Structure and Stability of Nucleoside Diphosphate Kinase from Haloalkaliphilic Archaeon Natronobacterium magadii
Alla KOSTYUKOVA*a,b, Yaroslava POLOSINAb, Dmitry ZAMYATKINb, Konstantin VASSILENKOb, Ken JARRELLc, and Oleg FEDOROVb
a International Institute for Advanced Research, Matsushita Electric Industrial Co., Ltd., 3-4 Hikaridai, Seika, Kyoto, 619-02, Japan
b Institute of Protein Research, Russian Academy of Sciences, Pushchino, 142292, Russia
c Department of Microbiology and Immunology, Queen's University, Kingston, K7L 3N6, Canada
The nucleoside diphosphate kinase, an enzyme whose main function is to exchange γ-phosphates between nucleoside triphosphates and diphosphates, has been firstly purified and characterized in the representative of Archaea -Natronobacterium magadii (optimal growth conditions 20% NaC1, pH 9.5). The N-terminal 20 amino acid sequence of the kinase showed a strong sequence similarity of this protein with nucleoside diphosphate kinases from different organisms. Comparison of the molecular weights of the NDP kinase monomer determined by SDS-PAGE (23,000) and of the oligomer determined by sedimentation equilibrium experiments (125,000) indicated that the oligomer is a hexamer. The structure and stability of the kinase were studied by CD and differential scanning calorimetry. It was found that protein is well structured and contains about 24% of α-helices and 33% of β-structure. The melting of the NDP kinase in the microcalorimeter in natural salt conditions, 20% NaC1, showed that it is more thermostable than eucaryotic and bacterial NDP kinases and does not denature up to 72℃. Repeated heating of the denatured samples showed 80% reversibility of the natronobacterial kinase unfolding that also differs it from other kinases. In melting curves of the NDP kinase in buffer solutions containing 10% and 5% NaC1 the only peak of heat absorption was observed, indicating a single step denaturation. The decrease of the NaC1 concentration resulted in a decrease of Tm, the temperature of the heat absorption maximum. It was shown that removal of Mg2+ did not influence the kinase thermal stability, but Mg2+ is necessary for its function. The enzyme is autophosphorylated in the presence of [γ-P32]-ATP and Mg2+ is required for the incorporation of phosphate. The kinase preserved the ability to transfer γ-phosphate from ATP to GDP in the range of NaC1 concentration from 0.5 to 20% and in the range of pH from 5 to 12. NDP kinase from haloalkaliphiles is a very interesting enzyme for studying its structure and stability and comparing these data with those for NDP kinases from mesophilic organisms. For this purpose conditions of the kinase crystallization have been found and hexagonal crystals (up to 150 μm in two dimensions and 100 μm in a third dimension) have been obtained.
