Structures of bacterial polynucleotide kinase in a Michaelis complex with GTP•Mg2+ and 5′-OH oligonucleotide and a product complex with GDP•Mg2+ and 5′-PO4 oligonucleotide reveal a mechanism of general acid-base catalysis and the determinants of phosphoacceptor recognition
نویسندگان
چکیده
Clostridium thermocellum polynucleotide kinase (CthPnk), the 5' end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from an NTP donor to a 5'-OH polynucleotide acceptor. Here we report the crystal structures of CthPnk-D38N in a Michaelis complex with GTP•Mg(2+) and a 5'-OH oligonucleotide and a product complex with GDP•Mg(2+) and a 5'-PO4 oligonucleotide. The O5' nucleophile is situated 3.0 Å from the GTP γ phosphorus in the Michaelis complex, where it is coordinated by Asn38 and is apical to the bridging β phosphate oxygen of the GDP leaving group. In the product complex, the transferred phosphate has undergone stereochemical inversion and Asn38 coordinates the 5'-bridging phosphate oxygen of the oligonucleotide. The D38N enzyme is poised for catalysis, but cannot execute because it lacks Asp38-hereby implicated as the essential general base catalyst that abstracts a proton from the 5'-OH during the kinase reaction. Asp38 serves as a general acid catalyst during the 'reverse kinase' reaction by donating a proton to the O5' leaving group of the 5'-PO4 strand. The acceptor strand binding mode of CthPnk is distinct from that of bacteriophage T4 Pnk.
منابع مشابه
Structures of bacterial polynucleotide kinase in a Michaelis complex with GTP Mg and 50-OH oligonucleotide and a product complex with GDP Mg and 5-PO4 oligonucleotide reveal a mechanism of general acid-base catalysis and the determinants of phosphoacceptor recognition
Clostridium thermocellum polynucleotide kinase (CthPnk), the 50 end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from an NTP donor to a 50-OH polynucleotide acceptor. Here we report the crystal structures of CthPnk-D38N in a Michaelis complex with GTP Mg and a 50-OH oligonucleotide and a product complex with GDP Mgand a 5-PO4 oligonucleotide. The O50...
متن کاملStructures of Bacterial Polynucleotide Kinase in a Michaelis Complex with Nucleoside Triphosphate (NTP)-Mg and 5=-OH RNA and a Mixed Substrate-Product Complex with NTP-Mg 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-Å crystal structure of CthPnk-D38N in a Michaelis complex with GTP-Mg and a 5=-OH RNA oligonucleotide. The RNA-binding...
متن کاملCrystal Structures of the G Protein GiR1 Complexed with GDP and Mg2+: A Crystallographic Titration Experiment‡
The effect of Mg2+ binding on the conformation of the inactive GDP-bound complex of the heterotrimeric G protein R subunit GiR1 has been investigated by X-ray crystallography. Crystal structures of the GiR1‚GDP complex were determined after titration with 5, 10, 100, and 200 mM Mg2+. Comparison of these structures with that of the Mg2+-free complex revealed Mg2+ bound at the same site as observ...
متن کاملA Thermodynamic Study of Complex Formation between 15-Crown-5 with Mg2+, Ca2+, Sr2+ and Ba2+ in Acetonitrile Methanol Binary Mixtures Using Conductometric Method
The complexation reactions between Mg2+, Ca2+, Sr2+ and Ba2+ metal cations with 15-crown-5 (15C5) were studied in acetonitrile (AN)-methanol (MeOH) binary mixtures at different temperatures using conductometric method. 15C54 forms 1:1 complexes with Mg2+, Ca2+ and Sr2+ cations in solutions. The Ma2+ cati...
متن کاملMutational analysis of the 5′-OH oligonucleotide phosphate acceptor site of T4 polynucleotide kinase
T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5'-kinase and 3'-phosphatase activities that function in nucleic acid repair. The N-terminal kinase domain belongs to the P-loop phosphotransferase superfamily. The kinase is distinguished by a tunnel-like active site with separate entrances on opposite sides of the protein for the NTP phosphate donor ...
متن کامل