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J. Biol. Chem., Vol. 276, Issue 41, 38002-38009, October 12, 2001

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Cloning and Verification of the Lactococcus lactis pyrG Gene and Characterization of the Gene Product, CTP Synthase^*

Steen L. L. Wadskov-Hansen, Martin Willemoës^§¶, Jan Martinussen, Karin Hammer, Jan Neuhard, and Sine Larsen^§

From the  Department of Microbiology, Technical University of Denmark, Building 301, DK-2800 Lyngby, the ^§ Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, and the  Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, Sølvgade 83H, DK-1307 Copenhagen, Denmark

Received for publication, January 19, 2001, and in revised form, August 7, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The pyrG gene of Lactococcus lactis subsp. cremoris, encoding CTP synthase, has been cloned and sequenced. It is flanked upstream by an open reading frame showing homology to several aminotransferases and downstream by an open reading frame of unknown function. L. lactis strains harboring disrupted pyrG alleles were constructed. These mutants required cytidine for growth, proving that in L. lactis, the pyrG product is the only enzyme responsible for the amination of UTP to CTP. In contrast to the situation in Escherichia coli, an L. lactis pyrG mutant could be constructed in the presence of a functional cdd gene encoding cytidine deaminase. A characterization of the enzyme revealed similar properties as found for CTP synthases from other organisms. However, unlike the majority of CTP synthases the lactococcal enzyme can convert dUTP to dCTP, although a half saturation concentration of 0.6 mM for dUTP makes it unlikely that this reaction plays a significant physiological role. As for other CTP synthases, the oligomeric structure of the lactococcal enzyme was found to be a tetramer, but unlike most of the other previously characterized enzymes, the tetramer was very stable even at dilute enzyme concentrations.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Any growing organism needs nucleotides for the synthesis of DNA, RNA, and several co-enzymes. This demand can be met in two ways, either by de novo synthesis of nucleotides or by exploiting nucleotides, nucleosides, and nucleobases taken up from the surroundings through the salvage pathways. The de novo synthesis of pyrimidines seems to be universal. The pathway consists of six enzymatic reactions leading to UMP, which is subsequently converted into UTP and CTP (see Fig. 1). Most prokaryotes for which the de novo synthesis of pyrimidines has been studied possess only one allele of each gene responsible for the six enzymatic reactions. In lactococci there are, however, two different pyrD genes encoding dihydroorotate dehydrogenase (1). Furthermore, the protein encoded by the pyrDb gene needs the activity of the protein encoded by the pyrK gene to be active and is organized as part of an operon, consisting also of pyrK and pyrF (2). The Lactococcus lactis carB gene encoding the catalytic subunit of carbamyl phosphate synthase has been cloned and was shown to be monocistronically transcribed (3). The pyrB gene encoding aspartate transcarbamylase and the carA gene encoding the glutaminase subunit of carbamyl phosphate synthase are members of a four cistronic operon including the genes encoding the PyrR (pyrR) regulator and the high affinity uracil transporter (pyrP) (4). Furthermore, the presence of the genes pyrC encoding dihydroorotase and pyrE encoding orotate phosphoribosyltransferase has been shown,¹ thus establishing that the universal pathway leading to synthesis of UMP is also present in L. lactis. In most other Gram-positive bacteria the pyrimidine biosynthetic genes are organized in one large operon (5-8).

Knowledge of the salvage pathways is important when studying the phenotype of auxotrophic pyr mutants. In contrast to the de novo synthesis of pyrimidines, the salvage pathways may vary between different organisms. However, the pathways by which uracil, uridine, deoxyuridine, cytidine, and deoxycytidine are metabolized in L. lactis seem to be quite similar to those found in Bacillus subtilis and Escherichia coli. Compared with E. coli, the only exceptions are that lactococci are unable to utilize cytosine and that L. lactis only contain one pyrimidine nucleoside phosphorylase encoded by pdp (9, 10). As part of the elucidation of the salvage pathways in L. lactis, mutants blocked in various pyrimidine salvage genes have been isolated using different 5-fluoropyrimidine analogues (10). These include mutations in genes encoding uracil phosphoribosyltransferase (upp), uridine/cytidine kinase (udk), pyrimidine nucleoside phosphorylase (pdp), cytidine/deoxycytidine deaminase (cdd), and thymidine kinase (tdk). Furthermore, the upp gene of L. lactis has been cloned and characterized (11). The pyrimidine salvage pathways, as verified in the present in L. lactis, are shown in Fig. 1.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Simplified representation of the pyrimidine biosynthesis in L. lactis. U, uracil; UR, uridine; CR, cytidine; PRPP, 5-phosphoribosyl-1--pyrophosphate; R-1-P, ribose 1-phosphate; udk, uridine kinase; cdd, cytidine deaminase; pyrH, UMP kinase; pyrG, CTP synthase; pdp, pyrimidine nucleoside phosphorylase; upp, uracil phosphoribosyltransferase.

