1 Laboratoire de Chimie Structurale des Macromolécules, Institut Pasteur, 75724 Paris Cedex 15, France
2 Plate-forme 3 Protéomique, Institut Pasteur, 75724 Paris Cedex 15, France
3 Department of Chemistry and Biochemistry, University of Regina, Regina, Saskatchewan, Canada S4S 0A2
Correspondence
Hiroshi Sakamoto
hiroshi{at}pasteur.fr
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ABSTRACT |
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INTRODUCTION |
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For several organisms, the genes encoding NMP kinases have been cloned and sequenced, and the corresponding enzyme studied with respect to biochemical properties and structural features. Recent studies indicate that these enzymes are potential targets for drug design leading to new therapeutic compounds active against pathogens (Munier-Lehmann et al., 2001). Adenosine monophosphate kinases and UMP/CMP kinases have been the subjects of study for some time (Dreusicke et al., 1988
; Müller-Dieckmann & Schultz, 1994
) and more recently, the scope has been expanded to include CMP and TMP kinases (Briozzo et al., 1998
; Munier-Lehmann et al., 2001
). However, much less is known about GMP kinases and bacterial UMP kinases. To date, UMP kinase or UMP/CMP kinase has been found in most organisms investigated and, in many cases, the corresponding gene has been defined through genomic sequencing programs. The latest members to be characterized were the UMP kinase from Lactobacillus lactis (Wadskov-Hansen et al., 2000
) and the human UMP/CMP kinase (Liou et al., 2002
).
Among the bacterial NMP kinases, UMP kinase deserves special attention as the enzyme shows a number of distinctive features. First, UMP kinase is the only NMP kinase known to be endowed with a second biological role besides its catalytic activity, namely, the participation of UMP kinase in regulating the transcription of the carAB operon (Kholti et al., 1998). Second, UMP kinase is a homohexamer, and its catalytic activity is allosterically regulated by GTP/UTP (Serina et al., 1996
). A third feature is that UMP kinase displays very low sequence similarity to other NMP kinases, which should allow for the design of highly specific inhibitors of its catalytic activity (Serina et al., 1995
).
The assertion that UMP kinase activity is essential for life is based in part on knowledge of the UTP biosynthetic pathway (Neuhard & Kelln, 1996). Genetic studies with Gram-negative bacteria have provided direct evidence that UMP kinase is an essential enzyme, as conditional-lethal mutants of pyrH (the gene encoding UMP kinase) have been isolated (Ingraham & Neuhard, 1972
; Smallshaw & Kelln, 1992
). The thermosensitive Escherichia coli mutant KUR1244, harbouring the pyrH88(ts) allele, was used in the first report on the cloning of the wild-type pyrH allele by complementation of the thermosensitive phenotype (Smallshaw & Kelln, 1992
).
In the present study, we report the characterization of the molecular basis leading to the thermosensitivity of the KUR1244 pyrH mutant, as well as the characterization of other pyrH(ts) mutants obtained in a new set of mutagenesis and selection experiments. Furthermore, three additional pyrH alleles from available Gram-negative pathogens were also characterized for purposes of comparison with existing data on E. coli. Implications in terms of using UMP kinase as a target for drug design or attenuation procedures are discussed.
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METHODs |
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General cloning methods were essentially as described by Sambrook et al. (1989). Genes encoding UMP or CMP kinases from various origins were amplified by thermocycling and cloned into vectors pET22b or pET24ma (Table 1
), providing plasmids for both complementation analysis and for enzyme overproduction. Cloned DNA was sequenced using the dideoxy chain-termination method.
Biochemical analytical procedures.
