3WX8

Purification, characterization and structure of nucleoside diphosphate kinase from Drosophila S2 cells

3WX8 の概要

• エントリーDOI: 10.2210/pdb3wx8/pdb
• 分子名称: Nucleoside diphosphate kinase (2 entities in total)
• 機能のキーワード: nucleotide binding specificity, nuclease activity, rossmann fold, catalyzation, ndp binding, phosphorylation, transferase
• 由来する生物種: Drosophila melanogaster (Fruit fly)
• 細胞内の位置: Cytoplasm : P08879
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 51704.87

構造登録者

Qian, L. (登録日: 2014-07-25, 公開日: 2015-06-10, 最終更新日: 2024-03-20)

主引用文献

Qian, L.,Liu, X.
Purification, characterization and structure of nucleoside diphosphate kinase from Drosophila melanogaster
Protein Expr.Purif., 103:48-55, 2014

PubMed Abstract: Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme found in all organisms and cell types, which catalyzes the transfer of the phosphoryl group from a nucleoside triphosphate to a nucleoside diphosphate. The gene encoding for NDPK from Drosophila melanogaster was amplified from the genomic DNA. The recombinant NDPK (rNDPK) was overexpressed in Escherichia coli and purified to homogeneity by Ni-NTA agarose affinity chromatography, HiTrap SP HP cation exchange chromatography and HiLoad 16/60 Superdex 200 gel filtration chromatography. The gel filtration chromatography and analytical ultracentrifugation showed that rNDPK was a trimer in solution. The binding affinity of NDPs with rNDPK, measured by isothermal titration calorimetry, indicated that the purines nucleotides show higher binding affinity compared with pyrimidines. The rNDPK had a definite nuclease activity in vitro, which could cleave supercoiled plasmid DNA, but had no effect on dsDNA and ssDNA. Furthermore, the structure for NDPK was determined by using the sitting drop vapor diffusion method. In the final model, the asymmetric unit is made of three molecules, each of which consists of a four-stranded anti-parallel β-sheets and seven α-helices. Sequence alignment and structure comparison illustrated that the simulated nucleotide-binding active site are conserved.

PubMed: 25195176
DOI: 10.1016/j.pep.2014.08.014

実験手法

X-RAY DIFFRACTION (1.952 Å)