Structure and mechanism of T4 polynucleotide kinase: An RNA repair enzyme

Abstract

T4 polynucleotide kinase (Pnk), in addition to being an invaluable research tool, exemplifies a family of bifunctional enzymes with 5′-kinase and 3′-phosphatase activities that play key roles in RNA and DNA repair. T4 Pnk is a homotetramer composed of a C-terminal phosphatase domain and an N-terminal kinase domain. The 2.0 Å crystal structure of the isolated kinase domain highlights a tunnel-like active site through the heart of the enzyme, with an entrance on the 5′ OH acceptor side that can accommodate a single-stranded polynucleotide. The active site is composed of essential side chains that coordinate the β phosphate of the NTP donor and the 3′ phosphate of the 5′ OH acceptor, plus a putative general acid that activates the 5′ OH. The structure rationalizes the different specificities of T4 and eukaryotic Pnk and suggests a model for the assembly of the tetramer.