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REGULATORY MECHANISMS DIFFER IN UMP KINASES FROMGRAM-NEGATIVE AND GRAM-POSITIVE BACTERIA

Cécile Evrin1, Monica Straut2, Neli Slavova-Azmanova1, Nadia Bucurenci2, Adrian Onu2,Liliane Assairi1,3, Mihaela Ionescu1, Nicolae Palibroda4, Octavian Bârzu1,

and Anne-Marie Gilles1

From 1Unité de Génétique des Génomes Bactériens, Institut Pasteur, 75724 Paris cedex, France,2Laboratory of Enzymology and Applied Microbiology, Cantacuzino Institute, Bucharest,Romania, 3INSERM U759, Institut Curie-Recherche, Centre Universitaire Paris-Sud, Bâtiments110-112, Orsay, France, and 4Institute of Isotopic and Molecular Technology, Cluj-Napoca,Romania.

Running Title: Regulation of bacterial UMP kinasesAddress correspondence to : Anne-Marie Gilles, Unité de Génétique des Génomes Bactériens, InstitutPasteur, 28 rue du Dr Roux, 75724 Paris cedex15, Tel. 33-1-45-68-89-68; Fax. 33-1-45-68-89-48; E-mail: amgilles@pasteur.fr

In this work, we have examined theregulation by GTP and UTP of the UMPkinase from eight bacterial species. Theenzyme from Gram-positive organismsexhibited cooperative kinetics with ATP assubstrate. GTP decreased this cooperativityand increased the affinity for ATP. UTP hasthe opposite effect as it decreased theenzyme affinity for ATP. The nucleotideanalogs 5Br-UTP and 5I-UTP were 5 to 10times stronger inhibitors than the parentcompound. On the other hand, UMP kinasesfrom the Gram-negative organisms did notshow cooperativity in substrate binding andcatalysis. The activation by GTP mainlyresulted from the reversal of the inhibitioncaused by excess of UMP, and the inhibitionby UTP was accompanied by a strongincrease of the apparent Km for UMP.Altogether, these results indicate thatdepending on the bacteria considered, theGTP and UTP interact with differentenzyme recognition sites. In Gram-positivebacteria, GTP and UTP bind to a single siteor largely overlapping sites, shifting the T R equilibrium either to the R form or to theT form, a scenario corresponding to almostall regulatory proteins, commonly called Ksystems. In Gram-negative organisms, theGTP binding site corresponds with theunique allosteric site of the Gram-positivebacteria. On the contrary, UTP interactscooperatively with a site, which overlaps thecatalytic center, i.e. the UMP binding siteand part of the ATP binding site. Thesecharacteristics make UTP an originalregulator of UMP kinase from Gram-

negative organisms, beyond the commonscheme of allosteric control.

Bacterial uridine monophosphate(UMP) kinases represent a particular subfamilyof nucleoside monophosphate (NMP) kinases(1,2). They do not share any significantsequence homology with other known NMPkinases and exist in solution as stablehexamers. A first structural model ofEscherichia coli UMP kinase (3) based on theconservation of the carbamate kinase and theN-acetyl glutamate kinase folds (4,5), helped tobetter rationalize previous site-directedmutagenesis experiments (6). The crystalstructure of E. coli UMP kinase (7) indicated asimilar fold between its monomers and N-acetyl glutamate kinase, a dimeric enzyme(4,5). However, the quaternary structureassembly of these two proteins is completelydifferent (7). Deposited crystal structures ofUMP kinase from other bacteria such asPyrococcus furiosus (8), Neisseria meningitidis(1YBD), Haemophilus influenzae (2AIF) andStreptococcus pyogenes (1Z9D) showed three-dimensional structures very similar to the E.coli enzyme. The residues essential for bindingof nucleotide substrates and catalysis areconserved among all bacterial UMP kinases(3,9) (Fig. 1). Consequently, the active site ofthese enzymes and the phosphoryl transfermechanism are most probably similar.

Comparison of the biochemicalproperties of recombinant UMP kinases fromthe Gram-negative E. coli (1,2) and the Gram-positive Streptococcus pneumoniae (10)indicated significant differences in their kinetic

http://www.jbc.org/cgi/doi/10.1074/jbc.M606963200The latest version is at JBC Papers in Press. Published on January 8, 2007 as Manuscript M606963200

Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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properties particularly in their regulation bynucleotides. UMP kinase from S. pneumoniae,unlike the E. coli enzyme, exhibitedcooperative kinetics with respect to ATP, andits activation by GTP resulted in the decrease ofcooperativity and the increase of affinity forATP.

In order to substantiate and eventuallyextend these observations to other UMP kinasesfrom Gram-negative or Gram-positive bacteria,the corresponding pyrH genes were cloned andthe recombinant proteins studied for theirkinetic properties in both forward and reversereaction. Thus, GTP and UTP are so fareffectors for all the investigated UMP kinases.They act on the kinetic parameters mostly viaconformational changes induced on the protein.Consequently, the regulating effects of GTPand UTP on UMP kinases from both Gram-negative and Gram-positive organisms arestrongly related to the quaternary structure ofthese proteins.

Experimental Procedures

Chemicals - Nucleotides, restriction enzymes,T4 DNA-ligase, Vent and Tfu DNApolymerase and coupling enzymes werepurchased from Roche-Applied Science, NewEngland Biolabs, Q-biogene or from Sigma.UTP and UMP analogs halogenated on theposition 5 of the heterocycle were purchasedfrom Jena Bioscience GmbH. NDP kinase fromDictyostelium discoideum (2000 U/mg ofprotein) was kindly provided by I. Lascu.Bacterial strains, plasmids, growth conditionsand DNA manipulations - General DNAmanipulations were performed as described bySambrook (11). Open reading frames from thepyrH gene from different organisms (E. coli, S.typhimurium, H. influenzae, N. meningitidis, B.subtilis, S. pneumoniae, S. aureus, and E .faecalis) were amplified from chromosomalDNA as template and using the correspondingprimers (Table I). The PCR products wereinserted in the vector pET24a (between theNdeI and EcoRI restriction sites) or in thevector pET28a (Novagen) (between the NdeIand XhoI or HindIII restriction sites). Theresulting plasmids were introduced into strainBL21(DE3)/pDIA17 (12) to overproduce theUMP kinase. The recombinant strains weregrown in 2YT medium supplemented withkanamycin (70 µg/ mL) and chloramphenicol

(30 µg/ mL) to an absorbance of 1.5 at 600 nm,then overproduction was triggered byisopropyl-β-D-thiogalactoside induction (1 mMfinal concentration) for 3 h at 37°C. The cellswere then harvested by centrifugation andserved as source for protein purification.

The single mutants T135A and N137Aand the double-mutant T135A-N137A of B.subtilis UMP kinase were constructed by theone-tube PCR-based mutagenesis method (13)using the plasmid harbouring the correspondingUMP kinase gene as template, Tfu DNApolymerase, the dNTPs and the followingmutagenic oligonucleotides: 3’- B. subtilis UMPkinase T135A, 5'-GAAATATGGGTTTCCAGCGCCCGCAGCGAAAAT-3’; 3’- B. subtilis UMP kinase N137A, 5'-AGTTGAGAAATATGGAGCTCCTGTGCCCGCAGC-3'; 3’- B. subtilis UMP kinase T135A-N137A, 5'-AGTTGAGAAATATGGAGCTCCAGCGCCCGCAGCGAAAAT-3'. The PCR product was cloned atthe NdeI and XhoI restriction sites of the vectorpET28a.

