Báo cáo khoa học: The role of Ureaplasma nucleoside monophosphate kinases in the synthesis of nucleoside triphosphates potx

The role of Ureaplasma nucleoside monophosphate kinases in the synthesis of nucleoside triphosphates Liya Wang Department of Molecular Biosciences, Swedish University of Agricultural Sciences, The Biomedical Centre, Uppsala, Sweden Mycoplasmas (Mollicutes) are wall-less bacteria and phylogenetically belong to the gram-positive bacteria. Mollicutes are pathogens affecting humans, animals and plants [1]. Ureaplasma is a human pathogen col- onizing the urogenital tract and is the most common cause of nonchlamydial nongonococcal urethritis. It has also been implicated in infertility, spontaneous abortion, stillbirth, and premature and perinatal mor- bidity and mortality [2–4]. Mollicutes, in general, have low G + C content of their genomes, and lack those genes necessary for the synthesis of precursors for DNA, RNA and pro- teins. For example, no de novo purine and pyrimi- dine biosynthesis pathway exists [1]. Of all the Mollicutes genomes sequenced to date, there is no annotated ndk gene, coding for nucleoside diphos- phate kinase, indicating that no homologue of any known ndk gene or catalytic domain is present in Mollicutes [5,6]. Nucleoside diphosphate kinase (NDK) catalyzes the final step in ribonucleoside triphosphate (NTP) and deoxynucleoside triphosphate (dNTP) biosynthesis. NDK is involved in multiple cellular processes including the control of cell growth and signalling by Keywords Mollicutes; nucleotide biosynthesis; nucleoside diphosphate kinase; nucleoside monophosphate kinase; Ureaplasma Correspondence L. Wang, Department of Molecular Biosciences, Section of Veterinary Medical Biochemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, PO Box 575, SE-751 23 Uppsala, Sweden Fax: +46 18550762 Tel: +46 184714119 E-mail: liya.wang@mbv.slu.se (Received 11 January 2007, revised 5 February 2007, accepted 14 February 2007) doi:10.1111/j.1742-4658.2007.05742.x Mollicutes are wall-less bacteria and cause various diseases in humans, ani- mals and plants. They have the smallest genomes with low G + C content and lack many genes of DNA, RNA and protein precursor biosynthesis. Nucleoside diphosphate kinase (NDK), a house-keeping enzyme that plays a critical role in the synthesis of nucleic acids precursors, i.e. NTPs and dNTPs, is absent in all the Mollicutes genomes sequenced to date. There- fore, it would be of interest to know how Mollicutes synthesize dNTPs ⁄ NTPs without NDK. To answer this question, nucleoside mono- phosphate kinases (NMPKs) from Ureaplasma were studied regarding their role in the synthesis of NTPs ⁄ dNTPs. In this work, Ureaplasma adenylate kinase, cytidylate kinase, uridylate kinase and thymidylate kinase were cloned and expressed in Escherichia coli. The recombinant enzymes were purified and characterized. These NMPKs are base specific, as indicated by their names, and capable of converting (d)NMPs directly to (d)NTPs. The catalytic rates of (d)NTPs and (d)NDP synthesis by these NMPKs were determined using tritium-labelled (d)NMPs, and the rates for (d)NDP syn- thesis, in general, were much higher (up to 100-fold) than that of (d)NTP. Equilibrium studies with adenylate kinase suggested that the rates of NTPs ⁄ dNTPs synthesis by NMPKs in vivo are probably regulated by the levels of (d)NMPs. These results strongly indicate that NMPKs could sub- stitute the NDK function in vivo. Abbreviations AdK, adenylate kinase; CMPK, cytidylate kinase; GMPK, guanylate kinase; NDK, nucleoside diphosphate kinase; NMPK, nucleoside monophosphate kinase; TMPK, thymidylate kinase; UMPK, uridylate