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 Journal Article
| Authors: | Das, U.; Wang, L. K.; Smith, P.; Munir, A.; Shuman, S. |
| Article 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-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 γ phosphate and the local architecture of the acceptor site is perturbed. |
| Keywords: | bacteria (microorganisms) |
| Journal Title: | Journal of Bacteriology |
| Volume: | 196 |
| Issue: | 24 |
| ISSN: | 0021-9193 |
| Publisher: | American Society for Microbiology |
| Date Published: | 2014-12-01 |
| Start Page: | 4285 |
| End Page: | 4292 |
| Language: | English |
| DOI: | 10.1128/jb.02197-14 |
| PROVIDER: | scopus |
| PMCID: | PMC4248851 |
| PUBMED: | 25266383 |
| DOI/URL: | |
| Notes: | Export Date: 2 January 2015 -- Source: Scopus |
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