All plasmids were sequenced to verifyeither their integrity or the incorporation of thedesired modifications.Purification of UMP kinases and activity assay- The different N-terminal His-tagged UMPkinases (E. coli D159N soluble variant, H.influenzae, N. meningitidis, B. subtilis, S.pneumoniae , S. a u r e u s , and E. faecalis)overproduced in E. coli were purified byN i c k e l - n i t r i l o a c e t i c a c i d a f f i n i t ychromatography using the QIA express system(14). The recombinant proteins (purity over95% as indicated by SDS-PAGE) were storedat +4°C in buffer (pH 8.0) containing 50 mMNa2HPO4, 150 mM imidazole and 300 mMNaCl. Recombinant S. typhimurium UMPkinase was purified as described previously forwild-type UMP kinase from E. coli (1). Proteinconcentration was measured according toBradford (15). Ion spray mass spectra ofpurified proteins were recorded on aquadrupole mass spectrometer API-365(Perkin-Elmer) equipped with an ion spray(nebulizer-assisted electrospray) source. SDS-PAGE was performed as described by Laemmli(16).

UMP kinase activity was determined at30°C using coupled spectrophotometric assays(0.5 mL final volume) on an Eppendorf ECOMphotometer (17). The reaction medium in the

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forward direction contained 50 mM Tris-HCl(pH 7.4) , 50 mM KCl, 1 mMphosphoenolpyruvate, 0.2 mM NADH, 2 unitseach of lactate dehydrogenase, pyruvate kinaseand NDP kinase, and various concentrations ofMgCl2, ATP and UMP. The UMP kinaseappropriately diluted in 50 mM Tris-HCl (pH7.4) was then added and the decrease ofabsorbance recorded at 340 nm. The reactionmedium in the reverse direction contained 50mM Tris-HCl (pH 7.4), 50 mM KCl, 1 mMglucose, 0.4 mM NADP+, 2 units each ofhexokinase and glucose-6-phosphatedehydrogenase and various concentrations ofMgCl2, ADP and UDP. The appropriatelydiluted UMP kinase was then added and theincrease of absorbance recorded at 340 nm.One unit of UMP kinase corresponds to 1 µmolof product formed per min.

The thermal stability of UMP kinaseswas tested by incubating the purified enzymes(1 mg/mL) in 50 mM Tris-HCl (pH 7.4 or 8.5)containing 0.1 M NaCl at a temperaturebetween 30° and 80°C for 10 min in thepresence or absence of various nucleotides. Theresults, expressed as the percentage of residualactivity as compared with unincubated controls,were used to calculate the temperature of half-inactivation (Tm).Calculation of Mg-nucleotide complexes andkinetic data analysis – The concentration ofMgCl2 in the assay medium in which coexistednucleotides differing in the number ofphosphate unit was found critical for severalreasons. The dissociation constant (Kd) ofmetal-nucleotide complexes varies within two-orders of magnitude, from 0.1 mM for MgNTP;1 mM for MgNDP and 20 mM for MgNMP(18,19). On the other hand, as some nucleotidesplayed multiple roles this resulted in mixedkinetic effects. Numerical simulations withdifferent concentrations of MgCl2 andnucleotides showed that an acceptablecompromise in the forward reaction was to usea 2 mM excess of MgCl2 over the concentrationof NTPs. Thus, for the range of ATP (0.2 to 25mM) and UMP (0.1 to 2 mM) concentrationsused in most experiments, the MgATPrepresented 95.7 ± 0.9 % of total ATP, and theMg-free UMP represented 89.6 ± 2 % of totalUMP (Table II). Furthermore, the free metalion (between 1.8 and 2.8 mM) was held at asufficiently high but non-inhibitoryconcentration. For the sake of simplicity, the

calculation of the kinetic constants in theforward reaction employed the actualconcentration of various nucleotides. In thiscase, a Km or K0.5 of 2 mM for ATPcorresponds approximately to a Km for MgATPof 1.9 mM. Similarly a Km for UMP of 0.1 mMcorresponds approximately to a Km of 0.09 mMfor Mg-free UMP. In the reverse reaction theconcentration of MgCl2 (mM) was related toconcentrations of UDP and ADP (or GDP whenpresent) by the following relationship: [MgCl2]t

= 4 + 0.8 [NDP]t. Under these conditions, theconcentration of MgNDPs represented 80% oftotal nucleotide concentration and theconcentration of free magnesium cation wasalways 4 mM. When GMP or GMP-PNP wasused this relation changed as follows: [MgCl2]t

= 4 + 0.8 [NDP]t + 0.1 [NMP]t and respectively[MgCl2]t = 4 + 0.8 [NDP]t + [NTP]t. With theseempirical adjustments, the [MgNDP]/[NDP]tratio varied by less than 1%, while theconcentration of MgNTP represented 98% ofthe total NTP.

The kinetic results were fitted to one ofthe following three equations, using the non-linear least-squares fitting analysis ofKaleidagraph software:

(1) v = Vm[S] / (Km + [S]);(2) v = Vm[S] / (Km + [S] + [S]2/KI);(3) v = Vm[S]n / (Kn

0.5 + [S]n);where v is the steady-state velocity, Vm themaximal rate, [S] the substrate concentration(i.e. ATP or UMP in the forward reaction andADP or UDP in the reverse reaction), Km theMichaelis-Menten constant, K0.5 the substrateconcentration at half-saturation, KI theinhibition constant and n (or nH) the Hillnumber, indicating the cooperativity index. Theaccuracy of the constants calculated by thesefittings (in average they varied within ± 10%)depended on the experimental errors (proteinconcentration and stability, purity of thecommercially available nucleotides, theefficiency of the coupling enzymes in the assaysystem), and the computed concentration of the“active” metal-free or metal-complexednucleotides from the corresponding dissociationconstants.

RESULTS

Purification and specific activity ofrecombinant UMP kinases - Since we did not

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observe significant differences in specificactivities of the wild-type or the His-taggedforms of E. coli (7), H. influenzae (this work)and B. subtilis (9) UMP kinases, therecombinant enzyme from the other bacterialspecies was overproduced with a N-terminalHis-tag, and purif ied by aff ini tychromatography on nickel nitriloaceticcolumns. We assumed that the His-tag does notaffect the activity of other bacterial UMPkinases. Gel permeation chromatography onSephacryl S-300 and ultracentrifugation bysedimentation equilibrium confirmed that allvariants exist as hexamers.

Table III indicates the specific activityof UMP kinase from eight bacterial species at asingle concentration of UMP (1 mM) and GTP(0.5 mM) and at two concentrations of ATP (2and 8 mM). The highest concentration ofnucleotides (8 mM ATP, 1 mM UMP and 0.5mM GTP) was selected arbitrarily to reach themaximal activity for all bacterial species. InGram-negative organisms (E. co l i , S.t yph imur ium , H. in f l uenzae and N .meningitidis) the ratio of UMP kinase activityin the presence and in the absence of GTP waspractically independent of the concentration ofATP, whereas in Gram-positive organisms (B.subtilis, S. pneumoniae, S. aureus and E .faecalis) the activating effect of GTP was muchhigher at low concentration of ATP. Furtherkinetic analysis of UMP kinase from variousspecies enlightened the origin of thisdifference.

Dependence of UMP kinase activity onATP concentration – E. coli and S.typhimurium UMP kinases were shown toexhibit hyperbolic dependence of activity as afunction of ATP concentration, both in theabsence or in the presence of GTP (1,20). Thesame was true for N. meningitidis UMP kinase.The H. influenzae UMP kinase slightly deviatesfrom this rule as the kinetics with ATP as thevariable substrate were best fitted by the Hillequation. However, the nH values did notexceed 1.30 (Table IV). In the case of UMPkinase from the Gram-positive bacteria, the plotof activity vs the concentration of ATP wasclearly sigmoidal in the absence of GTP, nHvarying from 1.7 for S. aureus), 2.0 for B.subtilis and 2.5 for S. pneumoniae. In thepresence of GTP the cooperativity indexdecreased to almost 1.00, and the K0.5 for ATPdecreased by a factor of 3 for S. aureus and 8for B. subtilis or S. pneumoniae. At saturating

concentrations of ATP, the Vm measured weresimilar with or without GTP (Table IV), inagreement with the previously published resultson S. pneumoniae UMP kinase (10).

