Intermolecular phosphotransfer is crucial for efficient catalytic activity of nucleoside diphosphate kinase

Abstract

NDK (nucleoside diphosphate kinase) is primarily involved in maintaining cellular nucleotide pools in both prokaryotes and eukaryotes. We cloned ndk from Salmonella typhimurium and expressed it in Escherichia coli as a histidine-tagged protein. The Ni-NTA (Ni2+-nitrilotriacetate)-purified protein (sNDK) was found to be tetrameric with a monomeric unit molecular mass of ~18 kDa. The sNDK exhibited bivalent-cation-dependent autophosphorylation at a wide range of pH values and the phosphorylation withstands acid or alkali treatment. Surprisingly, nucleoside diphosphates did not behave as ‘true inhibitors’ of autophosphorylation activity. The sNDK displayed phosphotransfer activity from nucleoside triphosphates to nucleoside diphosphates; however, it was Mg2+/Mn2+-dependent. Mutational analysis established His117 as the predominantly phosphorylating residue in sNDK. Although it is a histidine kinase, we found that substitution of Ser119 with alanine/glutamate significantly affected the autophosphorylation, as well as the NTP-synthesizing ability of sNDK. Interestingly, the mixture of inactive (H117A) and partially active (S119A) proteins was found to be catalytically more efficient than the presence of corresponding amounts of active population, advocating transfer of phosphate from phospho-His117 to Ser119. Consistent with this observation, the Ni-NTA-purified H117A protein, obtained following co-expression of both of the mutant constructs [His-tagged H117A and GST (glutathione transferase)-tagged S119A] in E. coli, exhibited autophosphorylation, thereby alluding to intermolecular phosphotransfer between His117 and Ser119. Although this housekeeping enzyme has long been discovered and characterized from different sources, the results of the present study portray how Ser119 in sNDK is phosphorylated. Furthermore, our findings illustrate for the first time that the intermolecular phosphotransfer is mandatory for the efficient NTP synthesis in any NDK.