors.forename: Paul
authors.initials: P
authors.lastname: Smith
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D008958:Models, Molecular; D016296:Mutagenesis; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism*; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D010710:Phosphates; D012329:RNA, Bacterial; D011116:Polynucleotide 5'-Hydroxyl-Kinase