Structures of bacterial polynucleotide kinase in a Michaelis complex with GTPMg2+ and 5′-OH oligonucleotide and a product complex with GDPMg2+ and 5′-PO4 oligonucleotide reveal a mechanism of general acid-base catalysis and the determinants of phosphoacceptor recognition Journal Article
| Authors: | Das, U.; Wang, L. K.; Smith, P.; Jacewicz, A.; Shuman, S. |
| Article Title: | Structures of bacterial polynucleotide kinase in a Michaelis complex with GTPMg2+ and 5′-OH oligonucleotide and a product complex with GDPMg2+ 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 GTPMg2+ and a 5′-OH oligonucleotide and a product complex with GDPMg2+ 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. © 2013 The Author(s). |
| Journal Title: | Nucleic Acids Research |
| Volume: | 42 |
| Issue: | 2 |
| ISSN: | 0305-1048 |
| Publisher: | Oxford University Press |
| Date Published: | 2014-01-01 |
| Start Page: | 1152 |
| End Page: | 1161 |
| Language: | English |
| DOI: | 10.1093/nar/gkt936 |
| PROVIDER: | scopus |
| PMCID: | PMC3902929 |
| PUBMED: | 24150947 |
| DOI/URL: | |
| Notes: | Export Date: 3 March 2014 -- CODEN: NARHA -- Source: Scopus |
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