Recombinant enzymes were produced and purified as follows. Strain BL21(DE3)/pDIA17 was used as the recipient for all recombinant plasmids used for overexpression. Cultures were grown at 30 °C in 2YT medium containing appropriate antibiotics until a value of 1 at OD600 was reached. IPTG was then added to a final concentration of 1 mM and incubation was continued for 3 h. Cells were harvested by centrifugation, resuspended in 20 mM Tris/HCl, pH 7·5, and disrupted by sonication (Landais et al., 1999). The recombinant enzyme was partially purified by several cycles of washing/centrifugation as described by Serina et al. (1995)
. Samples from non-overproducing strains were prepared as follows: strains were grown in 2YT medium at 30 °C to an OD600 of 4, then harvested, sonicated in 20 mM Tris/HCl, pH 7·5, and then the extract was heated for 10 min at 70 °C in the presence of 1 mM UTP. Bacterial debris was removed by centrifugation and UMP kinase activity was assayed in the supernatant as described below.
UMP kinase activity was assayed at 30 °C in a coupled system (Serina et al., 1995), with 1 mM ATP, 0·3 mM UMP, pH 7·4. Regulation by GTP/UTP was assayed in the presence of 0·5 mM effectors. One unit corresponds to 1 µmol UDP formed min1. Thermosensitivity of the enzymes was assayed after a 10 min heat denaturation treatment in a thermocycler, with the wild-type enzyme used as an internal control in each series. Indicated Tm values represent the denaturing temperature where 50 % of the UMP kinase activity was lost. Protein concentrations were determined by the method of Bradford (1976)
. All data come from triplicate experiments.
Western blots with polyclonal anti-UMP kinase were performed as previously described (Landais et al., 1999). Strains were grown in 2YT medium at 30 °C and harvested at an OD600 of 4. Strain KUR1244 was also grown at 30 °C to an OD600 of 0·3, then up-shifted to 42 °C until growth stopped. All samples were collected by centrifugation, sonicated, heated in the presence of 1 mM UTP and then centrifuged. Protein concentration was measured in each sample, then serial dilutions of the supernatants were subjected to SDS-PAGE (Laemmli, 1970
), transferred to a nitrocellulose membrane and immunodetected using polyclonal anti-UMP kinase antibodies. Immunoblots were scanned and the abundance of UMP kinase in the wild-type strain was considered as 100 %.
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RESULTS AND DISCUSSION |
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Other E. coli pyrH(ts) mutant strains
To broaden the information base regarding the E. coli enzyme, a collection of temperature-sensitive E. coli pyrH mutant strains was generated and characterized. As detailed in Methods, chemical mutagenesis was performed on strain W2915 coupled with selective and corresponding screening steps. Ultimately, three new pyrH(ts) strains were obtained: KUR1393, KUR1397 and KUR1398.
As described above for KUR1244, each new thermosensitive strain was tested for complementation by pyrH+. Complementation was observed in all cases with the wild-type pyrH allele from E. coli in either high or low copy number. Complementation with a low copy number of pyrH, or a high copy number of cmk from B. subtilis occurred as well.
All pyrH alleles were cloned by utilizing thermocycling amplification, then sequenced and their corresponding recombinant protein produced. The amino acid change in the enzyme from KUR1393 was the same as for KUR1244, namely R11H. The same change, along with two additional alterations (S106F, E196K) occurred in the enzyme from KUR1398. The enzyme from strain KUR1397 harboured a single distinct substitution at position 232, namely, a glycine to aspartate change.
Not unexpectedly, the properties of the recombinant UMP kinase variant from KUR1393 mimicked those observed with the enzyme from KUR1244. In the case of the UMP kinase variant from KUR1397, the G232D modification also led to biochemical features comparable to the R11H variant. Resistance to heat denaturation was altered, as the Tm value was lowered to 57 °C and catalytic activity was reduced to 17 % of the wild-type enzyme activity. Despite numerous attempts, the recombinant enzyme from KUR1398 could not be obtained in a soluble form.
