NUCLEOSIDE DIPHOSPHATE KINASE FROM HALOALKALIPHILIC ARCHAEON NATRONOBACTERIUM-MAGADII - PURIFICATION AND CHARACTERIZATION

An ATP-binding protein from the haloalkaliphilic archaeon Natronobacte rium magadii was purified and characterized by affinity chromatography on ATP-agarose and by fast protein liquid chromatography (FPLC) on a Mono Q column. The N-terminal 20 amino acid sequence of the kinase sho wed a strong sequence similarity of this protein with nucleoside dipho sphate (NDP) kinases from different organisms and, accordingly: we bel ieve that this protein is a nucleoside diphosphate kinase, an enzyme w hose main function is to exchange gamma-phosphates between nucleoside triphosphates and diphosphates, Comparison of the molecular weights of the NDP kinase monomer determined by sodium dodecyl sulfate polyacryl amide gel electrophoresis (SDS-PAGE) (23 000) and of the oligomer dete rmined by sedimentation equilibrium experiments (125 000) indicated th at the oligomer is a hexamer. The enzyme was autophosphorylated in the presence of [gamma-P-32]ATP, and Mg2+ was required for the incorporat ion of phosphate. The kinase preserved the ability to transfer gamma-p hosphate from ATP to GDP in the range of NaCl concentration from 90mM to 3.5M and in the range of pH from 5 to 13. It was found and confirme d by Western blotting that this kinase is one of the proteins that bin d specifically to natronobacterial flagellins. NDP kinase from haloalk aliphiles appeared to be simple to purify and to be a suitable enzyme for studies of structure and stability compared with NDP kinases from mesophilic organisms.