Dependence of UMP kinase activity onUMP concentration - At pH 7.4 and over, theactivity of E. coli UMP kinase with UMP asvariable substrate exhibited a biphasicbehaviour (1), (9), which was best fitted by theequation v = Vm UMP /(UMP + Km

UMP +UMP2/KI). In the presence of GTP the plot ofactivity vs the concentration of nucleotidemonophosphate became hyperbolic. Thecalculated Vm values using the two differentplots are closely similar, suggesting that themajor effect of GTP on E. coli UMP kinasewith UMP as variable substrate is the reversalof the inhibition caused by excess of nucleosidemonophosphate. The H. influenzae and N.meningitidis UMP kinases exhibited similarproperties, nevertheless for the latter enzyme,the GTP increased also significantly the Vm

(Table V). The “Vm” effect of GTP on N .m e n i n g i t i d i s UMP kinase was alsodemonstrated by measuring its activity atseveral constant concentrations of ATP and atvariable concentrations of UMP both in theabsence and the presence of GTP. Thecalculated Vm

UMP from each individualexperiments was plotted as a function of ATPconcentration (Fig. 2A). The resulting constants(Vm

ATP, UMP, KmATP) were 51.9 U/mg protein and

4.7 mM in the absence of GTP and respectively130 U/mg protein and 1.7 mM in its presence.

At saturating concentrations of ATP orin the presence of GTP, the Gram-positive B.sub t i l i s and S. pneumoniae UMP kinasesexhibited hyperbolic dependence of activitywith UMP as variable substrate. In the absenceof activator and at concentrations of ATPbelow K0.5, an inhibition by excess of UMP wasalso observed (Table V). Moreover, the Km forUMP increased significantly from low tosaturating concentrations of ATP, or in thepresence of GTP, suggesting a complexrelationship between the substrates and theregulatory nucleotide. To minimize the effectof one substrate or effector on the kineticparameters of the second substrate, the activityof B. subtilis UMP kinase was determined asfor the UMP kinase of N. meningitidis atseveral constant concentrations of ATP, and atvariable concentrations of UMP both in theabsence and the presence of saturating

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concentrations of GTP. Each individual serieswas fitted by the equation (2) and the calculatedVm

UMP (Fig. 2B) used for secondary plots withATP as variable substrate. The resultingconstants (Vm

ATP,UMP, K0.5ATP and nH) were 68

U/mg protein, 21 mM and 1.8 in the absence ofGTP and respectively 58 U/mg protein, 2.9 mMand 1.1 in its presence. The kinetics of S .aureus UMP kinase with UMP as variablesubstrate was hyperbolic both in the presenceor absence of GTP (data not shown).

Specificity of bacterial UMP kinasesfor GTP as activator – GTP appeared to be thecommon positive effector for all investigatedbacterial UMP kinases (Tables III, IV and V).The concentration of nucleotide required forhalf-maximal activation (Ka) was independenton the concentration of Mg2+ ions. At singleconcentration of substrates (2 mM ATP and 1mM UMP), the Ka of UMP kinases from E.co l i , H. influenzae, B. sub t i l i s , and S.pneumoniae varied between 70 and 120 µM.The N. meningitidis UMP kinase exhibited ahigher Ka for GTP (about 300 µM). When otherguanine nucleotides or related compounds, suchas dGTP, 7-deaza-dGTP, 3’-anthraniloyl-dGTP(Ant-dGTP), guanosine 5’-(β , γ-imido)triphosphate (GMP-PNP), inosine 5’-triphosphate (ITP), xanthosine 5’-triphosphate,(XTP), were tested as activators, a variety ofeffects were observed (data not shown). Thus,GMP-PNP and dGTP activated all forms ofUMP kinases but to a variable extent andaffinity as compared to GTP. The N .meningitidis UMP kinase was less sensitive tothis activation by GMP-PNP than the otherenzymes. GMP was ineffective on the UMPkinase from B. subtilis, S. pneumoniae or N.meningitidis, but did activate H. influenzae orE. coli UMP kinases. Ant-dGTP, a fluorescentanalogue of dGTP (9), was the strongestactivator of B. subtilis and S. pneumoniae UMPkinase with a four fold lower Ka than GTP butwas less effective on UMP kinase from theGram-negative organisms (data not shown).

Inhibition of UMP kinase activity byUTP - One of the earliest observations on E.coli UMP kinase, which made unique thisenzyme among the other NMP kinases, was theinhibition by UTP, and its reversal by GTP, orhigh concentrations of MgCl2 (1). These resultssuggested that the true inhibitor of bacterialenzyme was the Mg-free UTP and that GTPacted as antagonist of the former nucleotide. On

the other hand, high concentrations of UMPpartly protected the enzyme against inhibitionby UTP (2). These observations wereconfirmed on UMP kinase from N. meningitidis(Fig. 3A) or H. influenzae (not shown). The I50value for the inhibition by Mg-free UTP of N.meningitidis enzyme at 0.05 mM UMP was 10µM. A forty-fold increase of UMPconcentration shifted the I50 to 130 µM Mg-freeUTP. Under the same experimental conditionsthe inhibition of B. subtilis (Fig. 3B) or S .pneumoniae (not shown) UMP kinases by UTPwas very little affected by high concentrationsof UMP or Mg2+ ions.

To better understand these differencesbetween Gram-positive and Gram-negativespecies, the effect of UTP on individual kineticconstants was further investigated on E. coli, H.influenzae and B. subtilis UMP kinases. A firstseries of experiments was conducted at constantconcentrations of ATP (around the Km or K0.5values of individual enzymes) and UTP and atvariable concentrations of UMP (Fig. 4A, Band C). In the case of E. coli and H. influenzaeUMP kinases the curves converged at highconcentrations of UMP (Fig. 4A and B), inaccordance with the observed protective effectagainst inhibition by UTP of high UMPconcentrations (2). Until a 0.1 mMconcentration of UTP, i.e approximately 4.5µM Mg-free nucleotide, the apparent Km forUMP was almost unchanged. Over thisconcentration, a dramatic increase in theapparent Km for UMP was observed whichsuggested a strongly cooperative effect. Thus,in the presence of 0.25 mM or 0.50 mM of UTPthe apparent Km for UMP of E. coli UMPkinase increased by a factor of 6 or 34respectively. On the other hand, the inhibitionby an excess of UMP declined at highconcentrations of UTP as indicated by theincrease of the KI value. The cooperativity ofinhibition by UTP of UMP kinases from Gram-negative organisms was expressedquantitatively by an equation similar to thatdescribing the competitive inhibition: K’m=Km(1+[UTP]n/Kn

UTP). Km and K’m are theapparent Km

UMP in the absence or in thepresence of a given concentration of UTP, “n”is the cooperativity index and KUTP is a constantwhich corresponds to the concentration of UTPdoubling the apparent Km

UMP. Transformed in alinear form, log10 (K’m/Km - 1) = n.log10[UTP]

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- log10KnUTP this equation allows calculation of

the two constants. From the example describedin Fig. 4A for E. coli UMP kinase n = 2.7 andKUTP = 140 µM (i.e 6.3 µM in terms of Mg-freeUTP). It is obvious that for “n” equal or closeto unity we return to the common figure of“non-cooperative” competitive inhibition.