kinase. FEBS Journal 274 (2007) 1983–1990 ª 2007 The Author Journal compilation ª 2007 FEBS 1983 providing either all the NTPs or a specific NTP such as GTP, CTP or UTP [7–9]. NDK is a highly con- served enzyme and is widely expressed. In humans, eight ndk genes have been reported and the corres- ponding enzymes have different tissue distribution and subcellular localization. NDKs are also involved in cell growth, differentiation and tumour metastasis, etc. [8,9]. Null mutations in the ndk gene of Droso- phila caused abnormalities in the development of the larvae, leading to tissue necrosis and death at the pre- pupal stage [10]. In prokaryotes such as Escherichia coli, deletion of the ndk gene led to a mutator pheno- type due to abnormal dCTP and dGTP pools [11]. However, the growth rate was not affected, suggesting that other enzymes may be able to substitute the NDK activity [12,13]. So far, no NDK activity was detected in total cell ex- tracts or the chromatographic fraction of Mycoplasma pneumoniae in an attempt to isolate the NDK enzyme [14]. Thus, the question is how do Mollicutes synthesize their dNTPs and NTPs without the NDK enzyme? Glycolytic enzymes such as pyruvate kinase and phosphoglycerate kinase have been suggested to replace the NDK activity in Mollicutes [14], and, in that study, purine nucleoside diphosphates, e.g. ADP and GDP, were converted to triphosphates with relat- ively good efficiency; however, pyrimidine nucleoside diphosphates had very low relative activity. Therefore, there must be other alternative pathways that may contribute to the synthesis of NTPs ⁄ dNTPs, especially with regard to pyrimidine nucleotides. Nucleoside monophosphate kinases (NMPK) cata- lyze the reversible phosphorylation of a nucleoside monophosphate (NMP) using a nucleoside triphos- phate as phosphate donor, i.e. N 1 MP + N 2 TP ‹fi N 1 DP + N 2 DP. Adenylate kinase (AdK) has been suggested play a role in the synthesis of dNTPs and NTPs using the reverse reaction and it was proposed that AdK was the alternative enzyme in providing nucleoside triphosphates in NDK-deficient E. coli [12,13,15]. All Mollicutes species sequenced to date possess five nucleoside monophosphate kinases, which have been assigned as AdK, thymidylate kinase (TMPK), cyti- dylate kinase (CMPK), uridylate kinase (UMPK), and guanylate kinase (GMPK). In this work, four of the Ureaplasma parvum nucleoside monophosphate kinases, i.e. adenylate kinase, thymidylate kinase, cyti- dylate kinase, and uridylate kinase, were cloned and expressed in E. coli. The recombinant enzymes were affinity purified and their role in dNTP and NTP syn- thesis was investigated. Results Ureaplasma nucleoside monophosphate kinases: cloning, expression and purification Five NMPKs have been annotated in the genome U. parvum, i.e. AdK (adk, UU251), TMPK (tmk, UU020), CMPK (cmk, UU342), UMPK (pyrH, UU513) and GMPK (gmk, UU213), based on the sequence homology with NMPKs from other organ- isms (GenBank accession number AF222894). There is no experimental data regarding the functions of these genes reported [5]. Open reading frames coding for AdK (UU251), CMPK (UU342), UMPK (UU513), and TMPK (UU020) were PCR amplified using the U. parvum genomic DNA as template and cloned into the pET- 14b vector using the Nde I and BamH I restriction sites. A 6-His tag and a thrombin cleavage site were introduced to the N-terminus of the recombinant pro- teins. Tryptophans, coded by UGA codons in UMPK and AMPK, were mutated to UGG using the site- directed mutagenesis method in order to express these proteins in E. coli. Recombinant AdK, CMPK, UMPK and TMPK were expressed in E. coli and puri- fied by metal affinity