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Originally published In Press as doi:10.1074/jbc.M412415200 on December 8, 2004

J. Biol. Chem., Vol. 280, Issue 7, 5456-5467, February 18, 2005
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Catalytic Mechanism of Chlamydia trachomatis Flavin-dependent Thymidylate Synthase*

Jonathon Griffin{ddagger}, Christine Roshick{ddagger}§, Emma Iliffe-Lee{ddagger}, and Grant McClarty{ddagger}§

From the {ddagger}National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba R3E 3R2 and the §Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba R3E 0W3, Canada

Received for publication, November 3, 2004 , and in revised form, December 7, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Here we report on a Chlamydia trachomatis gene that complements the growth defect of a thymidylate synthase-deficient strain of Escherichia coli. The complementing gene encodes a 60.9-kDa protein that shows low level primary sequence homology to a new class of thymidylate-synthesizing enzymes, termed flavin-dependent thymidylate synthases (FDTS). Purified recombinant chlamydial FDTS (CTThyX) contains bound flavin. Results with site-directed mutants indicate that highly conserved arginine residues are required for flavin binding. Kinetic characterization indicates that CTThyX is active as a tetramer with NADPH, methylenetetrahydrofolate, and dUMP required as substrates, serving as source of reducing equivalents, methyl donor, and methyl acceptor, respectively. dTMP and H4folate are products of the reaction. Production of H4folate rather than H2folate, as in the classical thymidylate synthase reaction, eliminates the need for dihydrofolate reductase, explaining the trimethoprim-resistant phenotype displayed by thyA– E. coli-expressing CTThyX. In contrast to the extensively characterized thyA-encoded thymidylate synthases, which form a ternary complex with substrates dUMP and CH2H4folate and follow an ordered sequential mechanism, CTThyX follows a ping-pong kinetic mechanism involving a methyl enzyme intermediate. Mass spectrometry was used to localize the methyl group to a highly conserved arginine, and site-directed mutagenesis showed this arginine to be critical for thymidylate synthesizing activity. These differentiating characteristics clearly distinguish FDTS from ThyA, making this class of enzymes attractive targets for rational drug design.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Chlamydiae are obligate eubacterial intracellular parasites consisting of four species (1–4). Chlamydia trachomatis is primarily a human pathogen. There are numerous serovars with serovars D–K being a leading cause of bacterial sexually transmitted disease, and serovars A–C responsible for trachoma, which is the number one cause of infectious blindness (3). Chlamydia pneumoniae is a common human pathogen causing acute infection of the respiratory tract and has been identified as a potential risk factor for cardiovascular disease (5). Chlamydia psittaci and Chlamydia pecorum are responsible for a variety of diseases in avian and animal species (6, 7). Chlamydiae are extremely successful intracellular pathogens, in part due to their unique biphasic developmental cycle consisting of two morphologically and biochemically distinct forms. The elementary body is the metabolically inactive extracellular form capable of initiating infection. The reticulate body is the metabolically active intracellular form, which divides by binary fission within the confines of a membrane-bound vacuole, termed an inclusion (1).

Chlamydiae are capable of transporting NTPs but not dNTPs directly from the host cell (8–10). Chlamydiae contain nrdA and nrdB, encoding the two subunits of ribonucleotide reductase required for the conversion of ribonucleotides to deoxyribonucleotides. Ribonucleotide reductase accounts for the acquisition of three (dCTP, dGTP, and dATP) of the four deoxyribonucleotides needed for DNA biosynthesis. The fourth nucleotide, dTTP, is produced by two well known processes. Exogenous thymidine can be directly salvaged by thymidine kinase or dTMP can be synthesized de novo from dUMP, a reaction catalyzed by thymidylate synthase (ThyA) (11, 12).

In an earlier study, using mutant cell lines with deficiencies in thymidine kinase and dihydrofolate reductase (DHFR)1 as host, we reported that C. trachomatis was capable of incorporating exogenously added uridine into thymidine nucleotides, a result implying the existence of a thymidylate synthase and a dihydrofolate reductase (13). Most interestingly, subsequent in silico analyses of whole genome sequence data indicated that chlamydiae encode a DHFR homologue; however, there was no homologue for thymidine kinase or ThyA (14–18). This left open the question of how chlamydiae obtain thymidine nucleotides.

Until recently, ThyA was thought to represent the only enzyme capable of catalyzing the de novo formation of dTMP in vivo. ThyA carries out the reductive methylation of dUMP, using methylenetetrahydrofolate (CH2H4folate) as a one-carbon donor and source of reducing equivalents, generating dTMP and dihydrofolate (H2folate) as products. Because reduced folates are essential for many biochemical processes, H2folate is rapidly reduced to H4folate by DHFR with subsequent regeneration of CH2H4folate being catalyzed by serine hydroxymethyltransferase (11, 19). Together these reactions are known as the thymidylate cycle.

Recently the existence of a novel family of thymidylate-synthesizing enzymes, called thymidylate synthase complementing proteins or flavin-dependent thymidylate synthases (FDTS), encoded by thyX, has been described (19–24). All members of the family contain a conserved ThyX motif consisting of ((T/R)HRX7–8S) (19–21, 23). The first discovered member of this family was a gene encoding a protein shown to complement a thymidine-requiring mutant of Dictyostelium discoideum (25). In silico analyses indicate that homologues of FDTS are present in upwards of 30% of sequenced microbial genomes (19, 20, 26, 27). Most interestingly, many of the genomes containing ThyX lack DHFR (19, 26). This information coupled with the observation that NADH or NADPH are required for Helicobacter pylori FDTS dTMP synthesis activity has led to the suggestion that CH2H4folate acts solely as a one-carbon donor producing H4folate as the product, allowing for the conservation of reduced folates (19–21, 24, 26, 28). It has been demonstrated recently that the reduced pyridine nucleotide required in the FDTS reaction is involved in reducing the enzyme-associated FAD molecule prior to substrate interaction with the enzyme (24). This involvement of an alternative reducing agent in FDTS reactions could account for the lack of a DHFR homologue in many organisms encoding thyX.

