Enzymes used for adenosine triphosphate (ATP) synthesis play important roles in energy-dependent cascade reactions in vitro. In this study, two novel polyphosphate kinase 2 (PPK2) enzymes, HbPPK2 from Hydrogenophilaceae bacterium and NdPPK2 from Nocardioides dokdonensis, were characterized for ATP synthesis with the substrate polyphosphate (polyP). The optimum temperature and pH of both purified HbPPK2 and NdPPK2 were 30 degrees C and 6.5. HbPPK2 and NdPPK2 retained 30% and 14% of the initial activity at 30 degrees C for 12 h, respectively, whereas the presence of polyP significantly enhanced the stability of enzymes. The two PPK2s preferentially catalyzed the long-chain polyP hexametaphosphate as the phosphate donor. Adenosine monophosphate could not be used by HbPPK2 and NdPPK2 to synthesize ATP, indicating that they belonged to the class I subfamily of PPK2. HbPPK2 was used for ATP regeneration to produce glutathione by a two-enzyme cascade in vitro. 47.1 +/- 0.4 mM glutathione was synthesized with a productivity of 13.5 +/- 0.1 mM/h. ATP was regenerated approximately 471 times in the system within 3.5 h. HbPPK2 showed potential application for ATP regeneration in cascade reaction.