chromatography. SDS ⁄ PAGE analysis showed that the dominant band corresponded to the subunit molecular mass of each enzyme (Fig. 1). The purified enzymes were used directly in the assays without the removal of the His tag. Substrate specificity of Ureaplasma nucleoside monophosphate kinases The substrate specificities of AdK, CMPK, UMPK and TMPK were explored using a phosphoryl transfer Fig. 1. SDS ⁄ PAGE analysis of purified recombinant Ureaplasma nucleoside monophosphate kinases. Lanes 1–4, TMPK, UMPK, AdK and CMPK. MW, molecular mass markers. Ureaplasma nucleoside monophosphate kinases L. Wang 1984 FEBS Journal 274 (2007) 1983–1990 ª 2007 The Author Journal compilation ª 2007 FEBS assay with 32 P-labelled ATP as phosphate donor and natural (deoxy)ribonucleoside monophosphates as acceptors. AdK used AMP and dAMP as the preferred substrates (Fig. 2); both CMP and dCMP were effi- ciently phosphorylated by CMPK (Fig. 2). UMPK phosphorylated UMP and dUMP (Fig. 2) while TMPK phosphorylated dTMP and dUMP (Fig. 2). Products from the CMPK (with dCMP and CMP as substrates) and the TMPK (with TMP as substrate) reactions were re-analysed by TLC using a higher salt concentration to separate the other nucleotides from ATP. As shown in Fig. 3, substantial amounts of dCTP, CTP and TTP were formed in these reactions. Nucleoside di- and triphosphate synthesis catalyzed by AdK When tritium-labelled AMP was used as substrate in AdK catalyzed reactions, the reaction products, as ana- lysed by the TLC, were found to be [ 3 H]ADP and [ 3 H]ATP. [ 3 H]AMP was rapidly converted to [ 3 H]ADP, which reached a maximum after 3 min and then declined, while the formation of [ 3 H]ATP was increased linearly with time within the first 15 min. After 30 min, the reaction reached equilibrium (Fig. 4A). Further experiments were designed to study the extent of [ 3 H]ATP and [ 3 H]ADP synthesis using different phos- phate donors. The [ 3 H]ATP synthesis rates were slightly influenced by the phosphate donors used, approxi- mately two-fold; the highest rate was found with ATP as donor and the lowest with GTP as donor (Table 1). In order to measure the rate of [ 3 H]ADP synthesis, the reaction conditions was optimized, in terms of AdK concentration, so that a first order reaction was observed (Fig. 4B). As shown in Table 1, the rates of [ 3 H]ADP synthesis were approximately 40-fold higher than that of [ 3 H]ATP. As with [ 3 H]ATP synthesis, phosphate donors did not have major impact on the Fig. 2. Substrate specificities of Ureaplasma NMPKs. The reactions were performed as described in Experimental procedures and the products were separated by TLC and visualized by autoradiography. The concen- trations of nucleoside monophosphates and [c- 32 P]ATP were as follows: 0.1 mM 1. CMP; 2. dCMP; 3. AMP; 4. dAMP; 5. GMP; 6. dGMP; 7. UMP; 8. dUMP; 9. dTMP. The reaction products were marked as close to the product spot as possible. Fig. 3. Direct formation of nucleoside triphosphates in NMPK cata- lyzed reactions. Reaction mixtures 1 and 2 by CMPK and 9 by TMPK, described in Fig. 2, were reanalysed by TLC (developed using 0.32 M NaH 2 PO 4 ) and the radiolabelled nucleoside triphos- phate products were separated from ATP. L. Wang Ureaplasma nucleoside monophosphate kinases FEBS Journal 274 (2007) 1983–1990 ª 2007 The Author Journal compilation ª 2007 FEBS 1985 rates of [ 3 H]ADP synthesis, although the highest [ 3 H]ADP synthesis rate was obtained when ATP was the phosphate donor (Table 1). Reaction equilibrium for AdK In the reactions described above, the concentration of phosphate donors was 1 mm, which was 10-fold higher than that of [ 3 H]AMP; however, at equilibrium [ 3 H]ATP was the dominant product. Therefore, a ser- ies of reactions with varied ATP concentrations were carried out to study the equilibrium of labelled adenine nucleotides. As shown in Table 2, at equilibrium the concentration of [ 3 H]ATP was dependent on the con- centration of ATP used in the reaction; a higher ATP concentration yielded a higher concentration of [ 3 H]ATP. The equilibrium constants for labelled aden- ine nucleotides were lowest with highest ATP concen- tration tested (Table 2). The equilibrium constants of labelled adenine nucleo- tides with different phosphate donors at 1 mm concen- tration was two-fold lower with ATP as compared with CTP, UTP or GTP as phosphate donor (Table 3). 0 20 40 60 80 100 0 20 40 60 80 100 120 140 Nucleotide concentration (µ M ) Min ATP ADP AMP A 0 20 40 60 80 100 0 5 10 15 20 Nucleotide concentration (µ M ) Min ATP AMP ADP B Fig. 4. The synthesis of [ 3 H]ADP and [ 3 H]ATP from [ 3 H]AMP cata- lyzed by AdK. (A) The conditions for linear rate of [ 3 H]ATP synthesis were 0.2 l M AdK, 100 lM [ 3 H]AMP and 1 mM ATP ⁄ MgCl 2 in the reaction buffer, as described in experimental procedures. (B) The conditions for linear rate [ 3 H]ADP synthesis were 1.0 nM AdK, 100 l M [ 3 H]AMP and 1 mM ATP ⁄ MgCl 2 in the reaction buffer, as described in Experimental procedures. Table 1. Rate of nucleoside di- and triphosphate synthesis by Urea- plasma NMPKs (s )1 ). The concentrations of [ 3 H]AMP, [ 3 H]dCMP or [ 3 H]UMP were 100 lM, the concentrations of phosphate donors were 1 m M and the concentration of MgCl 2 was 10 mM. The values were the means of two to four measurements with < 10% varia- tion. AdK CMPK UMPK [ 3 H]ADP [ 3 H]ATP [ 3 H]dCDP [ 3 H]dCTP [ 3 H]UDP [ 3 H]UTP ATP 181.6 4.6 32.3 0.31 6.6 0.03 UTP 134.2 3.4 27.3 0.33 0.16 0.007 GTP 94.7 1.9 26.4 0.24 0.003 < 0.001 CTP 128.1 3.5 1.4 0.16 0.004 < 0.001 Table 2. The effect of ATP concentration on the equilibrium con- centration of labelled adenine nucleotides. The initial concentration of [ 3 H]AMP was 100 lM and the concentration of MgCl 2 was 10 m M. The values are the means of 2–4 measurements with < 10% variations. Equilibrium constants K eq of labelled adenine nucleotides were calculated according to the following equations: [ 3 H]ATP + [ 3 H]AMP fi 2[ 3 H]ADP; K eq ¼ ([ 3 H]ADP) 2 ⁄ ([ 3 H]ATP)([ 3 H] AMP). The theoretical K eq for AdK is close to unity, as determined by the equation. However, K eq values presented here refer to only the labelled adenine nucleotide at equilibrium but not the reaction equilibrium constants. ATP (m M) [ 3 H]ATP (l M) [ 3 H]ADP (l M) [ 3 H]AMP (l M) K eq 0.1 13 58 29 8.9 0.5 43 49 8 7.0 1.0 62 32 6 2.8 2.0 73 22 5 1.3 4.0 81 13 6 0.3 Table 3. Equilibrium concentrations of labelled adenine nucleotides in AdK-catalyzed reactions. Equilibrium constants K eq of labelled adenine nucleotides were calculated as described for Table 2. The initial concentrations of phosphate donors were 1 m M and the initial concentration of phosphate acceptor [ 3 H]AMP was 100 lM and the concentration of MgCl 2 was 10 mM. The values were the means of 2–4 measurements with < 10% variations. [ 3 H]ATP [ 3 H]ADP [ 3 H]AMP K eq ATP 62 32 6 2.8 UTP 46 47 7 6.9 GTP 47 45 8 5.4 CTP 45 47 8 6.1 Ureaplasma nucleoside monophosphate kinases L. Wang 1986 FEBS Journal 274 (2007) 1983–1990 ª 2007 The Author Journal compilation ª 2007 FEBS Nucleoside di- and triphosphate synthesis catalyzed by CMPK CMPK phosphorylated CMP and dCMP directly to their triphosphate forms in assays with radiolabelled ATP as phosphate donor (Fig. 3). The rate of [ 3 H]dCDP and [ 3 H]dCTP synthesis from [ 3 H]dCMP was further studied using ATP, UTP, GTP or CTP