Several years ago we isolated a clone from a C. trachomatis genomic DNA library that complemented an E. coli thyA mutant to thymidine prototrophy (29). The complementing open reading frame showed no homology to proteins deposited in the public data bases at that time. Results from the C. trachomatis serovar D genome sequencing project (14) indicated that the complementing open reading frame is encoded by CT632 and subsequent sequencing projects showed that CT632 is highly conserved in all chlamydiae species (14–18). Cross-species in silico analyses have shown that, although CT632 (60.9 kDa) is twice as large as typical FDTS ({approx}26–30 kDa) proteins and shows very low primary sequence homology to them, it does contain a partial sequence motif characteristic of FDTS proteins (19, 20). Here we report the kinetic characteristics of chlamydial FDTS.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals, Bacterial Strains, and Plasmids—[6-3H]dUMP (22 Ci/mmol) and [5-3H]dUMP(15 Ci/mmol) were purchased from Moravek Biochemicals, Brea, CA. H2folate, H4folate, dTMP, dUMP, NADPH, NADH, FMN, FAD, and thymidine were purchased from Sigma. CH2H4folate was donated by Merck. For H4folate and CH2H4folate, the biologically relevant R-stereoisomers were used in all assays. All other chemicals were of reagent grade or better. Escherichia coli DH5{alpha} [supE44{Delta}lacU169 ({Phi}80lacZ{Delta}M15) hsdR17recA1 endA1 gyrA96 thi-1 relA1] is routinely maintained in our laboratory, and E. coli MH2707 (LAM– e14– thi-1 relA1 thyA114(Stable)::Mu) was obtained from the E. coli Genetic Stock Center (New Haven, CT). The pQE80L plasmid was purchased from Qiagen.

Cloning of C. trachomatis ThyX and E. coli ThyA—C. trachomatis thyX was PCR-amplified from purified chromosomal DNA as described previously (30). The PCR primer sequences for CTThyX are forward, 5'-CCCCGGTACCATGTTGAGCAAAGAG-3', and reverse, 5'-CCCCAAGCTTTTAAGACTTTTTACG-3', and were designed to include unique KpnI and HindIII restriction sites (underlined) for cloning. The PCR products were gel-purified, restricted, and then ligated into the expression vector pQE80-L, which had been cut with the same restriction enzymes. Constructs were transformed into DH5{alpha} for screening, purified by miniprep, and then used to transform MH2720 E. coli for complementation and expression of recombinant His-tagged CTThyX.

Expression and Purification of Recombinant C. trachomatis FDTS— E. coli strains DH5{alpha} and MH2720 transformed with wild type or various mutant CTThyX plasmid constructs were grown in 1 liter of LB media containing 100 µg/ml ampicillin at 37 °C to an A of 0.6 at 595 nm. The plasmids were induced by the addition of IPTG to a concentration of 1 mM and were further incubated at 37 °C for 3 h. Cultures were harvested by centrifugation, and the pellet was resuspended in 20 ml of binding buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9) and frozen at –80 °C. After thawing on ice, the cells were lysed by sonication in the presence of lysozyme (at a final concentration of 350 µg/ml). Cell lysates were clarified by ultracentrifugation at 45,000 rpm in a Beckman Ti60 rotor for 2 h at 4 °C. All remaining steps were carried out at 4 °C. Recombinant His-tagged protein was purified from the supernatant by metal chelation chromatography. Briefly, clarified lysates were passed through a 3-ml activated nickel metal chelation column followed by washing with 15 ml of binding buffer and 15 ml of wash buffer (60 mM imidazole, 500 mM NaCl, 20 ml Tris-HCl, pH 7.9). The bound recombinant protein was then eluted off the column with elution buffer (200 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9). Glycerol was added to the purified protein at a final concentration of 10% to prevent precipitation. Purified CTThyX was dialyzed overnight at 4 °C in the dark against 50 mM Tris-HCl, pH 7.2, containing 5% glycerol. Samples were then aliquoted and stored at –80 °C.

Measurement of Protein Concentration—Protein concentrations were estimated by using the Bio-Rad protein assay based on the dye-binding procedure of Bradford (31) using bovine serum albumin as the standard.

Spectroscopic Analysis—The absorption spectrum of FAD bound to wild type and mutant CTThyX was determined. FAD was extracted from purified CTThyX by incubating at 95 °C for 10 min in the dark. Precipitated protein was pelleted by centrifugation at 10,000 rpm (Eppendorf centrifuge 5417C) for 10 min. The absorption spectra of the released flavin present in the supernatant was determined spectrophotometrically (Beckman Du62 Spectrophotometer) from 250 to 750 nm in a 1-cm quartz cuvette (19).

Site-directed Mutagenesis—The pQE80L plasmid containing wild type C. trachomatis thyX was used as a template for site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Primers containing the desired mutations were designed based on the consensus nucleotide sequence of the C. trachomatis serovar D CT632 gene (thyX) (GenBankTM accession number AE001334 [GenBank] ) and are shown in Table I. All plasmid constructs were confirmed for appropriate mutations by DNA sequencing.


