Structure of Escherichia coli UMP Kinase Differs from That ofOther Nucleoside Monophosphate Kinases and Sheds New Light on EnzymeRegulation

Abstract

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...

The pyrH Gene of Vibrio vulnificus Is an Essential In Vivo Survival Factor

“…To analyze the essential role of PyrH in the in vivo survival and growth of V. vulnificus, we introduced point mutations on the critical amino acid residues of the protein. Recently, Briozzo et al determined the crystal structure of UMP kinase (PyrH) from E. coli (2). They showed that the UMP kinase recognizes the UMP substrate through the simultaneous recognition of its base, sugar, and phosphate moieties: the side chain oxygen of Asp77 makes hydrogen bond with 2ЈOH of ribose and the terminal nitrogen of Arg62 interact with terminal oxygen of alpha-phosphate.…”

“…After sonication, recombinant tag-free Vv-PyrH was purified by using a chitin column and a 50 mM 1,4-dithiothreitol solution in accordance with the manufacturer's protocol, and the purity of the recombinant Vv-PyrH was confirmed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The UMP kinase activity of recombinant PyrH was determined as described elsewhere (2,3). The reaction mixture contained 50 mM Tris-Cl (pH 7.4), 50 mM KCl, 2 mM MgCl 2 , 2 mM ATP, 1 mM phosphoenolpyruvate, 0.2 mM NADH, 0.5 mM GTP, and 2 U each of pyruvate kinase, lactate dehydrogenase (LDH), and NDP kinase.…”

“…Bucurenci et al have elucidated critical amino acid residues of E. coli PyrH by using genetic and biochemical assays (3). Recently, molecular structure of PyrH encoded by E. coli and Pyrococcus furiosus was solved (2,24). In the present study, we introduced site-directed mutations on the chromosomal pyrH residues of Arg-62 and Asp-77, both involved in UMP binding, based on the comparison of sequence homologies with the E. coli pyrH.…”

Mutational Analysis of Intervening Sequences Connecting the Binding Sites for Integration Host Factor, PepA, PurR, and RNA Polymerase in the Control Region of the Escherichia coli carAB Operon, Encoding Carbamoylphosphate Synthase

“…The fact that P1 repressibility in the ins3 del8 double mutant, where all previously identified regulatory sites and the promoter are correctly positioned, is not fully restored suggests that this promoter-proximal region participates in a sequencespecific manner in the establishment of pyrimidine-dependent regulation. This region could be contacted by a pyrimidine- (3,19) have shown that the organization and complementary surface charge potentials of the hexameric UMP kinase and PepA enzymes might sustain their interaction with the threefold axes aligned (19). Most interestingly, the previously isolated pyrH41 mutant deficient for pyrimidine-specific repression of P1 but not affected in the catalytic activity of the UMP kinase bears a single-amino-acid substitution (A94E) that is localized in this contact area (18,19).…”

“…These observations lead us to propose the involvement of protein-protein interactions, likely with PyrH (7). Since then, the three-dimensional structures of E. coli PepA (24) and of E. coli and Pyrococcus furiosus PyrH (3,19) have been solved. On the basis of the complementarity of charge distribution on the surfaces of PepA and PyrH, Marco-Marín and coworkers (19) recently proposed the existence of a binding platform for PyrH on the PepA hexamer, but the interaction has yet to be demonstrated.…”