as phosphate donors. The rate for [ 3 H]dCDP synthesis, in general, is high, being 100-fold higher than that of [ 3 H]dCTP, except when CTP is used as a phosphate donor (Table 1). ATP, UTP and GTP were good phos- phate donors for both [ 3 H]dCDP and [ 3 H]dCTP syn- thesis, while CTP was a poor phosphate donor, i.e. 20-fold lower for [ 3 H]dCDP synthesis and two-fold lower for [ 3 H]dCTP synthesis (Table 1). Nucleoside triphosphate synthesis catalyzed by UMPK Using [ 3 H]UMP as substrate, the rates of [ 3 H]UDP and [ 3 H]UTP synthesis were determined. Similar to AdK and CMPK, the rate of [ 3 H]UDP synthesis was much higher than that of [ 3 H]UTP (Table 1). ATP was the most efficient phosphate donor for [ 3 H]UDP syn- thesis, while UTP, GTP and CTP were poor phosphate donors. There was detectable formation of [ 3 H]UTP in the reaction with GTP and CTP as phosphate donor, but the rates of [ 3 H]UTP synthesis were very low (Table 1). Mechanism of phosphoryl transfer Using [c- 32 P]ATP as phosphate donor, the mechanism of phosphoryl transfer during CTP synthesis by CMPK was studied. Initially, using either dCDP or CDP as the substrate and [c- 32 P]ATP as the phosphate donor, no radiolabelled dCTP formation was detected in the reac- tion with dCDP as the substrate, but it was observed when using CDP as the substrate. CTP was also formed in the control reaction using dCMP as sub- strate. However, as commercial CDP contains 4% CMP, and the level of radiolabelled product (CTP) was similar for both substrates (data not shown) and cor- responded to the CMP content in the CDP solution (4%), this accounted for the CTP product observed. When the reaction was repeated with purer CDP and run at shorter time intervals (90 s), no CTP was formed in the CDP reaction, while CTP was formed in the con- trol reactions with CMP as substrate. Thus, the CTP or dCTP synthesis carried out by CMPK was not a direct phosphorylation of CDP or dCDP by ATP, but rather CTP or dCTP was formed in the reverse reaction in two steps, i.e. (d)CMP + ATP* fi (d)CDP* + ADP; and (d)CDP* + (d)CDP fi (d)CTP* + (d)CMP. Discussion The aim of the present study was to define the role of Ureaplasma NMPKs in the synthesis of nucleo- side triphosphates. Four Ureaplasma NMPKs were cloned, expressed and the recombinant enzymes were affinity purified. These NMPKs were shown to have narrow substrate specificity regarding the phosphate acceptors, i.e. they are base specific and each enzyme has its own substrates sets (using the same nucleo- tides as their names indicated), with little overlapping activity. AdK, CMPK and UMPK phosphorylated both ribos- respective deoxyribos- forms of nucleo- tides efficiently. TMPK, however, was specific for dTMP and only dUMP had some activity. At the phosphate donor site, however, the specificities were broader, e.g. all natural nucleoside triphosphates were accepted as phosphate donors but with clear-cut pref- erences, especially in case of UMPK. The less strin- gent requirement for phosphate donors may be an advantage, since the enzymes can use any phosphate donors available. It is known that reactions catalyzed by NMPKs are reversible, which means that these enzymes can also synthesize nucleoside triphosphates via the reverse reaction. Using the tritium-labelled nucleoside mono- phosphates as substrates, the rates of (d)NDP and (d)NTP synthesis by these enzymes were investigated. The results clearly showed that Ureaplasma NMPKs are able to synthesize NTP ⁄ dNTPs via the reverse reaction, but not sequential phosphorylation of NMP by NTP as demonstrated here. This is also in agree- ment with a recent study using AdK, where it was clearly shown that