Studies of the pathways converting UMP to other pyrimidine derivatives in lactococci were initiated with the recent description of a pyrH-encoded UMP kinase as responsible for synthesis of UDP from UMP (12). These pathways are further elucidated with the present communication, which establishes the presence of a pyrG-encoded CTP synthase (EC 6.3.4.2.) as responsible for synthesis of CTP from UTP in L. lactis. In the presence of Mg²⁺ the enzyme catalyzes the reaction ATP + UTP + glutamine  ADP + P_i + CTP + glutamate. The glutamine-dependent amination of UTP to CTP is activated allosterically by GTP. Our studies included cloning and sequencing of pyrG from L. lactis and the construction of different pyrG disruption and deletion mutants as well as enzymatic characterization of the gene product.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

DNA and Genetic Methodology-- The methods described by Sambrook et al. (13) were used for the extraction and manipulation of plasmid DNA and general DNA in vitro methods. Chromosomal lactococcal DNA was prepared as described by Johansen and Kibenich (14). DNA sequences were determined by the dideoxy chain termination method using the Thermo Sequenase radiolabeled terminator cycle sequencing kit in accordance with the protocol of the manufacturer (Amersham Pharmacia Biotech) or using an ABI PRISM 310 DNA Sequencer as recommended by the supplier (PerkinElmer Life Sciences). PCR² amplification of DNA was performed using standard methods. E. coli cells were transformed after CaCl₂ treatment as described (13), and L. lactis was transformed by electroporation (15).

Plasmids, Bacterial Strains, Growth Media, and Assay of Cytidine Deaminase Activity-- The plasmids pSH105 and pSH106 (Fig. 2) were made in the following way. A 1.4-kilobase internal fragment of pyrG from L. lactis MG1363 (16) was obtained by PCR using the degenerate primers pyrG1a, 5'-CCCAAGCTTAYATHAAYGTNGAYCC-3', and pyrG2b, 5'-CGGGATCCRAAYTCNGGRTGRAAYTG-3', which were designed based on a primary sequence alignment of the CTP synthases from E. coli and B. subtilis. Subsequently the fragment was cloned in the EcoRV site of the commercial PCR cloning vector pMOSBlue T-vector (Amersham Pharmacia Biotech). From this construct the 1.4-kilobase fragment was excised with BamHI and ligated into BamHI-digested pRC1, and the plasmid was named pSH105. pRC1 (17) cannot replicate in L. lactis but contains an erm gene conferring erythromycin resistance in both E. coli and L. lactis. Plasmid pSH106 was constructed from pSH105 by deleting a 0.4-kilobase SacI fragment. Hence, pSH106 also cannot replicate in L. lactis but is carrying the erythromycin resistance gene.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Schematic representation of the open reading frames and the L. lactis DNA in the plasmids used in this study. The numbering refers to the DNA sequence submitted to the EMBL data library. A, pyrG with the two flanking open reading frames, orf81 and orf116. B, the PCR fragment obtained by degenerate PCR cloned in pSH105. C, the fragment in pSH106 obtained by a SacI deletion of pSH105. The bars indicate reading frames with names given above, solid and broken lines indicate chromosomal or pRC1 plasmid DNA, respectively.

The deoxy oligonucleotides PYRG LL5A, 5'-GGAATTCGAGGAGATTTAGATGTCAAC-3', that overlap the 5' end and PYRG LL3A, 5'-AAAACTGCAGTTATTTACTATTTTCAACAGC-3', that overlap the 3' end of L. lactis pyrG were used to amplify the gene by PCR using chromosomal DNA from MG1363 as a template. The sequences encoding the pyrG ribosome binding site, the start codon, and translation stop signal are indicated by italicized letters. The unique EcoRI and PstI restrictions sites of the PCR product inserted by the oligonucleotides are indicated in the sequence by underlining and were used for cloning of the pyrG fragment after the P_A1 promotor (18) of the vector pUHE23-2. The resultant plasmid pMW602 was verified by sequencing and transformed into SØ5393, a Leu⁺ derivative of the E. coli pyrG strain JF622 (19), which also carries an F' with lacI^Q and Tn5 in lacZ. The resulting plasmid-carrying strain SØ5399 was used for overexpression of the L. lactis pyrG.

Lactococcal cultures were grown either on M17 glucose broth (20) or on synthetic SA medium based on MOPS and containing 7 vitamins and 19 amino acids (21), in both cases supplied with glucose to 1% (w/v). E. coli cultures were grown either on LB medium (13) or synthetic AB medium (22). L. lactis was cultured at 30 °C in filled culture flasks without aeration. E. coli in batch cultures was grown at 37 °C with vigorous shaking. For all plates, agar was added to 15 g liter¹. When needed, the following was added to the different media: cytidine at 20 or 50 µg ml¹, uracil at 20 µg ml¹, erythromycin at 1 µg ml¹ for lactococci, and 150 µg ml¹ for E. coli, and ampicillin at 100 µg ml¹.

PyrG mutants were constructed in L .lactis subsp. cremoris MG1363 (12) and its cdd derivative MB109 (9) by transforming the strains with the plasmid pSH106, which contains a disrupted pyrG gene. Since the plasmid cannot replicate in L. lactis, selection for erythromycin results in transformants that harbor the nonreplicating plasmid integrated into the chromosome by homologous recombination in the pyrG region. The selections were performed using SA medium and cytidine 50 µg ml¹ and erythromycin at 1 µg ml¹. LKH 278 was derived in this way from MG1363, whereas LKH 280 was derived from MB109. Cytidine deaminase activity in crude extracts from L. lactis was assayed at 30 °C as previously described (9) using the spectroscopic assay developed by Beck et al. (24).

