Nucleoside diphosphate kinase from Escherichia coli

Abstract

Nucleoside diphosphate (NDP) kinase from Escherichia coli was purified to homogeneity and was crystallized. Gel filtration analysis of the purified enzyme indicated that it forms a tetramer. The enzyme was phosphorylated with [␥-32 P]ATP, and the pH stability profile of the phosphoenzyme indicated that two different amino acid residues were phosphorylated. Both a histidine residue and serine residues, including Ser-119 and Ser-121, appear to be phosphorylated. A Ser119Ala/Ser121Ala double mutant (i.e., with a Ser-toAla double mutation at positions 119 and 121), as well as Ser119Ala and Ser121Ala mutants, was isolated. All of these retained NDP kinase activity; also, both the Ser119Ala and Ser121Ala mutants could still be autophosphorylated. In the case of the double mutant, a slight autophosphorylation activity, which was resistant to acid treatment, was still detected, indicating that an additional minor autophosphorylation site besides Nucleoside diphosphate (NDP) kinase catalyzes the following transphosphorylation reaction, via the formation of a phosphoenzyme intermediate:The primary role of NDP kinase in the cell was considered the maintenance of a pool of nucleotide triphosphates for the synthesis of DNA and RNA (22). NDP kinase was reported to be the only enzyme responsible for the synthesis of nucleotides other than ATP in Salmonella typhimurium (7). Also, the ndk gene appears to be essential in the gram-negative bacterium Myxococcus xanthus (20). Recently the enzyme has been implicated as a tumor suppressor candidate in studies of highly metastatic human cells (14,26,27) and has been associated with the development of Drosophila larvae (3,25).NDP kinase genes (ndk) from prokaryotic and eukaryotic organisms have been cloned and sequenced (8,19). The deduced amino acid sequence of the enzyme from Escherichia coli indicated that NDP kinases are highly conserved throughout evolution; the E. coli and human (Nm23-H1) enzymes are 43% identical (8). The three-dimensional structures of NDP kinases from Dictyostelium discoideum (4), M. xanthus (33), and Drosophila melanogaster (2) have been determined. These studies again showed that NDP kinases from different sources share highly conserved three-dimensional structures.NDP kinases are known to function by a ping-pong mechanism which involves the formation of a high-energy phosphoenzyme intermediate. Most of the intermediates have been shown to be alkali stable and acid labile, suggesting the formation of phosphohistidine, which has been identified in NDP kinases from various sources (22). Recently, histidine 118 has been identified as the phosphorylation site in one of the human NDP kinases, Nm23 (6). This histidine residue is invariant among all the NDP kinases sequenced so far (8). However phospho-NDP kinases with drastically different stability characteristics have been reported (22). Phosphoserine formation has been reported to occur in NDP kinase from rat mucosal mast cells (11) and in NDP kinase from M. xanthus, in which the formation of phosphohistidine ha...

Molecular and Functional Interactions between Escherichia coli Nucleoside-diphosphate Kinase and the Uracil-DNA Glycosylase Ung

“…The origin of this residual uracil-cleaving activity of Ndk is not yet fully known, although on the basis of several biochemical observations and its dependence on the NDP kinase catalytic activity, one can reasonably assume that it is Ndk-derived. 4 Ndk and Ung Co-purify through Sequential Three-column Chromatography-To elucidate the nature of the robust UDG activity of recombinant Ndk purified from wild-type E. coli (Fig. 1, lanes 4 and 5) (28), we investigated the possibility that this activity may have arisen as a consequence of a physical interaction between Ung and Ndk.…”

“…NDP kinases catalyze the reversible transfer of ␥-phosphates between nucleoside di-and triphosphates at very high efficiencies through an evolutionarily conserved active site histidine residue (1)(2)(3). E. coli NDP kinase is encoded by a single gene, ndk (4), whereas the genetically distinct forms of human NDP kinases are encoded by multiple genes termed NM23-H1 through H8 (5). The name NM (nonmetastatic) 23 was initially accorded to the matriarch of the family, NM23-H1, on the basis of its reported action as a tissue-specific metastasis inhibitor (6).…”

Phosphoproteome Analysis of E. coli Reveals Evolutionary Conservation of Bacterial Ser/Thr/Tyr Phosphorylation

“…With respect to tyrosine phosphorylation, E. coli possesses two BY-kinases (15), Wzc and Etk (16), which are capable of auto-and substrate phosphorylation. Several E. coli proteins were also reported to autophosphorylate on Ser/Thr residues, including the molecular chaperone DnaK (17), an essential GTPase termed Era (18), and the nucleotide diphosphate kinase (19). Presently, available data include 12 phosphorylation sites on 6 proteins in E. coli, obtained from individual biochemical studies (20).…”

Elimination and Utilization of Oxidized Guanine Nucleotides in the Synthesis of RNA and Its Precursors

“…The enzyme is able to act on all types of RNAand DNA-related nucleotides (22). To determine whether this enzyme also acts on 8-oxo-Gua-containing nucleotides, a homogeneous preparation of NDK was obtained with the aid of the cloned gene (see Fig.…”