Abstract
Nucleoside-diphosphate (NDP) kinase (NTP:nucleoside-diphosphate phosphotransferase) catalyzes the reversible transfer of gamma-phosphates from nucleoside triphosphates to nucleoside diphosphates through an invariant histidine residue. It has been reported that the high-energy phosphorylated enzyme intermediate exhibits a protein phosphotransferase activity toward the protein histidine kinases CheA and EnvZ, members of the two-component signal transduction systems in bacteria. Here we demonstrate that the apparent protein phosphotransferase activity of NDP kinase occurs only in the presence of ADP, which can mediate the phosphotransfer from the phospho-NDP kinase to the target enzymes in catalytic amounts (approximately 1 nm). These findings suggest that the protein kinase activity of NDP kinase is probably an artifact attributable to trace amounts of contaminating ADP. Additionally, we show that Escherichia coli NDP kinase, like its human homologue NM23-H2/PuF/NDP kinase B, can bind and cleave DNA. Previous in vivo functions of E. coli NDP kinase in the regulation of gene expression that have been attributed to a protein phosphotransferase activity can be explained in the context of NDP kinase-DNA interactions. The conservation of the DNA binding and DNA cleavage activities between human and bacterial NDP kinases argues strongly for the hypothesis that these activities play an essential role in NDP kinase function in vivo.
Highlights
REACTION SCHEME where Mg1⁄7NDP and Mg1⁄7NЈTP are the Mg2ϩ complexes with nucleoside di- and triphosphates
The phosphorylation and dephosphorylation steps take less than 1 ms, whereas the phosphorylated form is stable for a few hours in the absence of a nucleoside diphosphate acceptor
It has been reported that the high-energy phosphorylated NDP kinase intermediate exhibits a protein phosphotransferase activity toward a number of important regulatory proteins, leading to the proposal that NDP kinases can participate in phosphorelay networks that regulate gene expression and metabolism
Summary
REACTION SCHEME where Mg1⁄7NDP and Mg1⁄7NЈTP are the Mg2ϩ complexes with nucleoside (or 2Ј-deoxynucleoside) di- and triphosphates. The enzyme is composed of four or six identical subunits (16 –20 kDa each) with an ␣/ sandwich or ferredoxin fold (reviewed in Ref. 2) In both crystals and solution, NDP kinases exist in two different quaternary structures: eukaryotic enzymes are hexamers (a trimer of dimers with dihedral D3 symmetry), and some bacterial enzymes are tetramers (a pair of dimers with pyramidal D2 symmetry) (3). It has been found that the Escherichia coli ndk gene is not essential and that deletion mutants are capable of normal growth (4) This result is consistent with the notion that the primary function of NM23/NDP kinase is not as a housekeeping nucleoside-diphosphate kinase. Among the reported targets of the phosphotransferase activity of NDP kinase were the histidine protein kinases CheA and EnvZ (15), members of the two-component signal transduction systems in bacteria (21).
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