Structural and Functional Characterization of Escherichia coli UMP Kinase in Complex with Its Allosteric Regulator GTP

Abstract

Bacterial UMP kinases are essential enzymes involved in the multistep synthesis of UTP. They are hexamers regulated by GTP (allosteric activator) and UTP (inhibitor). We describe here the 2.8 Å crystal structure of Escherichia coli UMP kinase bound to GTP. The GTP-binding site, situated at 15 Å from the UMP-binding site and at 24 Å from the ATP-binding site, is delineated by two contiguous dimers. The overall structure, as compared with those bound to UMP, UDP, or UTP, shows a rearrangement of its quaternary structure: GTP induces an 11°o pening of the UMP kinase dimer, resulting in a tighter dimerdimer interaction. A nucleotide-free UMP kinase dimer has an intermediate opening. Superposition of our structure with that of archaeal UMP kinases, which are also hexamers, shows that a loop appears to hamper any GTP binding in archeal enzymes. This would explain the absence of activating effect of GTP on this group of UMP kinases. Among GTP-binding residues, the Asp-93 is the most conserved in bacterial UMP kinases. In the previously published structures of E. coli UMP kinase, this residue was shown to be involved in hydrogen bonds between the subunits of a dimer. Its substitution by an alanine decreases the cooperativity for UTP binding and suppresses the reversal by GTP of UTP inhibition. This demonstrates that the previously described mutual exclusion of these two nucleotides is mediated by Asp-93.Uridine 5Ј-monophosphate kinases (UMPKs) 4 catalyze the reversible transfer of the ␥-phosphoryl group from ATP to UMP, according to the reaction, Mg⅐ATP ϩ UMP 7 Mg⅐ADP ϩ UDP. The resulting UDP is further phosphorylated by nucleoside diphosphate kinase to UTP, serving as a substrate for RNA polymerase or as a precursor for CTP. UDP can also be reduced by ribonucleoside diphosphate reductase to 2Ј-deoxy-UDP, which serves in the production of dTTP and dCTP used for DNA synthesis.The eukaryotic UMP/CMP kinases phosphorylate with comparable efficiency both UMP and CMP. Conversely, bacteria possess two distinct enzymes, each specific for either UMP or CMP. Bacterial UMPKs exist in solution as stable homohexamers, whereas eukaryotic UMP/CMP kinases are monomers. We previously solved three crystal structures of UMPK from Escherichia coli (UMPKeco) in complex with UMP, UDP, or UTP (1), using an enzyme variant (D159N) with similar stability and kinetic properties (2) but with significantly higher solubility than the wild-type protein. These structures showed that the overall fold of the monomer is different from that of eukaryotic UMP/CMP kinases. Besides, the three-dimensional structure of the UMPKeco hexamer is closely related to all of the other bacterial UMPKs structures deposited in the Protein Data Bank.Bacterial UMPKs are submitted to allosteric activation by GTP and to feedback inhibition by UTP, the major product of the reaction they catalyze (3). This contributes to balance the synthesis of purine versus pyrimidine nucleoside triphosphates. The crystal structure of UMPKeco in complex with UTP showed that the nucleotide, ...

Pyrimidine homeostasis is accomplished by directed overflow metabolism

“…Here we identify an alternative regulatory strategy that enables precise control of pyrimidine pathway end-product levels, even in the presence of dysregulated biosynthetic flux. The mechanism involves cooperative feedback regulation of the near-terminal pathway enzyme uridine monophosphate kinase 6 . Such feedback leads to build-up of the pathway intermediate uridine monophosphate, which is in turn degraded by a conserved phosphatase, here termed UmpH, with previously unknown physiological function 7,8 .…”

“…In analysing the regulatory architecture of the pathway, we noticed an additional potential feedback loop near the end of the pathway: UMP kinase (which catalyses the ATP-dependent phosphorylation of UMP to UDP), encoded by pyrH (Fig. 3a), is regulated in vitro by cooperative (ultrasensitive) inhibition by UTP (Hill-coefficient of 2.8, inhibition constant ( K i ) of 154 μM) 6 . This inhibition can be partially overcome by GTP activation 6,27 , and thus, like the regulation of ATCase by CTP and ATP, may function to achieve balance between pyrimidine and purine pools.…”

“…3a), is regulated in vitro by cooperative (ultrasensitive) inhibition by UTP (Hill-coefficient of 2.8, inhibition constant ( K i ) of 154 μM) 6 . This inhibition can be partially overcome by GTP activation 6,27 , and thus, like the regulation of ATCase by CTP and ATP, may function to achieve balance between pyrimidine and purine pools. Moreover, regulation at the end of the pathway would have the advantage of minimizing perturbations in UTP and CTP levels in response to alterations in any upstream pathway substrate (for example, not only of the de novo pathway, but also uracil, orotate, or their nucleosides).…”

Multistep Synthesis of UDP-Glucose Using Tailored, Permeabilized Cells of E. coli

“…As reported, magnesium is the preferred cofactor for UMPK and SPase, whereas no detailed information was found on the other enzymes [17][18][19]. Manganese activates the enzyme UDP-sugar diphosphatase (EC Fig.…”

A Nano-Chip-LC/MSn Based Strategy for Characterization of Modified Nucleosides Using Reduced Porous Graphitic Carbon as a Stationary Phase

“…The test-mixture contained the following 13 nucleosides in varying amounts (Table 1, [25], inorganic pyrophosphatase (hppA, EC 3.6.1.1) [26], and CTP synthetase (pyrG, EC 6.3.4.2) [27], were prepared from overexpression strains described. All standard analytical HPLC methods and stepwise purification methods are described in detail in Scott et al [28].…”