Structure and mechanism of the polynucleotide kinase component of the bacterial Pnkp-Hen1 RNA repair system.
Pnkp is the end-healing and end-sealing component of an RNA repair system
present in diverse bacteria from many phyla. Pnkp is composed of three catalytic
modules: an N-terminal polynucleotide 5'-kinase, a central 2',3' phosphatase,
and a C-terminal ligase. Here we report the crystal structure of the kinase
domain of Clostridium thermocellum Pnkp bound to ATP•Mg(2+) (substrate
complex) and ADP•Mg(2+) (product complex). The protein consists of a core
P-loop phosphotransferase fold embellished by a distinctive homodimerization
module composed of secondary structure elements derived from the N and C termini
of the kinase domain. ATP is bound within a crescent-shaped groove formed by the
P-loop ((15)GSSGSGKST(23)) and an overlying helix-loop-helix "lid."
The α and β phosphates are engaged by a network of hydrogen bonds from Thr23
and the P-loop main-chain amides; the γ phosphate is anchored by the lid
residues Arg120 and Arg123. The P-loop lysine (Lys21) and the catalytic Mg(2+)
bridge the ATP β and γ phosphates. The P-loop serine (Ser22) is the sole
enzymic constituent of the octahedral metal coordination complex.
Structure-guided mutational analysis underscored the essential contributions of
Lys21 and Ser22 in the ATP donor site and Asp38 and Arg41 in the phosphoacceptor
site. Our studies suggest a catalytic mechanism whereby Asp38 (as general base)
activates the polynucleotide 5'-OH for its nucleophilic attack on the γ
phosphorus and Lys21 and Mg(2+) stabilize the transition state.