title: 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.
abstract: 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.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
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: Agata
authors.initials: A
authors.lastname: Jacewicz
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017122:Bacteriophage T4 / Q000201:enzymology; D001665:Binding Sites; D055162:Biocatalysis; D020134:Catalytic Domain; D048013:Acetivibrio thermocellus / Q000201:enzymology; D003461:Crystallography / Q000379:methods; D006153:Guanosine Diphosphate / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D009841:Oligonucleotides; D010710:Phosphates; D006153:Guanosine Diphosphate; C036852:magnesium GTP; D006160:Guanosine Triphosphate; D011116:Polynucleotide 5'-Hydroxyl-Kinase