Comparative modelling and immunochemical reactivity of Escherichia coli UMP kinase

Labesse, Gilles; Bucurenci, Nadia; Douguet, Dominique; Sakamoto, Hiroshi; Landais, Stéphanie; Gagyi, Cristina; Gilles, Anne‐Marie; Bârzu, Octavian

doi:10.1016/s0006-291x(02)00450-3

Cited by 16 publications

(21 citation statements)

References 31 publications

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“…on May 10, 2018 by guest http://iai.asm.org/ microbial agents (2,3,9,20,24,30). Recently, two independent groups determined the molecular structure of PyrH encoded by E. coli and P. furiosus (2,24).…”

Section: Vol 75 2007 In Vivo-expressed Survival Factor Pyrh 2799mentioning

confidence: 99%

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

Lee

Kim

et al. 2007

Infect Immun

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We have suggested an important role of the pyrH gene during the infectious process of Vibrio vulnificus. Previously, we have identified 12 genes expressed preferentially during human infections by using in vivoinduced antigen technology. Among the in vivo-expressed genes, pyrH encodes UMP kinase catalyzing UMP phosphorylation. Introduction of a deletion mutation to the pyrH gene was lethal to V. vulnificus, and an insertional mutant showed a high frequency of curing. We constructed a site-directed mutant strain (R62H/ D77N) on Arg-62 and Asp-77, both predicted to be involved in UMP binding, and characterized the R62H/D77N strain compared with the previously reported insertional mutant. We further investigated the essential role of the pyrH gene in the establishment of infection using the R62H/D77N strain. Cytotoxicity was decreased in the R62H/D77N strain, and the defect was restored by an in trans complementation. The intraperitoneal 50% lethal dose of the R62H/D77N strain increased by 26-and 238,000-fold in normal and iron-overloaded mice, respectively. The growth of the R62H/D77N strain in 50% HeLa cell lysate, 100% human ascitic fluid, and 50% human serum was significantly retarded compared to that of the isogenic wild-type strain. The R62H/D77N mutant also had a critical defect in the ability to survive and replicate even in iron-overloaded mice. These results demonstrate that pyrH is essential for the in vivo survival and growth of V. vulnificus and should be an attractive new target for the development of antibacterial drugs and replication-controllable live attenuated vaccines.Vibrio vulnificus is an estuarine bacterium that opportunistically infects a human being through the consumption of contaminated seafood or wound infection. V. vulnificus septicemia preferentially occurs in patients with underlying hepatic diseases or other immunocompromised conditions and results in a rapid progress and high mortality rate of Ͼ50% (10,22,35). During the infectious process, the V. vulnificus is confronted with dramatic environmental changes, and the bacteria seem to cognitively sense the changes in the host milieu. For the successful infection, V. vulnificus should establish coordinated spatiotemporal expression of various virulence genes in vivo (11, 12). Our group has previously reported 12 in vivo expressed genes by using in vivo-induced antigen technology (17). Among them, pyrH encodes UMP kinase, which catalyzes phosphorylation of UMP to UDP (24, 26). It was reported that UMP kinase senses the environmental pyrimidine pool and directly regulates pyrimidine-specific CarP1 promoter of carbamoylphosphate synthetase of Escherichia coli responsible for the early stage de novo synthesis of pyrimidines (15). Klarsfeld et al. have reported that pyrE of Listeria monocytogenes, another de novo pyrimidine biosynthetic gene, preferentially expressed the intracellular milieu of host cells through a transposon mutant library screening experiment (18). These previous reports suggest that limited availability of pyrimidines i...