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  		TABLE I
Primers used for site-directed mutagenesis of C. trachomatis FDTS

 
Complementation—MH2720 cells transformed with pQE80L-CTThyX, pQE80L-CTThyXR124A, pQE80L-CTThyXS133A, pQE80L-CTThyXR397A, pQE80L-CTThyXR477A, or pQE80L (no insert, vector control) were grown overnight at 37 °C in LB containing 100 µg/ml ampicillin and 40 µg/ml thymidine. One ml of the overnight culture was centrifuged at 6000 rpm for 5 min (Eppendorf centrifuge 5417C), and the supernatant was removed. The pellet was then washed three times in ice-cold sterile phosphate-buffered saline. The cells were streaked onto minimal medium plates containing IPTG (200 µl of 100 mM IPTG added to the surface and allowed to dry) and either no thymidine or 40 µg/ml thymidine. The plates were incubated at 37 °C overnight and then photographed.

High Performance Liquid Chromatography—Nucleotides were separated by high performance liquid chromatography (HPLC) using a 12.5-cm C18 reverse phase column under isocratic conditions with a flow rate of 1 ml/min, the mobile phase consisted of 5 mM potassium phosphate buffer, pH 7.0, 5 mM tetrabutylammonium dihydrogen phosphate, and 5% (v/v) acetonitrile (32). Radioactive peaks were detected by monitoring in-line radioactive flow (171 detector; Beckman Instruments). The identities of the radioactive peaks were confirmed by simultaneously monitoring the A260 (1066 UV detector; Beckman Instruments) of dTMP and dUMP standards. Folates were separated using a 25-cm C18 column under isocratic conditions with a mobile phase consisting of 10 mM ammonium phosphate, pH 7.0, 5 mM tetrabutylammonium phosphate, 5% acetonitrile, 20% methanol and a flow rate of 1 ml/min. The buffer was extensively degassed and then maintained in a nitrogen gas environment in order to enhance the stability of the reduced folates. Peaks were detected by monitoring the A295. The identities of the individual peaks were determined by comparison with known H2folate, H4folate, and CH2H4folate standards.

Thymidylate Synthesis Assay—Thymidylate synthesizing activity was determined by monitoring the amount of tritium transferred to water using [5-3H]dUMP as substrate similar to that described for assaying thymidylate synthase activity and H. pylori FDTS activity (20, 33). All reactions were performed at 37 °C in a nitrogen gas environment to limit reduced folate decomposition. The optimized standard reaction mixture contained, in a total volume of 100 µl, 50 mM Tris-HCl, pH 7.2, 200 µM 5,10-CH2H4folate, 2 mM NADPH, 200 µM [5-3H]dUMP (15 µCi/ml), 5% glycerol. The reaction was initiated by the addition of 2.5 µg of purified CTThyX. After 3 min, the reaction was terminated by the addition of 300 µl of a 100 mg/ml activated charcoal suspension containing 2% trichloroacetic acid to remove the unused radiolabeled substrate [5-3H]dUMP. The samples were mixed at room temperature for 1 h and then centrifuged at 14,000 rpm (Eppendorf centrifuge 5417C) for 5 min to pellet the charcoal. Radioactivity in the supernatant was quantitated in a Beckman LS 5000 liquid scintillation counter after the addition of 5 ml of scintillation fluid (Universol, ICN Biomedical). One unit of enzyme activity corresponds to the production of 1 mmol of dTMP synthesized per min under optimum assay conditions. For the thymidylate-synthesizing half-reaction, a standard reaction mixture was used except that the dUMP was omitted. E. coli thymidylate synthase reaction was carried out as described previously (11).

Kinetic Analysis—Substrate saturation kinetics were determined for dUMP, CH2H4folate, and NADPH. The kinetic reaction for each substrate was carried out at fixed saturating concentrations of the other two substrates. In all cases, at least 11 different concentrations of the variable substrate were used in each set of experimental assays.

Multiple substrate kinetics were determined by varying the concentration of dUMP (0, 2.5, 10, 20, 40, and 75 µM) in the presence of various fixed concentrations of CH2H4folate (10, 25, 50, 100, and 200 µM). Product inhibition kinetics for dUMP were determined by varying the concentration of dUMP (0, 2.5, 10, 20, 40, and 75 µM) in the presence of varying fixed concentrations of either dTMP (0, 10, 25, 50, and 100 µM) or H4folate (0, 10, 25, 50, and 100 µM). CH2H4folate (200 µM) and NADPH (2 mM) were present at saturating concentrations. Product inhibition kinetics for CH2H4folate were determined by varying the concentration of CH2H4folate (0, 10, 20, 50, 100, and 200 µM) in the presence of varying fixed concentrations of dTMP (0, 25, 50, 100, and 200 µM) or H4folate (0, 25, 50, 100, and 200 µM). dUMP (200 µM) and NADPH (2 mM) were present at saturating concentrations.

All the measurements were made in triplicate, and the mean and S.E. were determined using GraphPad PRISM 3.0 software. The Michaelis-Menten equation was used when hyperbolic kinetics were obtained; Km = the substrate concentration giving one-half the maximal velocity (Vmax) (34). Lineweaver-Burk transformations were used for the determination of the inhibition mechanism. These calculations were fitted using a nonlinear least-squares regression and linear least squares regression computer kinetics program supplied by GraphPad PRISM 3.0 software.