the NDK-like activity of this enzyme is the result of the reverse reaction [11]. The capacity of Ureaplasma NMPKs to synthesize nucleo- side triphosphates in general is lower than that of diphosphates, which may implicate that the conversion of nucleoside diphosphates to triphosphates is the rate- limiting step. Equilibrium studies with AdK showed that the enzyme favours the reverse reaction, i.e. the highest level of [ 3 H]ATP formed from [ 3 H]AMP was achieved with the highest ATP concentration used in the assay. ATP was a better phosphate donor to bring about [ 3 H]ATP formation from [ 3 H]AMP as compared with other nucleoside triphosphates. At physiologically rele- vant ATP concentrations (2–4 mm), the level of labelled [ 3 H]AMP at equilibrium was the lowest among all labelled adenine nucleotides, suggesting that the L. Wang Ureaplasma nucleoside monophosphate kinases FEBS Journal 274 (2007) 1983–1990 ª 2007 The Author Journal compilation ª 2007 FEBS 1987 supply of nucleoside monophosphates may regulate the synthesis of nucleoside di- and triphosphates. In Mollicutes, other enzymes exist that are capable of synthesizing nucleoside triphosphates, e.g. pyvuvate kinase, phosphoglycerate kinase, etc. [14]. Together with NMPKs, they can provide cells with precursors for both DNA and RNA synthesis. The relatively low rates of (d)NTP synthesis may result in limited (d)NTPs supply. In the literature there are only few earlier studies regarding the level of ribonucleotides and deoxyribo- nucleotides in Mycoplasmas [16–18]. In Mycoplasma mycoides subsp. mycoides the levels of dNTPs were 100-fold lower as compared with NTPs [16–18]. Interestingly, the sum of ATP and UTP was two-fold higher than that of CTP and GTP, and the ratio of dATP and dTTP to dCTP and dGTP was also 2 : 1 [16,17]. This fact is in accordance with the genome composition of Mollicutes, which has a high A + T content at > 70%. Studies carried out in our labora- tory showed that the dTTP and dCTP levels were below the detection limit when Ureaplasma was cul- tured in the presence of tritium-labelled deoxycytidine or thymidine, even though > 20% of the radioacti- vity was recovered in the DNA fraction, indicating a very low dNTP pool in Ureaplasma [19]. Similarly, M. mycoides subsp. mycoides incubated with radiola- belled thymidine monophosphate resulted also in a high level of incorporation into DNA, but the level of labelled dTTP was very low [18]. In organisms where NDK is present, the rate-limit- ing step in the synthesis of dNTPs ⁄ NTPs is not the conversion of (d)NDPs to (d)NTPs, as the catalytic efficiency of NDK is high [7–9]. The regulatory mech- anism in dNTPs production relies on allosteric enzymes, e.g. ribonucleotide reductase, which to a large extent regulates dNDP production and thereby dNTP pools [20,21]. Disruption of the ndk gene in E. coli, Saccharomyces cerevisiae, and Saccharomyces pombe did not affect growth or morphology [11,22,23], suggesting that NDK is not essential. Although Molli- cutes lack the ndk gene, this work provided evidence that NMPKs, probably together other enzymes such as pyvuvate kinase [14], can replace NDK in providing the cells with NTPs and dNTPs. A recent study sug- gests that cells have a mechanism for arresting DNA synthesis when the dNTP pool size is limiting [24]. The doubling times for Mollicutes are usually much longer when compared with other bacteria such as E. coli, and the limitation of dNTP levels for DNA synthesis could be the reason. Mollicutes lack the de novo synthesis of purine and pyrimidine bases and have to rely on salvage path- ways for the biosynthesis of nucleotides required for cellular processes. The