The data provided herein show that the thermosensitive phenotype of KUR1244 is indeed linked to a defect of UMP kinase catalytic activity, an intrinsic property of a variant enzyme. Although the ability to affect transcription regulation of carAB was retained at the permissive temperature, the mutant enzyme displayed both a reduced catalytic activity and an increased thermosensitivity. The enzyme abundance was also found to be lowered in vivo. Therefore, the tenfold decrease in activity of the mutant strain in vivo as compared to the wild-type E. coli is a combination of two effects: a decrease in specific activity and a decrease of the protein abundance. Since the in vitro-measured Tm of the R11H mutant is still far from the non-permissive temperature of 42 °C, we infer that the low in vivo abundance of this variant reflects a folding and/or oligomerization defect. Hence KUR1244 may be more appropriately described as a mutant that is temperature-sensitive for synthesis, i.e., a tss mutant. In conjunction with the expanded study involving three newly isolated pyrH(ts) mutants of E. coli, the accumulated findings showed that the R11 residue is a key target for replacement when selection is based on 5-FOA resistance, uracil excretion, elevated ATCase activity and thermosensitivity. Another target residue is found at position G232, near the C terminus of the polypeptide.
S. enterica serovar Typhimurium pyrH mutants
In E. coli, a comprehensive set of pyrH mutant strains was characterized, both for recombinant UMP kinase catalytic activities and phenotypic features. We therefore investigated pyrH mutants from other related Gram-negative organisms to gain comparative insights on UMP kinases from one bacterial species to another.
For S. enterica serovar Typhimurium, strains defective in UMP kinase activity have been described since the early 1970s. Mutant strain pyrH1609 was shown to be cryo-sensitive and endowed with a reduced UMP kinase activity in vivo (Ingraham & Neuhard, 1972). Mutant strain pyrH1631 was described in a study of pyr gene expression in response to nucleotide pool shifts and was obtained by selection for the ability to use 2,6-diaminopurine as an exogenous purine source (Jensen, 1989
). Recently, the dum-1 mutation was demonstrated as a mutant pyrH allele, involved in establishing thymidine auxotrophy in a dcd cdd thyA+ background (Krogan et al., 1998
). The nature of the three corresponding mutations was not determined, nor were the biochemical properties of the respective UMP kinase variant enzymes assessed.
We undertook the cloning and sequencing of pyrH alleles from strains KR1633 (dum-1), KR1497 (pyrH1609) and KR1550 (pyrH1631), along with that of the wild-type LT2 strain, KR1639. The data in Table 3 include the UMP kinase activity of each strain and a summary of the amino acid differences in the variants, determined to be A122T (dum-1), D201N and E241D (pyrH1609) and D201G (pyrH1631).
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Key residues of bacterial UMP kinases
The R11 and G232 residues are highly conserved among UMP kinases from prokaryotes (Gagyi et al., 2003). This indicates that the R11 residue is at a key position, predisposed to replacement in the selection of pyrH(ts) mutants. For catalytic activity, the results obtained with S. enterica serovar Typhimurium confirm that the highly conserved D201 residue is important, as also applies to E. coli. 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. In the case of the D201 to G substitution, an increased flexibility of the polypeptide chain at this level might compensate for the loss of a negative charge at the expense of allosteric activation. Comparative crystallographic analysis of wild-type, D201N and D201G mutants would be able to provide a definitive answer.
What could be the lower limit of UMP kinase activity required to support life? Based on our data of the KUR1244 strain after upshift to 42 °C, and the catalytic activity of the corresponding recombinant protein, one can propose that such a threshold is in the range of an in vivo activity of 5 mU mg1. It is tempting to suggest that a conditional mutant of pyrH displaying a catalytic activity in the vicinity of this threshold would show a complete loss of viability in the non-permissive conditions.
The essential activity of UMP kinase is catalysis, and bacterial UMP kinases share significant sequence similarities. Based on the current data, we propose that a combination of the low catalytic activity substitutions (i.e. D146N or D201N) (Serina et al., 1995; Bucurenci et al., 1998
), along with mutations leading to thermosensitivity of UMP kinase (i.e. R11H or G232D) may provide a general method to construct attenuated bacterial strains, especially from Gram-negative pathogens.