Mass Spectrometric Analysis—To determine where chlamydial FDTS was methylated, highly purified CTThyX was used as a source of enzyme in a thymidylate-synthesizing half-reaction. Following incubation for 5 min at 37 °C, SDS sample buffer was added, and the sample was run on a 10% SDS-polyacrylamide gel. The FDTS band was excised and sent to the Scripps Center for Mass Spectrometry (La Jolla, CA, //masspec.scripps.edu) for analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of a C. trachomatis Thymidylate Synthase Complementing Protein—We employed functional complementation to clone a C. trachomatis gene that rescues the thymidine auxotrophy of an E. coli thyA mutant, MH2720 (Fig. 1a) (29). Subsequent sequencing and in silico analyses indicated that the encoded open reading frame corresponds to C. trachomatis CT632, a highly conserved 60.9-kDa chlamydial protein. The amino acid identity between CT632 homologues identified from all sequenced chlamydial genomes is between 71 and 91% (14–18). CT632 shows very low primary sequence homology with the newly identified ThyX family of folate-dependent thymidylate synthases (19, 20) (Fig. 2). CTThyX (60.9 kDa) is also approximately twice the molecular weight of most other FDTS proteins ({approx}26–30 kDa). From in silico analyses, it has been suggested that the CTThyX is a fusion of two individual subunits (20). Although much less common than the smaller FDTS proteins, which occur in upwards of 30% of all sequenced bacterial genomes, there are several homologues to CTThyX in the protein data base, and all contain at least a partial ThyX motif ((T/R)HRX7–8S) (19–21, 23, 27) (Fig. 2b). FDTS from C. trachomatis does not contain a fully conserved ThyX motif, as indicated in the amino acid sequence alignment (Fig. 2). There are two regions, one in the N-terminal region and one in the C-terminal region that have a partial ThyX motif and that may complement each other in the tertiary structure to form the complete motif. The N-terminal region contains DARX8S, whereas the C-terminal region contains RHRX7/8(L/L).



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  		FIG. 1.
Characteristics of C. trachomatis open reading frame CT632. a, analysis of CT632 protein function by genetic complementation. The gene-encoding open reading frame CT632 was cloned into E. coli expression vector pQE-80L. Vector without insert was used as a control. Constructs were transformed into E. coli MH2720 (thyA–), and growth of the transformants was assessed on M9 minimal agar plus IPTG with or without added thymidine. Sensitivity of E. coli MH2720 transformed with C. trachomatis CT632 or E. coli thyA, as a control, to trimethoprim (5 µg), sulfonamide (300 µg), or trimethoprim (1.25 µg)/sulfamethoxazole (3.75 µg) is shown. Cultures were grown overnight in LB medium, spun down, washed three times in phosphate-buffered saline, and then spread on Mueller-Hinton agar plates containing 1 mM IPTG and the indicated antibiotic disks. b, sensitivity of E. coli MH2720 transformed with C. trachomatis CT632 or E. coli thyA to 5-fluorodeoxyuridine. Cultures were grown overnight in LB broth and then diluted to an OD of ~0.2 in Mueller-Hinton broth plus 0.2 mM IPTG. The indicated concentrations of 5-fluorodeoxyuridine were added, and growth was monitored by measuring the OD. •, 0 µM; {square}, 0.62 µM; {blacksquare}, 1.25 µM; 224, 2.5 µM; {blacktriangleup}, 5 µM; and {circ}, 10 µM FdUMP.

 



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  		FIG. 2.
Comparison of various FDTS amino acid sequences. a, H. pylori, T. maritima, Chlorella virus-1, and C. trachomatis FDTS (GenBankTM accession numbers 026061, Q9WYT0, 041156, and NP220149, respectively) were aligned using ClustalW. C. trachomatis CT632 was divided into N- and C-terminal domains based on in silico analyses suggesting that a duplication event has occurred (28) b, C. trachomatis, Parachlamydia, Thermoplasma acidophilum, and Thermoanaerobacter tengcongensis (accession numbers NP220149, YP007154, NP623772, and CAC12549 [GenBank] respectively) were aligned using ClustalW. The residues mutagenized in this study are in boldface and marked with a # sign. The methyl-accepting arginine is in boldface and underlined. Putative thyX motifs ((T/R)HRX7–8S) are boldface italic.

 
In contrast with the phenotype displayed by recombinant E. coli expressing E. coli ThyA, CTThyX confers trimethoprim resistance to an E. coli thyA mutant strain (Fig. 1a), and a similar result was reported for H. pylori ThyX (19, 24). Similar to ThyA-expressing recombinant E. coli, CTThyX recombinants were still sensitive to sulfonamide and sulfamethoxazol-trimethoprim (Fig. 1a) and 5-fluorodeoxyuridine (Fig. 1b), characteristics typical of classical thymidylate synthases (11).

Expression, Purification, and Spectroscopic Characterization of C. trachomatis FDTS—Recombinant C. trachomatis FDTS was overexpressed in E. coli MH2720, using the pQE80L expression system. Subsequent purification by nickel affinity chromatography typically yielded 5–10 mg/liter of soluble purified CTThyX. On SDS-polyacrylamide gel electrophoresis, recombinant CTThyX showed one predominant band with an apparent molecular mass of {approx}61 kDa (Fig. 3a). The purified protein was bright yellow in color, suggesting the presence of an enzyme-bound flavin molecule, similar to that reported for H. pylori and Thermotoga maritima FDTS (19, 21). Spectroscopic studies on the flavin extracted from the enzyme indicated that it was in fact an oxidized flavin, with the characteristic flavin peak at 260 nm, and peaks at 375 and 450 nm indicating that it is in the oxidized form (Fig. 3b) (19, 21, 24). These results are analogous to those obtained for the H. pylori and T. maritima ThyX where an oxidized flavin is bound to the ThyX protein (19, 24). Further HPLC analysis comparing the isolated flavin to FAD and FMN standards demonstrated that the enzyme-bound flavin is in fact FAD (data not shown). Native PAGE revealed a native molecular mass of {approx}250 kDa, indicating that the native protein is in the form of a tetramer (data not shown). This is similar to the H. pylori FDTS that has also been shown to exist as a tetramer, in contrast E. coli ThyA forms dimers (11, 19, 21, 35).



