UMP kinase from Streptococcus pneumoniae : evidence for co-operative ATP binding and allosteric regulation

Străuţ

Slavova‐Azmanova

et al. 2007

In this work, we examined the regulation by GTP and UTP of the UMP kinases from eight bacterial species. The enzyme from Gram-positive organisms exhibited cooperative kinetics with ATP as substrate. GTP decreased this cooperativity and increased the affinity for ATP. UTP had the opposite effect, as it decreased the enzyme affinity for ATP. The nucleotide analogs 5-bromo-UTP and 5-iodo-UTP were 5-10 times stronger inhibitors than the parent compound. On the other hand, UMP kinases from the Gram-negative organisms did not show cooperativity in substrate binding and catalysis. Activation by GTP resulted mainly from the reversal of inhibition caused by excess UMP, and inhibition by UTP was accompanied by a strong increase in the apparent K m for UMP. Altogether, these results indicate that, depending on the bacteria considered, GTP and UTP interact with different enzyme recognition sites. In Grampositive bacteria, GTP and UTP bind to a single site or largely overlapping sites, shifting the T % R equilibrium to either the R or T form, a scenario corresponding to almost all regulatory proteins, commonly called K systems. In Gram-negative organisms, the GTP-binding site corresponds to the unique allosteric site of the Gram-positive bacteria. In contrast, UTP interacts cooperatively with a site that overlaps the catalytic center, i.e. the UMP-binding site and part of the ATP-binding site. These characteristics make UTP an original regulator of UMP kinases from Gram-negative organisms, beyond the common scheme of allosteric control.Bacterial UMP kinases represent a particular subfamily of NMP 2 kinases (1, 2). They do not share any significant sequence homology with other known NMP kinases and exist in solution as stable hexamers. A first structural model of Escherichia coli UMP kinase (3) based on the conservation of the carbamate kinase and N-acetylglutamate kinase folds (4, 5) helped to better rationalize previous site-directed mutagenesis experiments (6). The crystal structure of E. coli UMP kinase (7) indicated a similar fold between its monomers and N-acetylglutamate kinase, a dimeric enzyme (4, 5). However, the quaternary structure assembly of these two proteins is completely different (7). Deposited crystal structures of UMP kinases from other bacteria such as Pyrococcus furiosus (8), Neisseria meningitidis (Protein Data Bank code 1YBD), Hemophilus influenzae (code 2AIF), and Streptococcus pyogenes (code 1Z9D) show threedimensional structures very similar to that of the E. coli enzyme. The residues essential for binding nucleotide substrates and catalysis are conserved among all bacterial UMP kinases ( Fig. 1) (3, 9). Consequently, the active sites of these enzymes and the phosphoryl transfer mechanisms are most probably similar.Comparison of the biochemical properties of recombinant UMP kinases from Gram-negative E. coli (1, 2) and Gram-positive Streptococcus pneumoniae (10) indicated significant differences in their kinetic properties particularly in their regulation by nucleotides. Unlike the E. coli enzyme, U...

Section: Resultssupporting

confidence: 82%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Regulatory Mechanisms Differ in UMP Kinases from Gram-negative and Gram-positive Bacteria

Străuţ

Slavova‐Azmanova

et al. 2007

“…Thus, the nucleoside triphosphate inhibitor binds in the active site and not in an allosteric site presumably distant from it, as was suggested by previous publications (3,6,7). Overall, the enzyme-UTP interactions (Fig.…”

Section: Fig 1 Sequence Alignment Of Umpkeco With Other Kinases Almentioning

confidence: 54%

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

Briozzo

Meyer

et al. 2005

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...

“…For all bacterial species, UMPK appears as essential for growth (5)(6)(7)(8). Bacterial UMPKs represent a unique target for the design of antibacterial agents as underlined by recently published papers (8,9) and even a patent (10).…”

mentioning

confidence: 99%

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

Meyer

Briozzo

et al. 2008

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, ...