Concluding remarks
The 3D structure of bacterial UMP kinases is not yet solved, which hampers advances in rational drug design. While numerous variants of UMP kinase are available to decipher its structurefunction relationship for catalysis, very few variants address the issue with respect to overall folding and thermal stability. In the last few years impressive advances have been achieved in the design of oligopeptides of therapeutic interest (Gratton et al., 2003). Studies have shown that oligopeptides can be used for intracellular delivery, as well as in situ proteinprotein interaction. This new technology opens up the field for new antimicrobial compounds based on the disruption of essential catalytic activities. In this respect, the set of pyrH(ts) mutants described in this work may also find value for use in rational drug design.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 72, 248254.[CrossRef][Medline]
Briozzo, P., Golinelli-Pimpaneau, B., Gilles, A.-M., Gaucher, J.-F., Burlacu-Miron, S., Sakamoto, H., Janin, J. & Bârzu, O. (1998). Structures of Escherichia coli CMPK alone and in complex with CDP: a new fold of the nucleotide specificity. Structure 6, 15171527.[Medline]
Bucurenci, N., Serina, L., Zaharia, C., Landais, S., Danchin, A. & Bârzu, O. (1998). Mutational analysis of UMP kinase from Escherichia coli. J Bacteriol 180, 473477.
Dreusicke, D., Karplus, P. A. & Schulz, G. E. (1988). Refined structure of porcine cytosolic adenylate kinase at 2·1 Å resolution. J Mol Biol 199, 359371.[Medline]
Gagyi, C., Bucurenci, N., Sîrbu, O. & 7 other authors (2003). UMP kinase from the gram-positive bacterium Bacillus subtilis is strongly dependent on GTP for optimal activity. Eur J Biochem 270, 31963204.
Gratton, J.-P., Yu, J., Griffith, J. W., Babbitt, R. W., Scotland, R. S., Hickey, R., Giordano, F. J. & Sessa, W. C. (2003). Cell-permeable peptides improve cellular uptake and therapeutic gene delivery of replication-deficient viruses in cells and in vivo. Nature Med 9, 357363.[CrossRef][Medline]
Ingraham, J. L. & Neuhard, J. (1972). Cold-sensitive mutants of Salmonella typhimurium defective in uridine monophosphate kinase (pyrH). J Biol Chem 247, 62596265.
Jensen, K. F. (1989). Regulation of Salmonella typhimurium pyr gene expression: effect of changing both purine and pyrimidine nucleotide pools. J Gen Microbiol 135, 805815.[Medline]
Kelln, R. A., Foltermann, K. F. & O'Donovan, G. A. (1975). Location of the argR gene on the chromosome of Salmonella typhimurium. Mol Gen Genet 139, 277284.[Medline]
Kholti, A., Charlier, D., Gigot, D., Huysveld, N., Roovers, M. & Glansdorff, N. (1998). pyrH-encoded UMP kinase directly participates in pyrimidine-specific modulation of promoter activity in Escherichia coli. J Mol Biol 280, 571582.[CrossRef][Medline]
Krogan, N. J., Zaharik, M. L., Neuhard, J. & Kelln, R. A. (1998). A combination of three mutations, dcd, pyrH and cdd, establishes thymidine (deoxyuridine) auxotrophy in thyA+ strains of Salmonella typhimurium. J Bacteriol 180, 58915895.
Labesse, G., Bucurenci, N., Douguet, D., Sakamoto, H., Landais, S., Gagyi, C., Gilles, A.-M. & Bârzu, O. (2002). Comparative modelling and immunochemical reactivity of Escherichia coli UMP kinase. Biochem Biophys Res Commun 294, 173179.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Landais, S., Gounon, P., Laurent-Winter, C., Mazié, J.-C., Danchin, A., Bârzu, O. & Sakamoto, H. (1999). Immunochemical analysis of UMP kinase from Escherichia coli. J Bacteriol 181, 833840.