work presented here clearly showed that NMPKs are base specific and highly efficient in the synthesis of NTPs and dNTPs. NMPKs are essential enzymes for the survival of the organism, as demonstrated recently in Mycoplasma genitalium using transposon mutagenesis technique [25]. Therefore, inhibition of these enzymes, especially TMPK or UMPK, will most probably impair the synthesis of both DNA and RNA precursors, which eventually leads to cell death. Thus, these enzymes, especially TMPK and UMPK, are potential targets for future design of antibiotics against pathogenic Mycoplasmas. Experimental procedures Materials Radioactive substances [c- 32 P]ATP (3000 CiÆmmol )1 ) were purchased from PerkinElmer LAS Inc. (Boston, MA, USA). [2- 3 H]AMP (adenosine 5¢-monophosphate, 24.0 CiÆmmol )1 ) was from Amersham Biosciences (Uppsala, Sweden). [5- 3 H]dCMP (2¢-deoxycytidine 5¢-monophosphate, 21.9 CiÆmmol )1 ) and [5,6- 3 H]UMP (uridine 5¢-monophosphate, 32 CiÆmmol )1 ) were obtained from Moravek Biochemical, Inc (Brea, CA, USA). Non-radioactive nucleotides were from Sigma-Aldrich Sweden AB (Stockholm, Sweden). Cloning, expression and purification of Ureaplasma nucleoside monophosphate kinases Primers used in PCR amplification of Ureaplasma nucleo- side monophosphate kinases were designed according to the DNA sequence of the respective enzyme in the database (GenBank accession number of AE002122) and restriction sites (Nde IorBamH I) were introduced to the 5¢-sequence of the primers to facilitate subsequent cloning. PCR reac- tions were carried out by a standard procedure using the U. parvum genomic DNA (ATCC # 700970D) as template. The amplified PCR fragments were digested with Nde I and BamH I, purified on 1% agarose gel and cloned into the pET-14b vector (Novagen, Madison, WI, USA) that had been linearized with the same restriction enzymes. The recombinant plasmids carrying the Ureaplasma nucleoside monophosphate kinase genes were sequence verified using the Bigdye terminator kit and ABI Prisma 310 genetic Ana- lyzer (Applied Biosystems, Foster City, CA, USA). In order to express the recombinant protein in E. coli UGA codons, coding for Trp in AdK and UMPK, were mutated to UGG using site-directed mutagenesis as described previously [26] and sequence verified. Finally the plasmids carrying AdK, CMPK, UMPK and TMPK were transformed into the E. coli BL21 (DE3) bacteria for expression. Ureaplasma nucleoside monophosphate kinases L. Wang 1988 FEBS Journal 274 (2007) 1983–1990 ª 2007 The Author Journal compilation ª 2007 FEBS For production of recombinant protein, E. coli BL21 (DE3) harbouring the AdK, CMPK, UMPK or TMPK plasmids was cultured in LB media with 50 lgÆmL )1 carbe- nicilin at 37 °C until the optical density at 600 nm reached 0.6. The cultures were then changed to the induction temperature as indicated below and 0.1 mm isopropyl b-d-1-thiogalactopyranoside was added and the cultures were further incubated for 4 h. The temperature for induc- tion was 30 °C for AdK and CMPK; 37 °C for UMPK; 28 °C for TMPK. Bacteria were harvested by centrifugation and the pellets were resuspended in buffer containing 50 mm Tris ⁄ HCl, pH 7.5, 0.2 m NaCl, 0.2 mm phenylmethylsulfo- nyl fluoride and total proteins were extracted by sonication at 18 W for a total of 3 min, with a pulse every 5 s. The lysates were centrifuged at 30 000 g at 4 °C for 30 min (XL-70 ultracentrifuge, Beckman Coulter, rotor type RT50Ti) and the supernatant was loaded onto a metal affin- ity column (TALON resin, BD Biosciences Clontech, Palo Alto, CA, USA), which was equilibrated with 50 mm Tris ⁄ HCl, pH 7.5, 0.2 m NaCl and 5 mm imidazole. The column was subsequently washed