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Section: Vol 75 2007 In Vivo-expressed Survival Factor Pyrh 2799mentioning

confidence: 99%

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

Lee

Kim

et al. 2007

Infect Immun

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“…Measurements were performed with an Autosizer 4700 (MAL-VERN) instrument equipped with an argon ion laser operating The secondary-structure elements from the E. coli model of Labesse et al [14] are shown over the alignment. The residues cited in the text are in boldface.…”

Section: Dls (Dynamic Light Scattering)mentioning

confidence: 99%

“…The residues cited in the text are in boldface. Active-site motifs, as predicted in [14], are underlined in the E. coli sequence and noted below the alignment. Hypothetical GTP-binding site and subunit interface are underlined in the S. pneumoniae sequence and noted below the alignment.…”

Section: Dls (Dynamic Light Scattering)mentioning

confidence: 99%

“…Prokaryotic UMP kinase is instead more related by primary sequence comparison to the aspartokinase family [13]. In addition, molecular modelling studies predict that the E. coli UMP kinase structure shares similarities with carbamate kinases and acetylglutamate kinases [14]. To date, only UMP kinases from E. coli and B. subtilis have been overexpressed, purified and characterized [12,13,[15][16][17].…”

Section: Introductionmentioning

confidence: 99%

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

Fassy¹,

Krebs²,

Lowinski³

et al. 2004

Biochemical Journal

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UMP kinase catalyses the phosphorylation of UMP by ATP to yield UDP and ADP. In prokaryotes, the reaction is carried out by a hexameric enzyme, activated by GTP and inhibited by UTP. In the present study, Streptococcus pneumoniae UMP kinase was studied as a target for antibacterial research and its interest was confirmed by the demonstration of the essentiality of the gene for cell growth. In the presence of MnCl 2 or MgCl 2 , the saturation kinetics of recombinant purified UMP kinase was hyperbolic for UMP (K m = 0.1 mM) and sigmoidal for ATP (the substrate concentration at half-saturation S 0.5 = 9.4 + − 0.7 mM and n = 1.9 + − 0.1 in the presence of MgCl 2 ). GTP increased the affinity for ATP and decreased the Hill coefficient (n). UTP decreased the affinity for ATP and only slightly increased the Hill coefficient.The k cat (175 + − 13 s −1 in the presence of MgCl 2 ) was not affected by the addition of GTP or UTP, whose binding site was shown to be different from the active site. The hydrodynamic radius of the protein similarly decreased in the presence of ATP or GTP. There was a shift in the pH dependence of the activity when the ATP concentration was switched from low to high. These results support the hypothesis of an allosteric transition from a conformation with low affinity for ATP to a form with high affinity, which would be induced by the presence of ATP or GTP.

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“…In this respect it is worth mentioning that the D201N 'conservative' substitution first described by Yamanaka et al (1992) and explored enzymically by Bucurenci et al (1998) in E. coli, has a more dramatic effect on protein stability and catalysis than that observed with the newly described D201G variant. In the modelled structure of E. coli UMP kinase, D201 is situated at the N terminus of helix-7, which is predicted to provide residues to the catalytic centre (Labesse et al, 2002). The severe loss of catalytic activity upon substitution of this residue with asparagine suggests that formation of salt bridges is perhaps critical for stabilization of the active site.…”

Section: Key Residues Of Bacterial Ump Kinasesmentioning

confidence: 99%

Structure–function relationships of UMP kinases from pyrH mutants of Gram-negative bacteria

et al. 2004

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Bacterial uridine monophosphate (UMP) kinases are essential enzymes encoded by pyrH genes, and conditional-lethal or other pyrH mutants were analysed with respect to structure-function relationships. A set of thermosensitive pyrH mutants from Escherichia coli was generated and studied, along with already described pyrH mutants from Salmonella enterica serovar Typhimurium. It is shown that Arg-11 and Gly-232 are key residues for thermodynamic stability of the enzyme, and that Asp-201 is important for both catalysis and allosteric regulation. A comparison of the amino acid sequence of UMP kinases from several prokaryotes showed that these were conserved residues. Discussion on the enzyme activity level in relation to bacterial viability is also presented.

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Comparative modelling and immunochemical reactivity of Escherichia coli UMP kinase

Cited by 16 publications

References 31 publications

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

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

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

Structure–function relationships of UMP kinases from pyrH mutants of Gram-negative bacteria

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