Biomedical Enginnering Research Organization, Tohoku University* RIKEN, Genomic Sciences Center, Japan** Department of Chemisty, University of Tokyo, Japan***
○Kazutaka Murayama* Miyuki Murayama-Kato** Takaho Terada** Mikako Shirouzu** Shigeyuki Yokoyama***
Nucleosidediphosphate kinase (NDPK) transfers the g-phosphate of nucleosidetriphosphate to nucleosidediphosphate.
N1TP + N2DP → N1DP + N2TP
NDPK shows low selectivity for nucleotide bases. The enzyme catalyzes this reaction among variety of nucleosides as the phosphate donor and acceptor. We determined high-resolution structures of NDPK from Pyrococcus horikoshii to reveal the mechanism of the catalysis reaction.
The protein was synthesized by the cell-free system and co-crystallized with purin nucleotides (ADP, ATP, ATP analog, GDP, and GTP analog). Crystals were obtained from the same condition of the native crystal (unliganded form), although two space groups appeared, depending on the co-crystallized substrates. The reflection data were measured at 2.0-1.7Å resolution using in-house diffractometor with CuKa radiation. Structure determinations were conducted by molecular replacement method using the unliganded form of the enzyme (PDB code: 2CWK).
All crystal forms include two molecules in an asymmetric unit, forming a homodimer. Furthermore, the dimers are arranged by three-fold axis to form a hexamer. Analytical ultracentrifuge measurement also showed that the molecular weight of this protein is around 105kDa, which supports hexamer formation. In the complex structures, bases (guanine/adenine) were stabilized by stacking interaction with Phe63. The electron densities of diphospate groups in ADP or GDP were clear. However, b- and g-phospahe of NTP analogs could not identified from electron density. This result suggest that binding affinity for NTP and NDP are different, and the different affinity may play an important role for binding and release during catalytic reaction.