with the same buffer con- taining 30 mm imidazole and the recombinant proteins were eluted with 300 mm imidazole and 50 mm Tris ⁄ HCl, pH 7.5. Purified protein was analysed by SDS ⁄ PAGE and protein concentrations were determined by Bio-Rad protein assay with BSA as standard. Glycerol and dithiothreitol were added to the purified enzymes to 10% and 2 mm, respectively, and the enzymes were stored in aliquots at )70 °C. Enzyme assays Phosphoryl transfer assays were performed essentially as described previously [26]. Briefly, each reaction was per- formed in a total volume of 20 lL containing 50 mm Tris ⁄ HCl, pH 7.5, 0.5 mg.mL )1 BSA, 5 mm dithiothreitol, 2mm MgCl 2 ,15mm NaF, 0.1 mm nucleoside monophos- phate, 0.1 mm [g- 32 P]ATP and 100 ng purified enzyme at 37 °C for 20 min and was stopped by heating at 70 °C for 2 min. After brief centrifugation (13 000 g, Biofuge 13, Her- aeus Instruments, rotor type HFA 17.1, max 14 926 g), 1 lL of the supernatant was spotted onto a TLC plate (PEI- cellulose; MERCK, VWR International AB, Stockholm, Sweden) and dried. Authentic markers were also applied onto the same TLC plates. The TLC plates were then devel- oped in 0.2 m NaH 2 PO 4 for reactions with AdK and CMPK and 0.1 m NaH 2 PO 4 for UMPK and TMPK. To separate other nucleoside triphosphates from [g- 32 P]ATP, 0.32 m NaH 2 PO 4 was used. The reaction products were visualized by phosphoimagine analysis (Fuji ImageGause V3.1, Fuji Photo Film Co., Ltd., Tilburg, the Netherlands). Authentic markers were visualized under UV light. NMPK assay with [ 3 H]-labelled substrates were carried out on the reaction mixture containing 50 mm Tris ⁄ HCl, pH 7.5, 0.5 mgÆmL )1 BSA, 5 mm dithiothreitol, 5 mm MgCl 2 ,15mm NaF, 0.1 mm 3 H-labelled substrate in a total volume of 20 lL. The reaction was initiated by the addition the enzyme (100 ngÆreaction )1 for the determination of NTP synthesis rates and 0.5–1 ngÆreaction )1 for the deter- mination of NDP synthesis rates) and incubated at 37 °C. At each time point, 1 lL aliquot was withdrawn and spot- ted directly onto a TLC plate (PEI-cellulose) and dried. The TLC plate was then developed in 0.2 m NaH 2 PO 4 for assays with AdK and CMPK and 0.1 m NaH 2 PO 4 for assays with UMPK. Non-radioactive markers were spotted onto the TCL plate and identified under UV light. The reaction products were cut out and eluted with 0.5 mL of 0.1 m HCl and 0.2 m KCl, and then 2.5 mL of scintillation fluid was added and the radioactivity counted. The R f values for ATP, ADP, AMP, dCTP, dCDP and dCMP in 0.2 NaH 2 PO 4 were 0.01, 0.28, 0.61, 0.03, 0.39 and 0.67, respectively. The R f values for UMP, UDP, UTP, dTMP, dTDP and dTTP in 0.1 m NaH 2 PO 4 were 0.37, 0.21, 0.05, 0.36, 0.22, and 0.05, respectively. Acknowledgements This work was supported by grants from the Swedish research council for Environment, Agricultural Scien- ces, and Spatial Planning (FORMAS) and the Swedish Research Council (VR). References 1 Razin S, Yogev D & Naot Y (1998) Molecular biology and pathogenicity of mycoplasmas. 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The role of Ureaplasma nucleoside monophosphate kinases in the synthesis of nucleoside triphosphates Liya Wang Department of Molecular Biosciences, Swedish University of Agricultural. 1987 supply of nucleoside monophosphates may regulate the synthesis of nucleoside di- and triphosphates. In Mollicutes, other enzymes exist that are capable of synthesizing nucleoside triphosphates, . purification of Ureaplasma nucleoside monophosphate kinases Primers used in PCR amplification of Ureaplasma nucleo- side monophosphate kinases were designed according to the DNA sequence of the respective