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.
J Bacteriol
; 196(24): 4285-92, 2014 Dec.
Article
em En
| MEDLINE
| ID: mdl-25266383
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-Šcrystal 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Polinucleotídeo 5'-Hidroxiquinase
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RNA
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Clostridium thermocellum
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Guanosina Trifosfato
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Magnésio
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article