In the case of B. subtilis UMP kinase,the different curves obtained with UMP as thevariable substrate, evolved in parallel with onlya slight inhibition by an excess of UMP. Theapparent Vm a n d Km

U M P decreasedsimultaneously, while their ratio remainedalmost constant (Fig. 4C). No increase in theapparent Km

UMP was noticed even at thestrongest inhibitory concentrations of UTP.When ATP was the variable substrate, theinhibition by UTP resulted in the increase in theapparent Km or K0.5 for ATP. Thus, in thepresence of 0.5 mM UTP the apparent Km orK0.5 for ATP increased by a factor of 4 for E.coli, a factor of 3.2 for H. influenzae and afactor of 3.1 for B. subtilis UMP kinases (Fig.4D, E and F). GTP in excess over UTP restoredthe kinetic parameters of bacterial UMP kinasesto the values observed in the absence of UTP(Table VI).

Among the UTP analogues tested asinhibitors, dUTP was five times weaker thanthe corresponding ribonucleotide, whereasTTP, even in mM range of concentration, wascompletely ineffective. 5F-UTP mimicked theeffect of UTP with similar (E. coli, H.i n f l u e n z a e ) or lower (B. subtilis , S.pneumoniae ) affinity than the referencenucleotide (Fig. 5). 5Br-UTP and 5I-UTPexhibited the most interesting effects. Thus, B.subtilis and S. pneumoniae UMP kinases were5 to 10 times more sensitive to thesenucleotides than to the parent nucleotidewhereas E. coli and H. influenzae UMP kinaseswere much less sensitive to the inhibition by5Br-UTP and 5I-UTP (Fig. 5). It should also bementioned that the correspondingmonophosphates (5Br-UMP and 5I-UMP),unlike 5F-UMP (9) were not substrates ofbacterial UMP kinases.

UMP kinase activity in the reversereaction – An essential condition in achievingmeaningful quantitative data in the reversereaction was to maintain “controlled”concentrations of different nucleotide species,while varying one single nucleotide. Weassumed that ADP, UDP as well as GDP form

complexes with MgCl2 with similar Kd values,i.e. 1 mM (18). When GDP substitutedefficiently GTP or GMP-PNP as activator ofUMP kinase in the reverse reaction, we usedmixtures of these three nucleotides, andadjusted the concentration of MgCl2 accordingto the relationship indicated in Materials andMethods. Both N. meningitidis (Fig. 6A) and B.subt i l is (Fig. 6B) UMP kinases exhibitedbiphasic kinetics with Mg-free UDP as variablesubstrate. The apparent Km for the nucleotide inthe absence of activators was 4 µM (B. subtilis)and respectively 3.4 µM (N. meningitidis).GDP or GMP-PNP increased considerably thereaction rates, reversing almost completely theinhibition caused by an excess of Mg-freeUDP. As in the forward reaction GDP or GMP-PNP also increased the apparent Km for Mg-free UDP to 8.5 µM (N. meningitidis) andrespectively 24.1 µM (B. subtilis). In theabsence of GMP-PNP, the activity of B. subtilisUMP kinase with Mg-ADP as variablesubstrate was very low even at the highestconcentrations of nucleoside diphosphate (Fig.6C). The major effect of GMP-PNP on thereverse reaction rate was apparently to reversethe inhibition exhibited by both Mg-free andMg-complexed form of UDP and consequentlyto increase the affinity for MgADP.

Site-directed mutagenesis experiments -Structure analysis of E. coli UMP kinaseindicated that the vicinal amino acid residuesT138 and N140 are involved in the cross-talkbetween two adjacent dimers in the hexamericstructure (7). The main chain oxygen of T138from one subunit is hydrogen bonded with theside chain nitrogen of N140 from the neighboursubunit. The two residues also interact with thebase moiety of UMP. As expected the T138Aand N140A variants of E. coli UMP kinaseexhibited a much lower thermodynamicstability than the reference protein (7).Substituting T138, the side chain of which is H-bonded to uracil, results in a four times higherKm for UMP. In contrast, this Km is not alteredby the N140A substitution, as this residue onlybinds uracil throught its main chain carbonyl.The two single-residue mutations induced amoderate loss of sensitivity to inhibition byUTP (7). However, the cooperativity of thisinhibition appears significantly altered. Thus,the cooperativity index of the N140A variant ofE. coli UMP kinase declined to 1.5 and KUTPincreased to 300 µM. As in the case of

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reference enzyme, GTP restored the kineticconstants of UTP-inhibited N140A variant tothe values observed in the absence of UTP(Table VI).

Since T138 and N140 of E. coli UMPkinase were conserved as T135 and N137 in B.subtilis enzyme we investigated the kineticproperties of the similar variants obtained bysite-directed mutagenesis experiments. Allthree modified variants (T135A, N137A andthe T135A/N137A double mutant) of B. subtilisUMP kinase exhibited Tm values 10°C lowerthan the wild-type protein. The double mutantT135A/N137A was also the most affected in itsstability, since upon dilution in 50 mM Tris-HCl (pH 7.4) it was irreversibly inactivatedwithin several hours. The major kinetic changes(Table VII) are the following: a) loss ofcooperativity with ATP as variable substrate;all modified variants of B. subtilis UMP kinaseexhibited hyperbolic dependence of activityeither in the absence or in the presence of GTP;b) significant increase of the Km for UMP ofthe T135A variant in comparison with the wild-type enzyme or the N137A variant; and c) bothT135A and N137A variants of B. subtilis UMPkinase were still sensitive to activation by GTP,with a three-fold increase of Ka for activator ascompared to the wild-type enzyme.

DISCUSSION

Bacterial UMP kinases, an originalfamily of catalysts - Bacterial UMP kinases areunique members of NMP kinase family ofenzymes so far described in either prokaryoticor eukaryotic organisms: (i) the primary and thethree-dimensional structure of bacterial UMPkinases are divergent from the other studiedNMP kinases and related to the carbamate- orN-acetylglutamate kinases (1,3); (ii) bacterialUMP kinases are oligomers submitted to acomplex control of activity by GTP and UTP;(iii) the membrane proximity of UMP kinasesfrom E. coli (21) and B. subtilis (22) and mostprobably for all other bacterial species suggestsa specific role of these enzymes in the synthesisof membrane or cell-wall constituents. The cooperative kinetics with respect toATP of UMP kinase from S. pneumoniae (10),a Gram-positive organism, shed a new light onthis family of catalysts and prompted us toexplore or re-examine other UMP kinases fromeither Gram-positive and Gram-negative

bacteria. Our results showed that bacterialUMP kinases indeed can be classified in twosubfamilies, with significantly differentregulatory mechanisms. This is not anunprecedented case as, for instance, aspartatetranscarbamoylase (ATCase) from E. coli, theparadigm of allosteric enzymes, exhibits bothhomotropic and heterotropic interactions (23),whereas B. subtilis ATCase, a homotrimer,lacks both homotropic and heterotropicinteractions (24). Finding the structural basis ofthese differences in UMP kinases anddeciphering the mechanism of regulation is achallenging issue. For this purpose a selectionof several representative UMP kinases, somebelonging also to pathogenic strains forhumans, was a necessary step.

Common properties of UMP kinasesfrom Gram-positive and Gram-negativebacteria - Despite the diversity of responses tonucleotides, acting as substrates or effectors,the UMP kinases from Gram-negative andGram-positive bacteria share several commontraits: (i) GTP is the common positive effectorfor all explored enzymes. It reverses theinhibition of an excess of UMP (forward) orUDP (reverse) and increases the affinity forATP (forward) or ADP (reverse); (ii) UTP hasan opposite effect by decreasing the affinity forATP/ADP. Whereas in Gram-positiveorganisms the inhibition by UTP is independenton Mg2+ ions, in Gram-negative organisms, theinhibition by UTP occurs only via Mg-freenucleotide.