Sequencing of L. lactis pyrG-- The pyrG gene from L. lactis MG1363 was sequenced using the Easy Gene Walking method (25). For PCR with Chromosomal DNA from MG1363 two sets of oligonucleotides pyrG6a, 5'-GAAAAATGGTTCACGCCG-3', pyrG7a, 5'-TGCCAACGATGTGACCG-3', and pyrG8a, 5'-GGCAAAAAATTCTTCGTTGC-3', and pyrG6b, 5'-GAAATATAAGCATCTGGC-3', pyrG7b 5'-TTGGCACTTCACTGCGC-3', and pyrG8b, 5'-TCAGTTGTTGTTGCTGC-3', designed to anneal to each end of the internal pyrG fragment of pSH105, were used together with partly degenerate oligonucleotides containing an EcoRI (5'-NNNNNNNNNNGAATTC-3'), HindIII (5'-NNNNNNNNNNAAGCTT-3'), or Sau3AI (5'-NNNNNNNNNNGATC-3') restriction site. Fragments covering both ends of the DNA regions flanking pyrG were obtained in combination with all three degenerate oligonucleotides. Direct sequencing of the PCR fragments was done without prior cloning to eliminate the risk of errors caused by mutations in individual PCR fragments. Based on the sequences obtained, a PCR fragment covering the entire pyrG open reading frame and adjacent sequence was made using the oligonucleotides SLLH6, 5'-ACTTTGACAAAAAAGG-3', and SLLH7, 5'-TACAAAAGATTTTGGGC-3', and chromosomal DNA from MG1353 as a template. The direct sequencing of this PCR fragment on both strands revealed minor errors in the sequence determined by Easy Gene Walking (less than 0.5%). By combining the sequence data, the total sequence of pyrG and flanking regions was obtained.

Overproduction and Purification of CTP Synthase from L. lactis-- E. coli strain SØ5399 was grown in 1 liter of LB medium with the addition of ampicillin to an OD₄₃₆ of 0.8. Then isopropyl-1-thio--D-galactopyranoside was added to a final concentration of 1 mM, and cultivation was continued for 2.5 h. The cells were harvested by centrifugation at 8000 rpm for 5 min in a Sorvall rotor SLA3000. The cells were washed in 50 mM potassium phosphate, pH 7.5, divided in three portions of ~1 g of wet cell paste and stored at -20 °C. The purification was performed at 4 °C unless otherwise noted and is described for the amount of protein obtained from about 1 g of cell paste.

Cells were thawed on ice, resuspended in 15 ml of extraction buffer (50 mM potassium phosphate, pH 7.5 and 2 mM DTT), and opened using a Sonics Vibra-Cell ultrasonic processor. Cell debris was sedimented by centrifugation in a Sorvall SS34 rotor at 14,000 rpm for 15 min. The supernatant was made 1% (w/v) with streptomycin sulfate, and centrifugation was repeated as above. A fractionated ammonium sulfate precipitation was performed by first adding 6.3 g of ammonium sulfate (~ 35% saturated) to the supernatant while gently stirring the precipitate and centrifugation was repeated as above. To the supernatant, 4.2 g of ammonium sulfate (~60% saturated) was added, and centrifugation was repeated as above. The precipitate (about 10-15 mg of CTP synthase) was dissolved in 10 ml of extraction buffer and loaded on to a column (2.6 × 28 cm) of phenyl-Sepharose CL-4B (Amerham Pharmacia Biotech) equilibrated with the same buffer and placed at room temperature. With a flow rate of 3 ml min¹, the column was washed with 300 ml of extraction buffer followed by an isocratic elution of the protein with a solution containing 30% ethylene glycol, 0.5 mM potassium phosphate, pH 7.5, and 2 mM DTT. The fractions containing CTP synthase were pooled, and 200 mM potassium phosphate, pH 7.5, with 20 mM DTT was added to a final concentration of 20 mM potassium phosphate. The protein was precipitated by adding ammonium sulfate to 60% saturation and collected by centrifugation as above. The precipitate was dissolved in 5 ml of extraction buffer and loaded onto a column (2.6 × 7.5) of phenyl-Sepharose equilibrated with the same buffer. The protein started to elute after one column volume of buffer (40 ml), and the next 80 ml of eluent were collected. The CTP synthase-containing fractions were precipitated by 60% ammonium sulfate as above and redissolved in 2 ml of buffer (50 mM Hepes, pH 8.0, and 2 mM DTT). The protein was dialyzed against 2 × 1 liter of the same buffer for 24 h. Finally, the enzyme was loaded on a HiTrap desalting column (Amersham Pharmacia Biotech) and eluted with the same buffer as above with a flow rate of 1 ml min¹ in portions of 1 ml, and the protein fractions were collected. The highly reproducible purification procedure presented above involving two runs of hydrophobic chromatography appears to take advantage of a hysteretic effect on the retaining capacity of phenyl-Sepharose for L. lactis CTP synthase that depends on the protein concentration of the loaded sample. CTP synthase was prepared to near homogeneity as judged from SDS-PAGE (26). The overall purification fold was not determined but could be estimated from SDS-PAGE to be about 2-3-fold due to the very high overproduction of enzyme. The protein concentration was determined by the bicinchoninic acid procedure (27) with reagents provided by Pierce and with bovine serum albumin as a standard. The specific activity of the L. lactis CTP synthase ranged from 2.5 to 3 µmol min¹ mg¹ when determined as described below. The enzyme could be stored frozen at -20 °C for several months without loss of activity.

