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

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-angstrom crystal structure of CthPnk-D38N in a Michaelis complex with GTP-Mg2+ 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-Mg2+ and a 5 '-OH DNA yielded a mixed substrate-product complex with GTP-Mg2+ and 5 '-PO4 DNA, wherein the product 5 ' phosphate group is displaced by the NTP gamma phosphate and the local architecture of the acceptor site is perturbed.