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  		FIG. 3.
Biochemical analysis of recombinant C. trachomatis FDTS. a, purified sample of {approx}5 µg of C. trachomatis recombinant FDTS was run on a 10% SDS-polyacrylamide gel along with the molecular weight standards. Sizes are indicated in kilodaltons. b, spectroscopic analysis of flavin released from C. trachomatis FDTS. FAD was extracted from purified CTThyX by incubating at 95 °C for 10 min in the dark. The absorption spectrum of the released flavin present in the supernatant was determined spectrophotometrically.

 
Optimization of the dTMP Synthesizing Activity of C. trachomatis FDTS—To demonstrate conclusively that purified recombinant CTThyX was capable of catalyzing the formation of dTMP from dUMP, an in vitro assay was developed and optimized. CTThyX dTMP formation activity was absolutely dependent on dUMP, CH2H4folate, and a reduced pyridine nucleotide (NADH or NADPH). With regards to pyridine nucleotides, the maximal velocity with NADH (Vmax = 88.57) is less than half that with NADPH (Vmax = 192.84). Because NADPH is the preferred substrate, it was used in all subsequent assays. CTThyX showed maximal activity at pH 7.2, and activity was linear with respect to time and enzyme concentration (data not shown).

Folate Oxidation of C. trachomatis FDTS—It has been proposed that like FADH2-dependent ribothymidyl synthase (36), FDTS uses CH2H4folate solely as a one-carbon donor with enzyme-bound flavin serving as the source of reducing equivalents (19–22, 24, 26, 28). To determine directly whether H4folate is a product of the CTThyX-catalyzed reaction, we analyzed the folate products by HPLC. We compared the activity of CTThyX with that of E. coli ThyA, to demonstrate the difference between the two families of proteins. In contrast to the ThyA (Fig. 4a), which produces H2folate, CTThyX produces H4folate as a product of the thymidylate-synthesizing reaction (Fig. 4b).



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  		FIG. 4.
Analysis of folate products following thymidylate-synthesizing reaction. a, E. coli ThyA; b, C. trachomatis FDTS thymidylate-synthesizing reactions were run with and without added enzyme as described under "Experimental Procedures." Folate products were separated by HPLC using a 25-cm C18 reverse phase column under isocratic conditions with a flow rate of 1 ml/min. The identity of the peaks was confirmed by comparison to known standard CH2H4folate (17.5 min), H2folate (14.2 min), and H4folate (8.2 min). The reaction with enzyme is indicated by the solid line, and the dotted line represents the run in the absence of enzyme.

 
Saturation Kinetics—CTThyX displayed Michaelis-Menten kinetics with respect to dUMP, CH2H4folate, and NADPH. Fig. 5 shows that CTThyX exhibits hyperbolic kinetics with respect to varying concentrations of dUMP, CH2H4folate, or NADPH, respectively, under saturating conditions of the other two substrates. Apparent Km values for dUMP, CH2H4folate, and NADPH are 5.99 ± 0.54, 22.66 ± 2.02, and 60.80 ± 4.86 µM, respectively. Vmax is ~165 ± 3.3 7 mmol of dTMP produced per min/mg. The kcat was determined to be 39.98 ± 1.77 min–1.



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  		FIG. 5.
Michaelis-Menten saturation kinetics of C. trachomatis FDTS. Kinetics were determined for dUMP (a), CH2-H4folate (b), and NADPH (c). Enzyme activity was determined at various concentrations of the indicated substrates, with all other substrates at fixed saturating concentrations as described under "Experimental Procedures." The assays were run in triplicate, and the mean ± S.E. are shown.

 
Two-substrate Kinetics—The ThyA-catalyzed reaction follows an ordered sequential mechanism (11, 37). To determine the kinetic mechanism of CTThyX dTMP synthesis activity was measured at several fixed concentrations of CH2H4folate, while varying the concentration of dUMP. The result shown in the double-reciprocal plot is that of several parallel lines indicating that CTThyX follows a ping-pong kinetic mechanism (Fig. 6a). For comparison, the two-substrate kinetics of E. coli ThyA was carried out at several fixed concentrations of CH2H4folate while varying dUMP concentrations. The results presented in Fig. 6b show lines intersecting to the left of the y axis, indicating a sequential mechanism.



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  		FIG. 6.
Double-reciprocal plot of initial velocity data for C. trachomatis FDTS and E. coli ThyA with dUMP as the variable substrate. Enzyme activity was determined with various concentrations of CH2H4folate and dUMP, under saturating conditions of NADPH as described under "Experimental Procedures." a, C. trachomatis FDTS; the concentrations of CH2H4folate were 10 µM ({blacktriangledown}), 25 µM ({triangledown}), 50 µM ({diamond}), 100 µM ({blacktriangleup}), and 200 µM (•). b, E. coli ThyA; the concentrations of CH2H4folate were 5 µM ({blacktriangledown}), 10 µM ({triangledown}), 20 µM ({diamond}), 50 µM ({blacktriangleup}), and 100 µM (•). The assays were run in triplicate, and the means ± S.E. are shown.

 
Product Inhibition—To deduce the order of substrate binding and product exit from the active site, enzyme kinetics were carried out in the presence of varying concentrations of dTMP and H4folate. With dUMP as the variable substrate, hyperbolic saturation curves were obtained when CTThyX was assayed in the presence of various fixed concentrations of dTMP. Increasing the concentration of dTMP increased the Km for dUMP and decreased the Vmax. Double-reciprocal plots of the saturation data for the different dTMP concentrations gave a series of straight lines, which intersect at a point to the left of the y axis, indicating that dTMP is a mixed noncompetitive inhibitor with respect to dUMP (Fig. 7a). The inhibition constant (Ki) was determined to be 10 µM. Hyperbolic saturation curves were also obtained for CTThyX when dUMP was the variable substrate in the presence of several fixed concentrations of H4folate having a Ki of 170 µM. Increasing the concentration of H4folate increased the Km of CTThyX for dUMP but had no effect on the Vmax. The double-reciprocal plots of the saturation data for the different concentrations of H4folate gave a series of straight lines, which intersect at a point on the y axis (Fig. 7b), indicating competitive inhibition between H4folate and dUMP.