Assay of CTP Synthase Activity and Analysis of Enzyme Kinetic Data-- All chemicals were from Sigma. Enzyme activity was determined at 30 °C by following the increase in absorbance at 291 nm using a PerkinElmer Life Sciences Lambda 17 UV-visible spectrophotometer (28), and activities were calculated using the molar extinction coefficients  = 1338 M¹ cm¹ or  = 1664 M¹ cm¹ (29) for the conversion of UTP to CTP or dUTP to dCTP, respectively. The standard assay was performed in 150 µl of 50 mM Hepes, pH 8.0, 2 mM DTT, 27 mM MgCl₂, 1 mM ATP, 1 mM UTP, 0.1 mM GTP, and 10 mM glutamine. When nucleotide concentrations were varied, the MgCl₂ concentration was always maintained at an excess of 25 mM. CTP synthase was added to concentrations between 3 and 25 µg ml¹. All initial velocities were determined in duplicate at two different enzyme concentrations. Calculation of kinetic constants was performed by fitting the initial velocities to one of the five equations below using the computer program UltraFit (BioSoft, version 3.01). The reported standard errors are those calculated by the computer program. Equation 1 is for a two-substrate sequential mechanism where each substrate shows cooperative binding that is independent of the binding of the other. Equations 2 and 3 apply to hyperbolic and sigmoid saturation kinetics, respectively, Equation 4 applies to hyperbolic activation, and Equation 5 is for cooperative inhibition.

(Eq. 1)

(Eq. 2)

(Eq. 3)

(Eq. 4)

(Eq. 5)

where v is the initial velocity, V_app is the apparent maximal velocity, S_i0.5 and S_0.5 are the concentrations of substrates or activators, A or B, denoted when appropriate, at apparent half-maximal velocity at infinite small or saturating concentrations of the other substrate or activator, respectively, K_m and K_A are the apparent Michaelis-Menten constant for substrate or activator A, respectively, n is the Hill coefficient, denoted when appropriate, V₁ and V₂ are the velocities in the absence and in the presence of activator, respectively, IC₅₀ is the concentration of inhibitor I at half-maximal inhibition. Unless otherwise noted, all reported kinetic velocities are in µmol of CTP min¹ mg¹. The calculation of the free Mg²⁺ concentration was performed using a stability constant of 73,000 M¹ for the Mg²⁺ and nucleoside triphosphate complexes (30).

Molecular Size Determination by Gel Filtration, Chemical Cross-linking, and Dynamic Light-scattering of CTP Synthase-- For gel-filtration experiments, a prepacked Bio-Prep S.E.1000/17 column from Bio-Rad was equilibrated at room temperature with buffer (50 mM Hepes, pH 8.0, and 2 mM DTT). Fifty-microliter samples of (i) a calibration standard (Bio-Rad) containing thyroglobulin (670 kDa), -globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B-12 (1.35 kDa), (ii) a mix of thyroglobuline, catalase (232 kDa), and ovalbumin (Amersham Pharmacia Biotech), or (iii) CTP synthase (13-65 µg) was loaded onto the column with a flow rate of 0.5 ml min¹. Protein elution from the column was monitored at 280 nm. Furthermore, the position of elution of the individual proteins in the calibration standards was also evaluated by SDS-PAGE analysis of the collected fractions.

Chemical cross-linking experiments were performed in two ways. (i) CTP synthase (0.75 mg ml¹) was incubated at room temperature in 100 µl of 50 mM Hepes, pH 8.0, 2 mM DTT with either 0.1% or 1% of glutaraldehyde for 30 min. Apart from the difference in glutaraldehyde concentration four incubation conditions were used that contained enzyme alone, enzyme in the presence of 10 mM MgCl₂, enzyme in the presence of 10 mM MgCl₂ and 2 mM ATP, or enzyme in the presence of 10 mM MgCl₂ and 2 mM UTP. Samples were then removed and prepared for analysis by SDS-PAGE (26). (ii) CTP synthase (25 or 250 µg ml¹) was incubated in the absence of MgCl₂ and nucleotides with 0.1% or 1% glutaraldehyde as above, and the reactions were terminated by the addition of glycine to a final concentration of 200 mM. Subsequently, all samples were concentrated on Millipore ULTRAFREE-MC 30.000 NMWL filter units, and about 10 µg of protein from each sample was subjected to SDS-PAGE analysis. The molecular weight of the protein bands was calculated using a high range molecular weight marker from Bio-Rad.

Dynamic light-scattering was performed on a DynaPro MSXTC apparatus thermostated to 30 °C, and samples were prepared in 50 mM Hepes, pH 8.0, 2 mM DTT. Light-scattering was determined twice on 15-µl samples of CTP synthase at concentrations ranging from 5 to 0.05 mg ml¹. Each protein dilution was passed through an ULTRAFREE-MC 0.22-µm filter unit. One measurement consisted of 20 acquisitions of 10 s. Analysis of the light-scattering data was done with the software Dynamics V6 supplied with the DynaPro instrument using standard settings for a general aqueous buffer solution of protein (phosphate-buffered saline) with a refractive index at 589 nm and 20 °C of 1.33, a viscosity coefficient of 1.019 at 20 °C, a Cauchy coefficient of 3119 nm², a solvent intensity of 0, and a temperature model for an aqueous solution. From the peaks derived from CTP synthase, the calculated hydrodynamic radii and polydispersities are reported and compared with those obtained with catalase whose tetramer has a similar molecular mass as the CTP synthase tetramer.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The L. lactis pyrG Sequence-- The sequence of L. lactis PyrG was obtained as described under "Experimental Procedures." Southern blot analysis verified that the PCR fragment cloned into pSH105 (Fig. 2) originated from MG1363 (data not shown).