The inhibition caused by an excess ofUMP is variable from one enzyme to another,and might depend on pH, the concentration ofco-substrate or the presence of GTP. At pH 6,the inhibition by an excess of UMP was lessapparent or abolished for most examined UMPkinases. At saturating concentration of ATP, B.subti l is UMP kinase was insensitive toinhibition by an excess of UMP contrary to E.col i or H. influenzae UMP kinase. GTPreversed in all cases the inhibition by an excessof UMP. Since the inhibition by an excess ofnucleoside monophosphate was also observedwith other NMP kinases such as E. coliadenylate kinase (25,26) and CMP kinase (27),or yeast GMP kinase (28), several commoncauses might be invoked to explain thisphenomenon. Binding of UMP to the MgATPsite is excluded as inhibition is not competitivewith MgATP. Binding of UMP to the allostericsite also seems less probable as isothermal

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calorimetry (C. T. Craescu et al, unpublisheddata) showed that UMP binds to a single site ofE. coli and H. influenzae UMP kinases. Themost probable explanation would be theoccurrence of an abortive UMP kinase-MgADP-UMP complex, which slows down therelease of MgADP. Whatever the trueexplanation, the inhibition by an excess ofUMP of the bacterial UMP kinases alsodepends on their quaternary structure asdemonstrated by site-directed mutagenesisexperiments on E. coli (7) and on B. subtilis(this study) UMP kinases.

Another property common to variousUMP kinases (E. coli appears as an exception)is that activation by GTP occurs also by adecrease of Km or K0.5 for ATP. In other words,the positive effector acts simultaneously on thekinetic constants of both nucleotide substrates,irrespective of the cooperativity or non-cooperativity existing towards the phosphatedonor. As a corollary, the complex kineticeffects exhibited by the negative effector, UTP,i.e. a significant increase of the apparent Km orK0.5 for ATP and a change of the apparent Kmfor UMP was not surprising. The fact that atconcentrations of UTP over 0.1 mM theapparent Km for UMP of E. coli and H .influenzae UMP kinases increased dramaticallyis related to the cooperative binding of UTP toits site, which is consistent with thefluorescence properties of the E. coli UMPkinase - UTP complex (1,6).

Differences between UMP kinases fromGram-positive and Gram-negative organisms -The major difference between UMP kinasesfrom Gram-negative and Gram-positiveorganisms is the lack of cooperativity towardsATP in the former organisms. Although withH. influenzae UMP kinase the best fittings ofreaction rates with ATP as variable substratewere obtained using the Hill equation, the nHvalues never exceeded 1.3. On the other hand,the activation of Gram-negative N. meningitidisUMP kinase by GTP is a combination ofseveral effects: enhancement of Vm, increase inaffinity for ATP and reversal of the inhibitionby excess of UMP. In this respect, it is worthmentionning that cooperativity in allostericenzymes is mediated via changes in affinity forsubstrates (K systems) or via changes in themaximum velocity (V systems) (29). UMPkinases from Gram-positive organisms belongclearly to the K systems, i.e. both T and R

states have the same Vm values, but differentaffinities for ATP. In the absence of effectorsthe binding of ATP is cooperative, and thepositive homotropic interaction is lowered inthe presence of GTP or its analogs (10). Afactor, which might contribute to thecooperativity towards ATP of UMP kinasefrom Gram-positive bacteria might be thedissociation of active hexamers into lowermolecular mass oligomers. Such reversibledissociation of hexamers was never observedwith E. coli or H. influenzae UMP kinases.

Another major difference betweenUMP kinases from Gram-positive and Gram-negative organisms is related to their sensitivityto inhibition by UTP and its halogenatedanalogs. In Gram-positive bacteria theinhibition by UTP is not sensitive to highconcentrations of Mg2+ or UMP, whereas inGram-negative organisms the inhibition byUTP is reversed by high concentrations ofdivalent ion or UMP. On the other hand, thehalogen substituted UTP analogues expressedstrikingly different effects on UMP kinase fromGram-positive and Gram-negative organisms,suggesting that they interact with different sitesin UMP kinase from these two families ofbacteria.

The identity of the effector bindingsite(s) and the mechanism of regulation ofbacterial UMP kinases - The existence of eithera unique or two distinct binding sites for GTPand UTP, was raised at the very beginning ofour study of bacterial UMP kinases (1). Fromthe kinetic experiments described in this work,corroborated with previous spectroscopic andsite-directed mutagenesis experiments (2) (6),and the X-ray analysis of E. coli UMP kinase incomplex with GTP (Briozzo et al., unpublisheddata) we can confidently assume that eachsubunit of bacterial UMP kinase, irrespective ofits origin, has three distinct nucleotide bindingsites. The fundamental difference betweenGram-positive and Gram-negative organisms isrelated to the occupancy of these sites bynucleotides and their corresponding analogs.Two of these sites conserved throughoutdifferent bacterial species belong to thecatalytic center. They interact with ATP orADP, as either Mg-complexes or Mg-freenucleotides, and with UMP or UDP, only intheir Mg-free species. The third site, lessconserved than the previous ones, interactsprimarily with GTP, either as Mg-free or Mg-

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complexed species, and in some particularcases with GDP, GMP and even cGMP orguanosine (1). The GTP binding site, located atthe interface of two vicinal monomers, is mostprobably common to all bacterial speciessensitive to activation by guanine nucleotidesand/or related analogs. Comparison of a E. coliUMP kinase-GTP complex with otherstructurally known bacterial UMP kinases (H.influenzae, S. pyogenes) indicates an identicalfold and distribution of amino acid residuescritical for binding of this effector (Briozzo etal ., unpublished data). In Gram-positiveorganisms, the GTP binding site corresponds tothe single allosteric site, commonly describedfor the vast majority of regulatory proteins. Itcan, therefore, be designed as “GTP/UTP site”or “effector site”. Binding of effectors to thissite shifts the T R equilibrium either to the R-form (GTP) or T-form (UTP), i.e. either to the“high-” or “low-affinity” forms for ATP. Thebulky substituents (Br and I) in the UTPheterocycle can be accommodated in therelatively large GTP binding pocket with aneven better affinity than the natural nucleotide.TTP, unlike its closest derivative dUTP, doesnot inhibit the UMP kinase activity indicatingthat the hydrophobic methyl group in theposition 5 of the pyrimidine ring precludesbinding to the allosteric site.

In Gram-negative organisms, GTP andUTP, although mutually exclusive, bind todifferent sites. The allosteric regulation impliesa conformational adjustment caused by aneffector, which affects indirectly the bindingability for the other effector. The binding site ofUTP in Gram-negative bacteria overlaps theUMP/UDP site and part of the ATP/ADP site,as indicated by the X-ray data of E. coli UMPkinase in complex with UTP (7). This explainsalso why UTP increases simultaneously andcooperatively the apparent Km for bothnucleotide substrates. Among the UTP analogs,only 5F-UTP satisfies the structuralrequirements to fit to the catalytic site and tosubstitute with similar efficiency the naturalnucleotide. The fact that 5Br-UTP and 5I-UTPare still inhibitors of E. coli and H. influenzaeUMP kinases might be explained by their“promiscuous” interaction with the GTPbinding site as suggested by fluorescenceexperiments conducted with H. influenzae

UMP kinase (Evrin, PhD thesis, November2006).