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  		FIG. 7.
Double-reciprocal plot of product inhibition data for C. trachomatis FDTS. a and b, dUMP; c and d, CH2-H4folate as the variable substrates; a and c, dTMP; or b and d, H4folate the product inhibitors. The reactions were conducted as described under "Experimental Procedures." a and b, the concentrations of dTMP or H4folate were 0 µM ({blacktriangledown}), 10 µM ({triangledown}), 25 µM ({diamond}), 50 µM ({blacktriangleup}), and 100 µM (•). c and d, the concentrations of dTMP or H4folate were 0 µM ({blacktriangledown}), 25 µM ({triangledown}), 50 µM ({diamond}), 100 µM ({blacktriangleup}), and 200 µM (•). The assay was run in triplicate, and the means ± S.E. are shown.

 
With CH2H4folate as the variable substrate, hyperbolic saturation curves were obtained when CTThyX was assayed in the presence of fixed dTMP or H4folate concentrations. Increasing the concentration of dTMP increased the Km of CTThyX for CH2H4folate and had no effect on the Vmax of the enzyme, giving a Ki of 290 µM. A double-reciprocal plot of the saturation data for the different concentrations of dTMP shows a series of straight lines intersecting at the y axis (Fig. 7c). These results show that dTMP is a competitive inhibitor with respect to CH2H4folate. Increasing concentrations of H4folate increased the Km value for CH2H4folate but decreased the Vmax value of CTThyX, giving a Ki of 115 µM. The double-reciprocal plot again produced a series of straight lines, but the lines intersect the x axis to the left of the y axis (Fig. 7d), indicating that H4folate follows classical noncompetitive inhibition with respect to CH2H4folate. The pattern of product inhibition for CTThyX is again consistent with a ping-pong kinetic mechanism.

C. trachomatis FDTS Half-reaction—A characteristic feature of ping-pong kinetic mechanisms is that a secondary product is released from the enzyme, prior to the final substrate entering the active site. In the case of the CTThyX, H4folate would be generated and released from the enzyme active site, prior to dUMP entering. To verify that CTThyX does follow a ping-pong mechanism as demonstrated by the enzyme kinetics, a standard CTThyX assay was carried out in the absence of dUMP, the methyl acceptor. The production of H4folate was followed by HPLC. As shown in Fig. 8, H4folate is produced in an enzyme-dependent manner in the absence of dUMP.



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  		FIG. 8.
C. trachomatis FDTS half-reaction. The standard C. trachomatis FDTS assay reaction was carried out in the absence of dUMP with saturating amounts of NADPH (2 mM) and CH2H4folate (200 µM). Folate products were separated by HPLC using a 25-cm C18 reverse phase column under isocratic conditions with a flow rate of 1 ml/min. Positions of peaks were determined by comparison with standard CH2H4folate (12.5 min) and H4folate (6.5min). The dotted line indicates the reaction incubated with C. trachomatis FDTS, and the solid line represents a reaction incubated without enzyme. Inset, blow up of the CH2H4folate peak spanning 12–14 min.

 
Site-directed Mutagenesis—As indicated in Fig. 2, CTThyX has two regions, each containing a portion of the conserved ThyX motif. To determine the functional role of these two regions in FDTS catalysis, we employed site-directed mutagenesis. Several site-specific mutations were constructed as summarized in Table II and shown in Fig. 2. Arg-124 and Ser-133 of the N-terminal region (T. maritima Arg-80 and Ser-88) as well as Arg-397 (equivalent to T. maritima Arg-80) in the C-terminal ThyX motif were all mutated to alanine.


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  		TABLE II
Functional complementation and in vitro activity of CTThyX mutant proteins

 
The ability of the various mutant CTThyX proteins to functionally complement the growth defect of an E. coli thyA mutant was assessed. The results presented in Fig. 9 indicate that all of the mutant ThyX proteins lost their ability to complement the thymidine auxotrophy of a thyA– strain of E. coli. To explore the loss of activity in more detail, we overexpressed and purified the CTThyX mutant proteins. Two of the mutant proteins (R124A and R397A) lacked the yellow color characteristic of flavin-containing enzymes. Absorption spectroscopy confirmed that mutation of these conserved arginine residues resulted in a loss of enzyme-bound flavin (data not shown). In vitro dTMP synthesizing activity was assessed, and as shown in Table II, R124A and R397A CTThyX proteins were inactive, confirming the essential role of FAD for CTThyX activity. The S133A mutant protein retained its ability to bind flavin; however, the protein was inactive in vitro.



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  		FIG. 9.
Analysis of mutant C. trachomatis FDTS enzymatic function by genetic by complementation. In vitro mutagenized C. trachomatis FDTS constructs pQE80L-CTThyXR124A, pQE80L-CTThyXS133A, pQE80L-CTThyXR397A, pQE80L-CTThyXR477A, and pQE80L vector control were transformed into E. coli MH2720 (thyA–), and growth of the transformants was assessed on M9 minimal agar plus IPTG with or without added thymidine.

