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Bacterial UMP kinases are essential enzymes involved in the multistep synthesis of nucleoside triphosphates. They are hexamers regulated by the allosteric activator GTP and inhibited by UTP. We solved the crystal structure of Escherichia coli UMP kinase bound to the UMP substrate (2.3 Å resolution), the UDP product (2.6 Å), or UTP (2.45 Å). The monomer fold, unrelated to that of other nucleoside monophosphate kinases, belongs to the carbamate kinase-like superfamily. However, the phosphate acceptor binding cleft and subunit assembly are characteristic of UMP kinase. Interactions with UMP explain the high specificity for this natural substrate. UTP, previously described as an allosteric inhibitor, was unexpectedly found in the phosphate acceptor site, suggesting that it acts as a competitive inhibitor. Site-directed mutagenesis of residues Thr-138 and Asn-140, involved in both uracil recognition and active site interaction within the hexamer, decreased the activation by GTP and inhibition by UTP. These experiments suggest a cross-talk mechanism between enzyme subunits involved in cooperative binding at the phosphate acceptor site and in allosteric regulation by GTP. As bacterial UMP kinases have no counterpart in eukaryotes, the information provided here could help the design of new antibiotics.Nucleoside monophosphate kinases are key enzymes in the metabolism of nucleotides. They represent a relatively homogeneous family of catalysts (1) thought to derive from a common ancestor. They catalyze the reversible transfer of the ␥-phosphoryl group from a nucleoside triphosphate, generally ATP, to a particular nucleoside monophosphate. The resulting nucleoside diphosphates will be further phosphorylated (and eventually reduced) to produce nucleoside triphosphates, precursors of the major biological molecules RNA, DNA, and phospholipids. Eukaryotic UMP/CMP kinases represent an exception to the otherwise generally observed specificity of NMP 1 kinases for the base moiety of the phosphate acceptor nucleotide; they phosphorylate with comparable efficiency both UMP and CMP. Conversely, bacteria possess two distinct enzymes, specific to UMP or CMP. Bacterial CMP kinases are monomers, like most NMP kinases, and their overall fold is similar to that of other enzymes of the latter family (2). In contrast, bacterial UMP kinases are hexamers (3). They specifically phosphorylate UMP according to the scheme: UMP ϩ Mg 2ϩ ⅐ATP 7 UDP ϩ Mg 2ϩ ⅐ADP. They are activated by GTP and inhibited by UTP. This contributes to equilibrating the synthesis of purine versus pyrimidine nucleoside triphosphates.Genes coding for UMP kinases have been identified in all bacterial genomes investigated to date. They have no closely related counterpart in eukaryotes and have proved to be essential for growth in both Gram-negative species (Escherichia coli (4, 5)) and some Gram-positive species (Streptococcus pneumoniae (6)). They code for enzymes that have strong sequence similarity. Therefore, bacterial UMP kinases represent not only an interesting model of...

Centrins are well-conserved calcium binding proteins from the EF-hand superfamily implicated in various cellular functions, such as centrosome duplication, DNA repair, and nuclear mRNA export. The intrinsic molecular flexibility and the self-association tendency make difficult the structural characterization of the integral protein. In this paper we report the solution structure, the Ca2+ binding properties, and the intermolecular interactions of the N-terminal domain of two human centrin isoforms, HsCen1 and HsCen2. In the absence of Ca2+, the N-terminal construct of HsCen2 revealed a compact core conformation including four almost antiparallel alpha-helices and a short antiparallel beta-sheet, very similar to the apo state structure of other calcium regulatory EF-hand domains. The first 25 residues show a highly irregular and dynamic structure. The three-dimensional model for the N-terminal domain of HsCen1, based on the high sequence conservation and NMR spectroscopic data, shows very close structural properties. Ca2+ titration of the apo-N-terminal domain of HsCen1 and HsCen2, monitored by NMR spectroscopy, revealed a very weak affinity (10(2)-10(3) M(-1)), suggesting that the cellular role of this domain is not calcium dependent. Isothermal calorimetric titrations showed that an 18-residue peptide, derived from the N-terminal unstructured fragment, has a significant affinity (approximately 10(5) M(-1)) for the isolated C-terminal domain, suggesting an active role in the self-assembly of centrin molecules.