Analysis of the DNA sequence using the GeneMark program (31) indicated a consensus Shine-Dalgarno motif (GAGGAG) spaced six nucleotides upstream of the deduced translational initiation site of the pyrG reading frame (nucleotide 898-2505) encoding a 537-amino acid polypeptide with a M_r of 59,455. When the deduced amino acid sequence of the L. lactis CTP synthase was submitted to a BLAST search, it revealed identities between 36 and 77% with that of other CTP synthases. Of particular interest is maybe the E. coli sequence, which showed 47% identity with that of L. lactis CTP synthase.

The lactococcal pyrG is flanked by two open reading frames. The upstream reading frame orf81 (nucleotides 1-248) encodes 81 C-terminal amino acids of a polypeptide that shows homology to several aspartate aminotransferases. The downstream open reading frame (nucleotide 2717-3052) encoding polypeptide of 116 amino acids, named orf116, is preceded with a spacing of 5 base pairs by a good ribosome binding site motif (AGGAAA). The orf116 does not show homology to any known open reading frames in the data bases. The DNA sequence was submitted to the EMBL data library and was assigned the accession number AJ010153.

The pyrG Gene of L. lactis Encodes the Only CTP Synthase-- If pyrG is the only functional gene encoding CTP synthase in L. lactis, a gene disruption would result in a cytidine requirement, i.e. the cell would be restricted to acquire CTP through uptake and subsequent phosphorylation of cytidine. In E. coli and Salmonella typhimurium, exogenously added cytidine is rapidly deaminated to uridine by cytidine deaminase encoded by cdd, and hence, a cdd inactivation is needed to isolate a pyrG mutant (32, 33). Consequently, the pyrG mutation in L. lactis was established in MB109 (10), a cdd derivative of MG1363. Plasmid pSH106 was transformed into MB109 and, by selecting for erythromycin resistance, transformants were obtained in which this nonreplicating plasmid had integrated into the chromosome. 12 transformants were tested for the ability to grow without cytidine, and all 12 were found to have a cytidine requirement, which, as expected, could not be satisfied by the addition of uracil. These results strongly indicate that the cloned pyrG gene encodes the only physiological relevant CTP synthase in L. lactis. Chromosomal DNA from one of the pyrG mutants was extracted, and the pyrG mutation due to the integration of the plasmid was verified by PCR. The strain was kept as LKH280 (Fig. 2).

A cdd Mutation Is Not a Prerequisite for Isolating pyrG Mutants in L. lactis-- Next we tested whether a pyrG mutant could be established in L. lactis without an additional mutation in cdd. The wild type strain MG1363 was transformed with pSH106, and erythromycin-resistant colonies were readily obtained. Again all transformants were shown to have a cytidine requirement, thus showing that the pyrG mutation could be established in L. lactis even in the presence of a wild type cdd gene. The mutated pyrG region was verified by PCR on chromosomal DNA extracted from the strain. Based on the observations made in E. coli (33, 34), we speculated that such mutants would require a high external cytidine concentration to avoid cytidine depletion due to the deamination to uridine. Hence, mutants were isolated using a cytidine concentration of 50 µg ml¹ but were later proved able to grow even at a cytidine concentration of 20 µg ml¹. One of these mutants was kept as LKH278 (Fig. 2).

In E. coli it has only been possible to isolate a mutant defective in pyrG if the strain also contained a cdd mutation. This difference could be explained if the activity of cytidine deaminase varies between the two bacterial genera. Without added cytidine, the levels of cytidine deaminase are of the same order of magnitude in the two genera (9). However, the presence of cytidine in the medium induces the expression of cdd 30-fold in E. coli (36) and ensures a very rapid degradation of cytidine in this organism. In fact, cytidine is the effector for the CytR repressor in E. coli (37), which in turn regulates most of the nucleoside catabolic enzymes. In crude extracts from L. lactis grown in defined medium in the presence and absence of a high concentration of cytidine (200 µg ml¹), the cytidine deaminase activity was assayed. The enzyme activity was determined to be 26 ± 3 and 20 ± 6 nmol min¹mg¹ in the absence and presence of cytidine, respectively. Apparently, the L. lactis cdd gene expression is not induced by cytidine. In B. subtilis it has also been found that cytidine deaminase activity is not induced by addition of exogenous cytidine (38).

Kinetic Properties of the L. lactis CTP Synthase-- A linear correlation of the increase in CTP formation with time was observed for all enzyme concentrations used. Also, the specific activity was unchanged with the used enzyme concentrations both when the ATP and UTP concentration was saturating (2 mM each) or unsaturating (each 0.5 mM or 0.25 mM) (data not shown).