Physiological relevance of regulatoryeffects of GTP and UTP on bacterial UMPkinases - UMP kinase is first of the threeenzymes involved in the conversion of UMP toUTP and CTP, the last two being NDP kinaseand CTP synthetase. Both UMP kinase andCTP synthetase are oligomeric proteinspositively regulated by GTP and usingnucleotides as substrates (1,30-32). Thestructural and functional complexity of thesetwo bacterial enzymes and the inhibition oftheir activity by the end products UTP andrespectively CTP indicate that they might besubmitted also in vivo to a closely similarcontrol of activity by nucleotides and Mg2+

ions. Assuming that in bacteria like E. coli andB. subtilis the concentration of ATP, GTP, UTPand UMP oscillate around 2-3 mM (ATP), 0.8-1.2 mM (GTP and UTP) and 0.050-0.1 mM(UMP (33,34) and that the concentration ofsoluble Mg2+ is around 15 mM (35,36) wemight speculate about a role of thesenucleotides in modulating the activity ofindividual UMP kinases. In the case of Gram-positive bacteria, whose UMP kinases exhibitlow affinity for ATP in the absence of GTP, itis obvious that the latter nucleotide is a majorplayer besides the two substrates. Once theUTP pool is saturated it competes with GTP forthe allosteric site lowering the UMP kinaseactivity. In Gram-negative organisms thesituation appears different. Thus, in bacterialike H. influenzae or N. meningitidis the Km forATP of the corresponding UMP kinases is ofthe same order of magnitude as the cellularconcentration of this nucleotide. Consequentlythe cooperative inhibition by Mg-free UTP over10 µM might be physiologically relevant. Therole of GTP would be rather compensating as“antagonist of the inhibitor”. E. coli hasapparently the most “buffered” UMP kinasesystem, the enzyme operating always undersaturating concentrations of ATP (ten times thecorresponding Km). One of the future tasksbeside the precise identification of the allostericsite of UMP kinase from Gram-positiveorganisms will be to determine the in vivocoupling of the UMP kinase and CTPsynthetase activities, as much as both enzymesrepresent valuable targets for antibacterialagents.

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FOOTNOTES

*We thank Pierre Briozzo (INRA Paris-Grignon) for constructive criticisms and for providing us withmanuscript ahead of publication, Yves Janin for carefully reading this manuscript and helpfulsuggestions, Cristina Gagyi and Ovidiu Sirbu, for participation in the preliminary experiments andJean-Claude Rousselle and Pascal Lenormand for mass spectrometry measurements. This work wassupported by grants from the Centre National de la Recherche Scientifique (URA 2185 and URA2171), Institut Pasteur (ACO2), the Institut National de la Recherche Agronomique (UMR206) andAstraZeneca R & D, Boston, Inc.

1The abbreviations used are : ATCase, aspartate transcarbamoylase ; 5Br-UTP, 5-Bromo-uridine-5’-triphosphate ; 5F-UTP, 5-Fluoro-uridine-5’-triphophate ; GMP-PNP, guanosine 5’-(β , γ-imido)-triphosphate ; 5I-UTP, 5-Iodo-uridine-5’-triphosphate ; NDP, nucleoside 5’-diphosphate ; NMP,nucleoside 5’-monophosphate ; NTP, nucleoside 5’-triphosphate.

FIGURE LEGENDS

Fig. 1. Sequence alignment of eight bacterial UMP kinases explored in this work and belonging toGram-negative (E. coli, S. typhimurium, H. influenzae and N. meningitidis) and Gram-positive (B.subtilis, S. pneumoniae, S. aureus and E. faecalis) organisms. Conserved residues are framed in grey.The residues deduced in E. coli UMP kinase interacting with different nucleotides are labelled inyellow (ATP), blue (UMP) or magenta (GTP). Asterisks on the top of E. coli UMP kinase sequenceindicated residues modified by site-directed mutagenesis, either in the past or in the present work.

Fig. 2. Secondary plot illustrating the dependence of N. meningitidis (A) and B. subtilis (B) UMPkinase activity, on the concentration of ATP in the absence () or the presence () of 0.5 mM GTP.The primary plots were obtained at several constant concentrations of ATP (between 0.5 and 12 mMfor N. meningitidis UMP kinase and between 0.2 and 30 mM for B. subtilis UMP kinase) and variableconcentrations of UMP. The calculated kinetic constants are indicated in the text. Notice the “V”effect of GTP on N. meningitidis UMP kinase and the “K” effect on B. subtilis UMP kinase

Fig. 3. Inhibition of N. meningitidis (A) and B. subtilis (B) UMP kinases by variable concentrations ofUTP at constant concentrations of ATP (2 mM), UMP and MgCl2. ( ) 0.05 mM UMP and 4 mMMgCl2; () 0.05 mM UMP and 20 mM MgCl2; () 2 mM UMP and 4 mM MgCl2.

Fig. 4. Inhibition of E. coli (A and D), H. influenzae (B and E) and B. subtilis (C and F) UMP kinasesby UTP at variable concentrations of UMP or ATP. When constant, the concentration of ATP was 0.2mM (A), 2 mM (B) and 16 mM (C). In D, E, and F, the concentration of UMP was always 0.3 mM.

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() no UTP; () 0.05 mM UTP; () 0.1 mM UTP; () 0.2 mM UTP; () 0.25 mM UTP; () 0.5mM UTP.

Fig. 5. Comparative inhibitory effects of UTP and its 5 halogenated analogs on H. influenzae (A) andB. subtilis (B) UMP kinases at constant concentrations of ATP (2 mM in A; 15 mM in B) and UMP(0.1 mM in A; 0.3 mM in B). () UTP; () 5F-UTP; () 5Br-UTP; () 5I-UTP.

Fig. 6. Dependence of N. meningitidis (A) and of B. subtilis (B and C) UMP kinase activity in thereverse reaction on Mg-free UDP and MgADP concentrations. The experimental conditions aredescribed under Materials and Methods. The activator in A is GDP and in B and C is GMP-PNP. ()no activator; () 0.2 mM activator; () 1 mM activator.

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Table I. Primers used for cloning the various pyrH genes.

Organism Flanking primers Restriction sites Ref.

E. coli 5’-GGAATTCCATATGGCTACCAATGCAAAACCCGT-3’5’-CGGCGCTCGAGTTATTCCGTGATTAAAGTCCCTTCT-3’ NdeI and XhoI (1)

S. typhimurium 5’-GGGGCATATGGCTACCAATGCAAAACCCGTCTAT-3’5’-CCCCAAGCTTATTCCGTGATTAACGTCCCTTCTTTTTCGCC-3’ NdeI and HindIII This study

H. influenzae 5’-CCCCCCGGGATGAGCCAACCAATTTATAAACGT-3’5’-GGAATTCTAACAAATAGTGGTGCCTTCTT-3’ NdeI and EcoRI This study

N. meningitidis 5' - GGGATCTCATATGACACAGCAAATCAAATACAAAC-3'5' - GGATAAGCTTTCAGCAGTGAACCAGCGTTCC-3' NdeI and HindIII This study

B. subtilis 5’-GGGGCATATGGAAAACCAAAATACAAACGTATCGTATTA-3’5’-CCCCCTCGATTATTTCCCCCTCACGATCGTTCCATTGATTCAC-3’ NdeI and XhoI (9)

S. pneumoniae 5'-GGAATTCCATATGAAAATGGCGAATCCCAAGTAT-3'5'-CGGCGCTCGAGTTATTCCTTTTCTTCGATATTATTTGAAA-3' NdeI and XhoI This study

S. aureus 5' -GGGATCTCATATGGCTCAAATTTCTAAATATAAAC-3'5' -GGGATAAGCTTTTATTTTGTAATTAACGTACCTATC-3' NdeI and HindIII This study

E. faecalis 5' -GGGATCTCATATGATGGTTAAACCTAAGTATCAAC-3'5' -GGCATTAAGCTTTTATTTCCCCCTTACAGTTGTTC-3' NdeI and HindIII This study

Table II. Concentration (mM) of free (ATPf, UMPf), and metal-complexed (MgATP, MgUMP)nucleotides as a function of their total concentration (ATPo, UMPo) and total concentrations of MgCl2(Mgo)a.