Liljelund, P. & Lacroute, F. (1986). Genetic characterization and isolation of the Saccharomyces cerevisiae gene coding for uridine monophosphokinase. Mol Gen Genet 205, 7481.[Medline]
Liou, J. Y., Dutschman, G. E., Lam, W., Jiang, Z. & Cheng, Y. C. (2002). Characterization of human UMP/CMP kinase and its phosphorylation of D- and L-form deoxycytidine analogue monophosphates. Cancer Res 62, 16241631.
Miller, J. (1992). A Short Course in Bacterial Genetics: Laboratory Manual and Handbook for Escherichia Coli and Related Bacteria. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Müller-Dieckmann, H.-J. & Schultz, G. E. (1994). The structure of uridylate kinase with its substrates, showing the transition state geometry. J Mol Biol 236, 361367.[CrossRef][Medline]
Munier-Lehmann, H., Chafotte, A., Pochet, S. & Labesse, G. (2001). Thymidylate kinase of Mycobacterium tuberculosis: a chimera sharing properties common to eukaryotic and bacterial enzymes. Protein Sci 10, 11951205.
Neuhard, J. & Kelln, R. A. (1996). Biosynthesis and Conversions of Pyrimidines in Escherichia Coli and Salmonella: Cellular and Molecular Biology. Washington, DC: American Society for Microbiology.
O'Donovan, G. A. & Gerhart, J. C. (1972). Isolation and partial characterization of regulatory mutants of the pyrimidine pathway in Salmonella typhimurium. J Bacteriol 109, 10851096.[Medline]
Raleigh, E. A., Murray, N. E., Revel, H., Blumenthal, R. M., Westaway, D., Reith, A. D., Rigby, P. W. J., Elhai, J. & Hanahan, D. (1988). McrA and McrB restriction phenotypes of some E. coli strains and implications for gene cloning. Nucleic Acids Res 16, 15631575.[Abstract]
Roovers, M., Charlier, D., Feller, A., Gigot, D., Holemans, F., Lissens, W., Piérard, A. & Glansdorff, N. (1998). carP, a novel gene regulating the transcription of the carbamoylphosphate synthetase operon of Escherichia coli. J Mol Biol 204, 857865.[CrossRef]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Serina, L., Blondin, C., Krin, E., Sismeiro, O., Danchin, A., Sakamoto, H., Gilles, A.-M. & Bârzu, O. (1995). Escherichia coli UMP-kinase, a member of the aspartokinase family, is a hexamer regulated by guanine nucleotides and UTP. Biochemistry 34, 50665074.[Medline]
Serina, L., Bucurenci, N., Gilles, A.-M. & 7 other authors (1996). Structural properties of UMP-kinase from Escherichia coli: modulation of protein solubility by pH and UTP. Biochemistry 35, 70037011.[CrossRef][Medline]
Smallshaw, J. C. & Kelln, R. A. (1992). Cloning, nucleotide sequence and expression of the Escherichia coli K-12 pyrH gene encoding UMP kinase. Genetics (Life Sci Adv) 11, 5965.
Wadskov-Hansen, S. L., Martinussen, J. & Hammer, K. (2000). The pyrH gene of Lactococcus lactis subsp. cremoris encoding UMP kinase is transcribed as part of an operon including the frr1 gene encoding ribosomal recycling factor 1. Gene 241, 157166.[CrossRef][Medline]
Yamanaka, K., Ogura, T., Niki, H. & Hiraga, S. (1992). Identification and characterization of the smbA gene, a suppressor of the mukB null mutant of Escherichia coli. J Bacteriol 174, 75177526.[Abstract]
Received 18 December 2003;
revised 26 February 2004;
accepted 23 March 2004.
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