When ATP was varied at increasing fixed concentrations of UTP, sigmoid saturation curves were observed with n values of about 2 when the data for each UTP concentration was fitted to Equation 3. Subsequently, the data were fitted to Equation 1 (Fig. 3), and the calculated kinetic constants are reported in Table I. As for the non-cooperative sequential mechanism (39), the validity of the S_i0.5 constants in Equation 1 will depend on the exact binding mechanism and should be interpreted with caution. Assuming a random binding of ATP and UTP to the enzyme, the S_i0.5 was 0.90 ± 0.13 mM for ATP and 0.8 ± 0.2 mM for UTP. However, the values of S_i0.5 that represent the binding of the nucleotide to the enzyme in the absence of the other nucleotide correlates well with the results from equilibrium binding of ATP or UTP to the E. coli CTP synthase (40).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effect on initial velocity of L. lactis CTP synthase of varying the ATP concentration at different fixed concentrations of UTP. Assay conditions were as described under "Experimental Procedures." ATP concentrations varied as indicated at fixed UTP concentrations of 1 mM (), 0.5 mM (), 0.25 mM (), 0.125 mM (), and 0.0625 mM (). Data were fitted to Equation 1, and the calculated kinetic constants are reported in Table I.

Ammonium ions, or more likely ammonia (41, 42), could substitute for glutamine in the absence of GTP. The saturation curves for both glutamine and ammonium were hyperbolic, and the calculated kinetic constants are reported in Table I.

GTP is an allosteric activator of CTP synthase and enhances the glutamine-dependent rate of CTP production (41, 43). For the L. lactis enzyme activation, curves were hyperbolic both at near saturating (1 mM) and unsaturating (0.25 mM) concentrations of ATP and UTP. The kinetics showed an increase in activation constant with increasing ATP and UTP concentrations (Fig. 4A and Table I). The increase in velocity of the glutamine-dependent reaction due to GTP activation was between 25- and 50-fold, increasing with the ATP and UTP concentration. The V_app (the sum of V₁ and V₂ in Equation 4) for the glutamine-dependent reaction as determined from varying the GTP concentration at 1 mM ATP and 1 mM UTP (Fig. 4A) was 65% of the ammonia-dependent reaction under otherwise similar conditions (Table I).

View larger version (10K): [in this window] [in a new window]   Fig. 4.   Effect on initial velocity of L. lactis CTP synthase of varying the GTP (A) and (B) free Mg2+ concentration at saturating and unsaturating concentrations of ATP and UTP. Assay conditions were as described under "Experimental Procedures." A, GTP varied as indicated at 1 mM ATP and 1 mM UTP () and 0.25 mM ATP and 0.25 mM UTP (). Data were fitted to Equation 4, and the calculated kinetic constants are reported in Table I. B, the concentration of free Mg2+ was calculated as described under "Experimental Procedures" and varied as indicated at 1 mM ATP and 1 mM UTP () and 0.25 mM ATP and 0.25 mM UTP (). Data were fitted to Equation 3, and the calculated kinetic constants are reported in Table I.

The CTP synthase reaction requires the presence of free Mg²⁺ in addition to Mg²⁺ complexed with nucleotides (44). However, the activation curve for the free Mg²⁺ concentration calculated as described under "Experimental Procedures" was greatly dependent on whether the concentrations of ATP and UTP were near saturating (1 mM) or unsaturating (0.25 mM) (Fig. 4B). A decrease in both the activation constant and Hill coefficient was observed with increasing ATP and UTP concentrations (Table I).

The Saccharomyces cerevisiae CTP synthase encoded by the URA7 gene has recently been shown to catalyze the conversion of dUTP to dCTP (29), in contrast to what was found previously for the E. coli enzyme (45). As shown in Fig. 5A, the L. lactis enzyme was fully capable of aminating dUTP. The V_app for the dUTP-dependent reaction was about half that found for the UTP-dependent reaction under similar conditions (Table I). The S_0.5 for dUTP was about 6-fold higher than that found for UTP, but with a similar degree of cooperativity (Table I).

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Conversion of UTP to CTP and dUTP to dCTP by the L. lactis CTP synthase. Assay conditions were as described under "Experimental Procedures." A, saturation of CTP synthase with UTP () or dUTP (). Data were fitted to Equation 3. B, inhibition by the products CTP () or dCTP () in the presence of 0.25 mM UTP. Data were fitted to Equation 5. The calculated kinetic constants are reported in Table I.

The product CTP was an effective inhibitor of the L. lactis enzyme and showed cooperative inhibition. dCTP, the product of dUTP amination, was a very weak inhibitor. The IC₅₀ value for dCTP should be considered very approximate, since it relies on a extreme extrapolation as the spectrophotometric assay prevented the use of dCTP concentrations exceeding 1 mM (Fig. 5B).