ATPo UMPo Mgo MgATP MgUMP ATPf UMPf Mgf

0.2 0.1 2.2 0.1905 0.0091 0.0095 0.0909 2.0004

1.0 0.1 3.0 0.9532 0.0092 0.0468 0.0908 2.0375

5.0 0.1 7.0 4.7833 0.0099 0.2167 0.0901 2.2068

25.0 0.1 27.0 24.1492 0.0124 0.8508 0.0876 2.8384

0.2 2.0 2.2 0.1897 0.1686 0.0103 1.8314 1.8417

1.0 2.0 3.0 0.9495 0.1717 0.0505 1.8283 1.8788

5.0 2.0 7.0 4.7671 0.1857 0.2329 1.8143 2.0472

25.0 2.0 27.0 24.0968 0.2354 0.9032 1.7646 2.6678a The dissociation constants (Kd) of MgATP and MgUMP complexes (0.1 mM and respectively 20 mM)were taken from Alberty (18,19) and assuming that only the phosphate chain contributes to the strengthof the metal-nucleotide complex.

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Table III. UMP kinase activity of eight different bacterial strains in the absence and presence of GTP (0.5 mM)at a single concentration of UMP (1 mM) and two concentrations of ATP. The reaction rates are expressed asU/mg of protein.

2 mM ATP 8 mM ATPMicroorganism

- GTP + GTP ratio - GTP + GTP ratio

E. coli 26.1 65.9 2.52 28.1 71.1 2.53

S. typhimurium 46.8 134.3 2.87 47.5 124.2 2.61

H. influenzae 9.1 52.8 5.80 11.8 60.5 5.73

N. meningitidis 5.1 57.7 11.30 10.2 103.1 10.11

B. subtilis 2.7 28.6 10.60 18.1 39.1 2.16

S. pneumoniae 3.7 68.5 18.50 38.9 87.4 2.24

S.aureus 4.6 23.5 5.10 12.8 23.6 1.84

E. faecalis 1.7 9.7 5.71 9.1 19.4 2.13

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Table IV. Kinetic parameters of UMP kinase from three Gram-negative and three Gram-positive organisms withATP as variable substrate and fixed concentration of UMP.

No GTP 0.5 mM GTPSource of

UMP kinaseUMP as fixedsubstrate (mM)

Vm Km or K0.5 nH Vm Km or K0.5 nH

0.1 66.5 ± 1.5 0.23 ± 0.02 - 88.3 ± 1.7 0.27 ± 0.02 -E. coli

1.0 42.6 ± 1.3 0.15 ± 0.02 - 100 ± 1.8 0.20 ± 0.02 -

0.05 35.3 ± 1.7 2.98 ± 0.41 - 66 ± 3.3 0.83 ± 0.18 -N. meningitidis

1.0 16.2 ± 0.6 3.22 ± 0.36 - 125 ± 6.4 1.98 ± 0.39 -

0.1 56.9 ± 1.7 1.57 ± 0.12 1.28 ± 0.09 64.2 ± 1.2 0.46 ± 0.03 1.19 ± 0.13H. influenzae

1.0 30.1 ± 0.9 1.62 ± 0.11 1.30 ± 0.10 96.6 ± 1.7 0.60 ± 0.03 1.10 ± 0.006

0.1 18.2 ± 3.3 10.4 ± 2.7 1.65 ± 0.2 22.8 ± 1.1 0.93 ± 0.13 1.08 ± 0.13B. subtilis

1.0 29.5 ± 4.5 14.5 ± 1.9 2.04 ± 0.32 34.0 ± 0.83 1.79 ± 0.11 1.14 ± 0.07

S. pneumoniae 1.0 57.7 ± 2.8 9.8 ± 0.6 2.55 ± 0.30 76.0 ± 1.3 0.92 ± 0.04 1.40 ± 0.09

S. aureus 1.0 38.6 ± 2.6 2.64 ± 0.16 1.74 ± 0.28 38.2 ± 2.2 0.90 ± 0.22 1.06 ± 0.05

The reaction rates were fitted according to the Michaelis-Menten equation (v= Vm [ATP]/Km +[ATP]) or the Hill equation(v=Vm [ATP]n/K0.5

n+[ATP]n), where Vm is the maximal rate (µmol/min.mg of prot.), Km is the Michaelis-Menten constant (mM), nH the Hillnumber, and K0.5 the ATP concentration (mM) at half-maximal activity.

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Table V. Kinetic parameters of UMP kinase from three Gram-negative and two Gram-positiveorganisms with UMP as variable substrate and fixed concentration of ATP.

No GTP 0.5 mM GTPSource of

UMP kinaseATP as fixed

substrate (mM)Vm Km KI Vm Km

0.2 45.9 ± 3.6 49.2 ± 8.6 0.60 ± 0.04 62.2 ± 1.4 47.0 ± 3.2E. coli

2.0 92.5 ± 1.9 46.0 ± 4.2 0.44 ± 0.03 100.3 ± 2.2 51.0 ± 4.4

2.0 15.7 ± 3.3 8.7 ± 4.7 0.11 ± 0.05 46.4 ± 2.1 10.0 ± 2.8N. meningitidis

10.0 35.1 ± 5.6 15.6 ± 6.7 0.23 ± 0.08 110.0 ± 2.4 57.4 ± 4.7

1.0 50.8 ± 6.4 40.0 ± 10 0.31 ± 0.08 61.8 ± 0.5 40.0 ± 1.0H. influenzae

12.0 109.2 ± 15.5 100.0 ± 20 0.17 ± 0.04 75.9 ± 1.0 50.0 ± 2.0

2.0 3.2 ± 0.1 22.2 ± 2.9 4.40 ± 0.98 69.3 ± 0.94 99.7 ± 5.6S. pneumoniae

30.0 59.1 ± 1.3 150.0 ± 12.2 No inhibition 80.1 ± 2.4 105.6 ± 12.5

2.0 5.3 ± 0.4 10.0 ± 2.1 0.66 ± 0.13 27.6 ± 0.4 27.4 ± 1.9B. subtilis

30.0 41.8 ± 1.0 131.0 ± 13.0 No inhibition 52.0 ± 1.4 155.0 ± 14.5

The reaction rates were fitted according to the Michaelis-Menten equation or to the equation v=Vm [UMP](Km

UMP+[UMP]+[UMP]2/KI), where Vm is the maximal reaction rate (µmol/min.mg of prot.), Km is the Michaelis-Menten constant (µM) and KI corresponds to the inhibition constant (mM).

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Table VI. Reversal by GTP of the inhibition caused by UTP on E. coli UMP kinaseConstant ATP (0.2 mM),

variable UMPConstant UMP (0.3 mM),

variable ATPEnzyme Effector(0.5 mM) Vm

(U/mg prot)Km UMP

(µM)Vm

(U/mg prot)Km

ATP

(mM)Reference(D159N) - 46.1 50.0 62.4 0.21

GTP 51.9 59.3 99.6 0.24

UTP 36.8 1690 23.8 0.77

GTP+UTP 46.6 207 91.7 0.26

N140Avariant - 48.9 50.0 81.9 0.25

GTP 58.2 73.9 110.5 0.21

UTP 30.4 181.2 78.3 0.53

GTP+UTP 55.7 108.6 98.2 0.23The results are mean value of two separate experiments.