 
From the kinetic analysis CTThyX follows a ping-pong kinetic mechanism, which implies that there is a methyl-enzyme intermediate formed during the reaction. Mass spectrometry was employed to elucidate the peptide fragment that harbors the methyl group. MALDI-TOF was carried out on trypsin-digested CTThyX after the thymidylate-synthesizing protein half-reaction. Results from the analysis revealed a peptide corresponding to amino acid residues 469–477 (GLQWLCELR), which showed a mass shift of 14 atomic mass units following the methyl-donating half-reaction (Fig. 10, peak 1188.6). The only residue within the identified peptide that is appropriately located in the active site to be a methyl donor, conserved in all FDTS proteins deposited in the public data bases, and that can accept a methyl group is Arg-477. Arg-477 was changed to alanine by site-directed mutagenesis. Fig. 9 shows that the R477A CTThyX protein lost its ability to complement an E. coli thyA– mutant to thymidine prototrophy. The mutant protein was overexpressed and purified and was shown to retain the ability to bind FAD. In agreement with the complementation results, the mutant protein lacked in vitro dTMP synthesizing activity (Table II).



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  		FIG. 10.
MALDI-TOF mass spectrometric analysis of purified recombinant C. trachomatis FDTS following the thymidylate-synthesizing half-reaction. The thymidylate-synthesizing half-reaction was carried out as described under "Experimental Procedures." MALDI-TOF was carried out on trypsin-digested CTThyX after the thymidylate-synthesizing protein half-reaction. Only those peaks with a mass between 1000 and 2400 are displayed. Peptide 1188.6 represents residues 469–477 (GLQWLCELR) and shows an increase of 14 atomic mass units in calculated mass as compared with the expected mass.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In silico analyses indicate that C. trachomatis open reading frame CT632 encodes a protein that is highly conserved (66% identity and 88.9% similarity) in all five chlamydial genomes (C. trachomatis serovar D, Chlamydia muridarium, Chlamydia caviae, Chlamydia abortus, and C. pneumoniae) sequenced to date (14–16, 18), including the more distantly related parachlamydia, a chlamydiae-like symbiont of free living amoeba (38) and that it is distantly related to the FDTS family of proteins. Here we show that CT632 can complement a thyA mutant strain of E. coli, and in vitro assays confirmed that the protein possesses thymidylate synthesizing activity. Purified recombinant CT632 contains bound flavin. Thus, taken together it is clear that CT632 encodes a chlamydial FDTS homologue. We demonstrated that CTThyX uses CH2H4folate strictly as a one-carbon donor and employs an enzyme-bound FAD molecule as source of reducing equivalents. Results from in vitro mutagenesis studies indicate that the highly conserved arginines (Arg-124 and Arg-397) are essential for flavin binding and FDTS activity. This is in agreement with structural studies with the T. maritima FDTS which show that the counterpart arginine (Arg-80) interacts with FAD (21). Mutagenesis studies with H. pylori FDTS indicate that the earlier arginine in the ThyX motif (Arg-74) is also essential for FAD binding and thymidylate synthesizing activity (20). For CTThyX the counterpart of H. pylori Arg-74 is conserved in the C-terminal half (Arg-395) but not in the N-terminal half (Asp-122) of the enzyme. In addition, similar to studies with H. pylori FDTS (20), our in vitro mutagenesis results indicate that CTThyX Ser-133, the conserved serine in the N-terminal ThyX motif ((T/RHRX7–8S) H. pylori Ser-84, T. maritima Ser-88) is essential for thymidylate synthesizing activity. It has been proposed that this serine activates dUMP by nucleophilic attack at its C-6 position (20, 24). Most interestingly, a leucine occupies the seventh and eighth amino acid positions downstream of the C-terminal ThyX motif in chlamydial FDTS.

In using CH2H4folate solely as a one-carbon donor, H4folate is produced, and the need for reduced folate recycling by DHFR is eliminated (Fig. 11a). This would account for the absence of folA in many of the thyX-containing genomes (19) and the trimethoprim-resistant phenotype displayed by thyA mutant E. coli strains expressing recombinant H. pylori FDTS and CTThyX. Most interestingly, all chlamydiae encode a DHFR homologue (14, 15, 18). This is not surprising as C. trachomatis can carry out de novo folate synthesis with DHFR being an essential enzyme in this pathway (39). Presumably, other organisms that contain an FDTS homologue, but lack DHFR, are auxotrophic for reduced folates or use an alternate enzyme, with little or no homology to DHFR, for reducing folate.



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  		FIG. 11.
Thymidylate synthase cycle and Cleland plots of the reaction sequence of C. trachomatis FDTS and E. coli ThyA. a, schematic representation of proposed C. trachomatis thymidylate synthesis cycle. Schematic representation of the order of substrate binding and product release during E. coli ThyA (11) (b), and C. trachomatis FDTS catalytic cycle (c).

 
Substrate saturation kinetics demonstrated that CTThyX follows standard Michaelis-Menten kinetics with respect to dUMP, CH2H4folate, and NADPH. The apparent Km values for CTThyX value for dUMP was determined to be 5.99 ± 0.54 µM, which is comparable with FDTS of Chlorella virus-1 (Km = 15 µM) (40) and ThyA of E. coli (Km = 10 µM) (41) and Lactobacillus casei (Km = 0.7 µM) (37). The CTThyX Km value for CH2H4folate was determined to be 22.66 ± 2.02 µM, which is similar to that reported for Chlorella virus-1 (Km = 20 µM) FDTS and L. casei (Km = 14 µM) ThyA (37) but much lower than that reported for T. maritima FDTS (Km = 4mM) (24) and ThyA of E. coli (Km = 14 mM) (41). The CTThyX Vmax was estimated to be165 ± 3.3 7 µmol of dTMP produced per min/mg, which is significantly less than in other organisms, with that of E. coli being 1.45 µmol of dTMP produced per min/mg and that of L. casei being 4.39 µmol of dTMP produced per min/mg (37, 41). The kcat of CTThyX was determined from the saturation kinetics to be ~40.0 min–1 (0.67 s–1), similar to that of Chlorella virus-1 (40), whereas the kcat for ThyA has been reported to be ~300 min–1 (5 s–1) (11). For comparison with another slow growing organism, the kcat for the H. pylori ThyX has been reported to be 0.46 min–1 (20). The difference in the specific activity may be attributed to the growth rate of the individual organisms. Faster growth rates such as that for E. coli, with a doubling time of 20 min, would require more efficient thymidylate synthesis to prevent a shortage of dTTP during DNA replication. However, C. trachomatis has a much slower growth rate with a doubling time of ~2 h. The slower growth rate could account for the reduced need for rapid dTMP synthesis, allowing for an enzyme with much lower specific activity.