Quaternary Structure of L. lactis CTP Synthase-- To investigate the quaternary structure of L. lactis CTP synthase, we performed gel filtration experiments (Fig. 6), chemical cross-linking with glutaraldehyde (Fig. 7), and dynamic light-scattering (Table II). In gel filtration only one protein peak was observed whether the protein concentration of the loaded sample was 1.3 or 0.26 mg ml¹. The CTP synthase peak migrated slightly faster than the catalase tetramer of about 230 kDa (Fig. 6A), suggesting a molecular mass for L. lactis CTP synthase of 234 kDa, in agreement with a tetramer of 60-kDa subunits.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Determination of the oligomeric state of L. lactis CTP synthase by gel filtration. The experimental conditions are described under "Experimental Procedures." A, elution profile (10-20 min) of calibration standards and L. lactis CTP synthase. Profile a, calibration standard I (thyroglobulin, -globulin, ovalbumin, myoglobin, and vitamin B-12); profile b, calibration standard II (thyroglobulin, catalase, and ovalbumin); profile c, CTP synthase (1.3 mg ml1); profile d, CTP synthase (0.26 mg ml1). The arrows point to peaks representing protein aggregates (1), thyroglobulin (2), CTP synthase (3), catalase (4), -globulin (5), and ovalbumin (6). B, the relative point of elution is indicated for CTP synthase (×) and calibration standard proteins (). The arrow points to the point of elution of CTP synthase with a calculated molecular mass as shown.

View larger version (42K): [in this window] [in a new window]   Fig. 7.   Determination of the oligomeric state of L. lactis CTP synthase by chemical cross-linking. The experimental conditions are described under "Experimental Procedures." A, the CTP synthase concentration was 0.75 mg ml1; arrows point to the individual protein bands. Chemical cross-linking was performed with 0.1% glutaraldehyde (lanes 2, 4, and 6) or 1% glutaraldehyde (lanes 3, 5, 7, and 8). Lane 1, HMW marker; lanes 2 and 3, CTP synthase; lane 4 and 5, CTP synthase and 10 mM MgCl2 and 2 mM ATP; lane 6 and 7, CTP synthase and 10 mM MgCl2 and 2 mM UTP; lane 8, CTP synthase and 10 mM MgCl2. B, chemical cross-linking was performed with 1% glutaraldehyde (lanes 2 and 5) or 0.1% glutaraldehyde (lane 3 and 6). Lane 1, HMW marker; lanes 2 and 3, 0.025 mg ml1 CTP synthase; lane 4, untreated CTP synthase; lane 5 and 6, 0.25 mg ml1 CTP synthase. C, the relative migration in panel A is indicated for HMW marker proteins () and CTP synthase cross-linking products (×). The arrows point to the point of migration of cross-linked CTP synthase species with a calculated molecular weight as shown.

From chemical cross-linking experiments with 0.75 mg ml¹ (Fig. 7A), we identified a maximum of four protein bands, of which the largest band was dominant at high glutaraldehyde concentrations. A similar pattern was observed when cross-linking was performed on samples containing 0.25 and 0.025 mg ml¹ CTP synthase (Fig. 7B), indicating that the extent of cross-linked species are independent of the protein concentration when varied from 0.75 mg ml¹ to 0.025 mg ml¹. The slowest-migrating protein band was estimated to migrate at about 266 kDa, which would be in accordance with a tetramer (Fig. 7C). The protein bands migrating between the monomer of 60- and the 266-kDa band could be derived from either trimer (177 kDa) and another form of the tetramer with altered migration pattern due to a difference in the extent of cross-linking (234 kDa) (Fig. 7C). Another interpretation could be that the 177- and 234-kDa bands represent dimer and trimer, respectively, but that the migration in the gel of these species reflects not only size but also shape and, thereby, deviates from that expected of 120 and 180 kDa, respectively. The analysis of chemically cross-linked CTP synthase obtained both in the presence and absence of Mg²⁺, Mg²⁺ and ATP, or Mg²⁺ and UTP gave similar results and seemed primarily to depend on the glutaraldehyde concentration in the incubation (Fig. 7A).

The results from dynamic light-scattering experiments performed on L. lactis CTP synthase are presented in Table II. The hydrodynamic radius, R_H, remains fairly constant over a 100-fold dilution of the protein, as does the degree of polydispersity, %Pd, that measures the broadness of the peak. The results indicate that the enzyme is on the same oligomeric form at the concentrations tested. It should be noted that a concentration of 0.05 mg ml¹ is close to the limit for a 240-kDa protein that can be determined reliably with the apparatus used. The molecular mass of the enzyme corresponding to the obtained R_H when calculated with the DynaPro software using the settings described under "Experimental Procedures" is about 180 kDa. Since this size is intermediate between dimer and tetramer, we performed an experiment with catalase to correlate the hydrodynamic radius of this protein with a known mass to that of CTP synthase. This comparison of hydrodynamic radii of catalase and CTP synthase (Table II) suggests that L. lactis CTP synthase is a tetramer at the tested protein concentrations, in agreement with the results from gel filtration and chemical cross-linking.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The enzyme CTP synthase encoded by pyrG plays a major role in the growth and physiology of cells, since this enzyme catalyzes the only reaction in which the uracil moiety is aminated to the corresponding cytosine derivative. Hence, CTP synthase is essential for the synthesis of CTP de novo, and in contrast to the findings for E. coli, a pyrG mutation can be established in L. lactis in a wild type cdd gene background.

The L. lactis enzyme is apparently the first CTP synthase from a Gram-positive bacterium to be characterized. The enzyme appeared to have kinetic properties with respect to substrates and activators that were very similar to that found for other CTP synthases, especially the E. coli counterpart, although there were some significant differences.