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Table VII. Kinetic parameters of three modified forms of B. subtilis UMP kinase obtained by site-directedmutagenesis

Experimentalconditions Parameters Wild-type T135A N137A T135A/N137A

Vm (U/mg) 29.5 ± 4.5 4.9 ± 0.2 3.0 ± 0.3 5.4 ± 1.5

K0.5 (mM) 14.5 ± 1.9 9.5 ±0.8 10.5 ± 2.1 10.0 ± 1.3

UMP fixedsubstrate(1 mM), no GTP

nH 2.04 ± 0.31 No cooperativity No cooperativity No cooperativity

Vm (U/mg) 34.0 ± 0.83 19.0 ± 0.6 14.7 ± 0.4 11.3 ± 0.5

K0.5 (mM) 1.79 ± 0.11 1.20 ± 0.2 1.0 ± 0.1 2.2 ± 0.4

UMP fixedsubstrate (1mM), 0.5 mMGTPa nH 1.14 ± 0.07 No cooperativity No cooperativity No cooperativity

Vm (U/mg) 5.3 ± 0.4 0.88 ± 0.035 0.65 ± 0.023 1.09 ± 0.03

Km (µM) 10.0 ± 2.1 47.5 ± 8.1 12.7 ± 1.9 53.1 ± 6.5ATP fixedsubstrate (2mM), no GTP KI (mM) 0.66 ± 0.13 No inhibition 7.2 ± 2.5 No inhibition

Vm (U/mg) 27.6 ± 0.4 9.52 ± 0.11 6.75 ± 0.12 12.5 ± 0.33

Km (µM) 27 .4 ± 1.9 80. 7 ± 4.1 17.8 ± 1.6 143.0 ± 15.0

ATP fixedsubstrate (2mM), 0.5 mMGTPa KI (mM) No inhibition No inhibition No inhibition No inhibitionaAs GTP is at sub-saturating concentrations in the case of variants obtained by site-directed mutagenesis, thecorresponding Vm values were underestimated with respect to the wild-type protein.

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Figure 1

* * *UMPK-Ec MATNAKPVYKRILLKLSGEALQGTEGFGIDASILDRMAQEIKELVELGIQVGVVIGGGNLFRGAG 65UMPK-St MATNAKPVYKRILLKLSGEALQGTEGFGIDASILDRMAQEIKELVELGIQVGVVIGGGNLFRGAG 65UMPK-Hi MSQPIYKRILLKLSGEALQGEDGLGIDPAILDRMAVEIKELVEMGVEVSVVLGGGNLFRGAK 62UMPK-Nm MTQQIYKRVLLKLSGESLMGSDPFGINHDTIVQTVGEIAEVVKMGVQVGIVVGGGNIFRGVS 62UMPK-Bs MEKPKYKRIVLKLSGEALAGEQGNGINPTVIQSIAKQVKEIAELEVEVAVVVGGGNLWRGKT 62UMPK-Sp MKMANPKYKRILIKLSGEALAGERGVGIDIQTVQTIAKEIQEVHSLGIEIALVIGGGNLWRGEP 62UMPK-Sa MAQISKYKRVVLKLSGEALAGEKGFGINPVIIKSVAEQVAEVAKMDCEIAVIVGGGNIWRGKT 63UMPK-Ef KPKYQRVVLKLSGEALAGEDGFGIKPPVIKEIVQEIKEVHELGIEMAIVVGGGNIWRGQI 60

* *UMPK-Ec LAKAGMNRVVGDHMGMLATVMNGLAMRDALHRAYVNARLMSAIPLNGVCDSYSWAEAISLLRNNR 130UMPK-St LAKAGMNRVVGDHMGMLATVMNGLAMRDALHRAYVNARLMSAIPLNGVCDNYSWAEAISLLRNNR 130UMPK-Hi LAKAGMNRVVGDHMGMLATVMNGLAMRDSLFRADVNAKLMSAFQLNGICDTYNWSEAIKMLREKR 127UMPK-Nm AQAGSMDRATADYMGMMATVMNALALKDAFETLGIKARVQSALSMQQIAETYARPKAIQYLEEGK 127UMPK-Bs GSDLGMDRATADYMGMLATVMNSLALQDSLETLGIQSRVQTSIEMRQVAEPYIRRKAIRHLEKKR 127UMPK-Sp AAEAGMDRVQADYTGMLGTVMNALVMADSLQQVGVDTRVQTAIAMQQVAEPYVRGRALRHLEKGR 127UMPK-Sa GSDLGMDRGTADYMGMLATVMNALALQDSLEQLDCDTRVLTSIEMKQVAEPYIRRRAIRHLEKKR 128UMPK-Ef GAQMGMERAQADYMGMLATVMNALALQDTLENLGVPTRVQTSIEMRQIAEPYIRRRAERHLEKGR 125

* * * * * *UMPK-Ec VVILSAGTGNPFFTTDSAACLRGIEIEADVVLKATK-VDGVFTADPAKDPTATMYEQLTYSEVLE 194UMPK-St VVILSAGTGNPFFTTDSAACLRGIEIEADVVLKATK-VDGVFTADPAKDPSATMYDQLTYSEVLD 194UMPK-Hi VVIFSAGTGNPFFTTDSTACLRGIEIEADVVLKATK-VDGVYDCDPAKNPDAKLYKNLSYAEVID 191UMPK-Nm VVIFAAGTGNPFFTTDTAAALRGAEMNCDVMLKATN-VDGVYTADPKKDPSATRYETITFDEALL 191UMPK-Bs VVIFAAGTGNPYFSTDTTAALRAAEIEADVILMAKNNVDGVYNADPRKDESAVKYESLSYLDVLK 192UMPK-Sp IVIFGAGIGSPYFSTDTTAALRAAEIEADAILMAKNGVDGVYNADPKKDKTAVKFEELTHRDVIN 192UMPK-Sa VVIFAAGIGNPYFSTDTTAALRAAEVEADVILMGKNNVDGVYSADPKVNKDAVKYEHLTHIQMLQ 193UMPK-Ef VVIFAGGTGNPYFSTDTTAALRAAEVDADVILMAKNNVDGVYSADPRVDETATKFEELTHLDVIS 190

*UMPK-Ec KELKVMDLAAFTLARDHKLPIRVFNMNKPGALRRVVMGEKEGTLITE------ 241UMPK-St KELKVMDLAAFTLARDHKLPIRVFNMNKPGALRRVVMGEKEGTLITE------ 241UMPK-Hi KELKVMDLSAFTLARDHGMPIRVFNMGKPGALRQVVTGTEEGTTIC------- 237UMPK-Nm KNLKVMDATAFALCRERKLNIVVFGIAKEGSLKRVITGEDEGTLVHC------ 237UMPK-Bs DGLEVMDSTASSLCMDNDIPLIVFSIMEEGNIKRAVIGESIGTIVRGK----- 239UMPK-Sp KGLRIMDSTASTLSMDNDIDLVVFNMNQPGNIKRVVFGENIGTTVSNNIEEKE 245UMPK-Sa EGLQVMDSTASSFCMDNNIPLTVFSIMEEGNIKRAVMGEKIGTLITK------ 240UMPK-Ef KGLQVMDSTASSLSMDNDIPLVVFNLNEAGNIRRAILGENIGTTV-------- 235

Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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GillesLiliane Assairi, Mihaela Ionescu, Nicolae Palibroda, Octavian Barzu and Anne-Marie Cécile Evrin, Monica Straut, Neli Slavova-Azmanova, Nadia Bucurenci, Adrian Onu,

Gram-positive bacteriaRegulatory mechanisms differ in UMP kinases from Gram-negative and

published online January 8, 2007J. Biol. Chem. 

  10.1074/jbc.M606963200Access the most updated version of this article at doi:

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