Since the binding of both substrates, ATP and UTP, appeared cooperative and since the cooperativity seemed independent of the concentration of the other, it was possible to describe the saturation kinetics for these nucleotides by a sequential model outlined in Equation 1 (Fig. 3). Similar homotropic cooperativity of ATP and UTP binding has been reported for the yeast enzyme encoded by URA7 (46). However, in general, the degree of cooperativity of nucleotide binding appears to cease at saturating concentrations of the nonvaried nucleotide, as found for E. coli CTP synthase (40) and the yeast isozyme encoded by URA8 (47).

In contrast to what has been reported for the E. coli CTP synthase (45), the L. lactis enzyme was capable of aminating dUTP (Fig. 5A and Table I). However, unlike the yeast isozyme encoded by the URA7 gene (29), which has previously been shown to carry out this reaction, the S_0.5 for dUTP found for the lactococcal enzyme is so high that the reaction is unlikely to have a significant role at physiological conditions. The inhibitory effect of the products CTP or dCTP was similar to that observed for the yeast enzyme (29).

The role of free Mg²⁺ in the reaction appeared more complex than to just form the actual substrates MgATP and MgUTP and the MgGTP activator complex (Fig. 4B). The activation of L. lactis CTP synthase by free Mg²⁺ in the presence of high ATP and UTP concentrations (1 mM) was very similar to that of the E. coli enzyme, but at unsaturating concentrations (0.25 mM) of ATP and UTP the increase in the activation constant and Hill coefficient (Table I), both, were about half that found for the E. coli enzyme under similar conditions (44).

The activation of the glutamine-dependent reaction by GTP is a general feature of the CTP synthase reaction and has recently been suggested to act by stabilizing the enzyme form that binds the tetrahedral intermediate of glutamine during its hydrolysis (41). The GTP activation of the E. coli enzyme was originally reported to be very complex, involving hyperbolic binding of GTP to the dimer and negative cooperativity for the binding to tetramer (43). Later studies found that GTP activation was hyperbolic under conditions where the enzyme would be on the tetrameric form (44), as has also been shown for the yeast isozymes (47). In agreement with these later reports, we found that GTP activation was hyperbolic for the L. lactis CTP synthase at both near-saturating (1 mM) and unsaturating (0.25 mM) ATP and UTP concentrations (Fig. 4A and Table I). The difference in V_app of the GTP-activated, glutamine-dependent reaction and the ammonium-dependent reaction is similar to what have been found for the E. coli enzyme (41, 44).

One striking difference between the L. lactis enzyme and the enzymes from E. coli (48-50), yeast (51), rat (52), and human (9) is the observation that the lactococcal enzyme remains a tetramer at dilute enzyme concentrations even in the absence of Mg²⁺, ATP, and UTP (Figs. 6 and 8 and Table II). Under similar conditions, at least one of these two nucleotides, together with Mg²⁺, appear to be essential for tetramerization of most previously characterized CTP synthases. The only exception known to us is the enzyme from the parasite Giardia intestinales, which also appears to be a tetramer in the absence of nucleotides (35). The E. coli CTP synthase will even dissociate to monomers at 4 °C and, in the absence of ATP and UTP, has been shown only partially to form tetramers at high enzyme concentrations (48, 50). Together these observations suggest that, apart from being substrates, the varying effect of ATP and UTP in activating CTP synthases from different organisms is caused by a difference in the equilibrium constant for the tetramer formation of the various enzymes. The activation by ATP and/or UTP by binding to the tetramer may then drive this equilibrium toward tetramer formation at low enzyme concentrations. Hysteretic behavior of the E. coli enzyme with respect to initial velocity that depends on enzyme concentration and preincubation conditions before activity measurement has been explained by an equilibrium between monomer, dimer, and tetramer (48). The lactococcal CTP synthase appeared to be a tetramer at dilute enzyme concentrations similar to those used under assay conditions (Fig. 7B and Table II). Also, we observed no nucleotide concentration-dependent hysteretic behavior with respect to initial velocities and specific activity in the range of enzyme concentrations used in this study. Together, these results seems to indicate that the observed cooperativity of nucleotide and free Mg²⁺ binding for this enzyme is not an effect of an equilibrium between different oligomeric states of the enzyme. We suggest for L. lactis CTP synthase that the cooperativity observed for ATP and UTP reflects true homotropic interactions (Fig. 3), and for Mg²⁺ activation, the dependence on the nucleotide concentration (Fig. 4B) may suggest some heterotropic interactions between nucleotide binding and Mg²⁺ binding as well (40). The apparent stability of the tetramer found for the L. lactis CTP synthase is a welcome simplification and makes the L. lactis enzyme an attractive candidate for the study of the structure-function relationship of the catalytic and regulatory properties of CTP synthase.

	ACKNOWLEDGEMENTS

We sincerely appreciate the expert technical assistance of Dorthe Boelskifte, Janni Juul Jørgensen, Lisbeth Stauning, and Lise Sørensen. We also thank Michael Gajhede for use of the DynaPro light-scattering instrument.

	FOOTNOTES

^* This work was supported by the Danish National Research Foundation and the Danish Government Program for Food Science and Technology through the Center for Advanced Food Studies.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AJ010153.

^¶ To whom correspondence should be addressed. Tel.: 45 35 32 02 39; Fax: 45 35 32 02 99; E-mail: martin@xray.ki.ku.dk.

Published, JBC Papers in Press, August 10, 2001, DOI 10.1074/jbc.M100531200

¹ Jan Martinussen, unpublished results.

	ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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