One distinguishing characteristic of ThyA is its ability to form a stable ternary complex between the enzyme CH2H4folate and 5-fluoro-dUMP (FdUMP), a mechanism-based inhibitor that prevents catalysis. This observation is consistent with the ordered sequential catalytic mechanism of ThyA (11, 33, 37, 42). A Cleland plot depicting the ordered reaction sequence for ThyA is shown in Fig. 11b (33, 37). In contrast, our kinetic analyses demonstrate that the dTMP synthesis reaction catalyzed by CTThyX follows a ping-pong (double displacement) mechanism, which differs markedly from that of ThyA. From the studies of Agrawal et al. (24) with T. maritima FDTS, it has been determined that the first substrate to bind to the enzyme is NADPH (see below). From our studies with CTThyX, we propose that the next substrate to enter the enzyme active site is CH2H4folate, at which point the methyl group is transferred from CH2H4folate to the enzyme, producing H4folate and a methyl enzyme intermediate. Through the use of MALDI-TOF, we have demonstrated that Arg-477 is the most likely methyl group acceptor. In silico analysis indicates that the counterpart of CTThyX Arg-477 is absolutely conserved in all FDTS sequences deposited in the protein data bases. Furthermore, our in vitro mutagenesis results indicate that Arg-477 is essential for thymidylate synthesizing activity as assessed by complementation and in vitro enzyme assay.

The crystal structure of T. maritima FDTS indicates that Arg-477 (T. maritima Arg-174) is located in the active site of the enzyme (21). The only rearrangement that has to take place is a 180° rotation of the dUMP base to put Arg-174 (Chlamydia Arg-477) within 2.8 Å of the carbon (dUMP C-5) that is going to be methylated. Following the transfer of the methyl group from CH2H4folate, H4folate exits the active site, allowing dUMP to enter with subsequent transfer of the methyl group from Arg-477 to the C-5 position of dUMP producing dTMP. dTMP then exits the active site. A ping-pong mechanism is consistent with our inability to detect a ternary complex when purified recombinant CTThyX was incubated with CH2H4folate and 5FdUMP (data not shown), because both substrates are never in the active site at the same time during catalysis. The ping-pong mechanism has also been confirmed by carrying out the enzyme assay in the absence of dUMP (half-reaction) and following the formation of H4folate. With the half-reaction, the formation of H4folate was absolutely dependent on the presence of enzyme and NADPH (data not shown). The unique situation that arises with the ping-pong kinetic mechanism is that there is a methyl enzyme intermediate being formed.

In a recent study it was shown that Chlorella virus-1 FDTS-catalyzed reaction occurs by a different mechanism (40). Although the reaction requires the same three substrates (dUMP, CH2H4folate and NADPH), the order of binding is different, and the mechanism is reported to be sequential rather than ping pong, as shown here for CTThyX. In the Chlorella virus FDTS reaction, dUMP is the first substrate to bind, and its binding is required for NADPH oxidation and subsequent reduction of the enzyme-bound FAD. Following NADP+ release, CH2H4folate binds to the active site, allowing for transmethylation of dUMP. In contrast, with T. maritima FDTS, it has recently been shown that NADPH binding occurs first; enzyme-bound FAD is reduced; NADP exists (first half-reaction); then dUMP and CH2H4folate bind in a sequential fashion (random or ordered), followed by ordered release of H4folate and then dTMP (second half-reaction); thus the overall reaction is ping-pong (24). The first half-reaction was supported by experimental evidence, and the second half-reaction was proposed based on analogy to the classical ThyA reaction (24). Therefore, unlike the CTThyX reaction, there is no methyl-enzyme intermediate, and dUMP and CH2H4folate are proposed to bind in the active site at the same time. Most interestingly, despite different proposed reaction mechanisms and low overall primary sequence homology, all the FDTS enzymes have conserved the counterpart of the CTThyX methyl-accepting Arg-477 (H. pylori Arg-174, T. maritima Arg-174, and Chlorella virus Arg-182).

In summary, the results presented indicate that C. trachomatis contains an active thymidylate-synthesizing enzyme that has been grouped into a novel family of proteins termed FDTS (19–22, 24, 26, 28, 40). We have demonstrated CTThyX employs CH2H4folate solely as a one-carbon donor, and we use an enzyme-bound FAD molecule and an external NADPH as the reducing agents required for reduction of the methyl moiety needed for dTMP biosynthesis. CTThyX catalytic reaction follows a ping-pong mechanism involving a methyl enzyme intermediate whereby Arg-477 accepts the methyl group. Taking all the experimental data into consideration, we propose the order of substrate binding and product release for CTThyX as depicted in the Cleland plot presented in Fig. 11c.


   FOOTNOTES
 
* This work was supported by Grant GR-13301 from the Canadian Institutes of Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington St., Winnipeg, Manitoba R3E 3R2, Canada. Tel.: 204-789-6097; Fax: 204-789-2097; E-mail: mcclart{at}cc.umanitoba.ca.

1 The abbreviations used are: DHFR, dihydrofolate reductase; FDTS, flavin-dependent thymidylate synthases; IPTG, isopropyl 1-thio-{beta}-D-galactopyranoside; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; H4folate, tetrahydrofolate; CH2H4folate, methylenetetrahydrofolate; H2folate, dihydrofolate; FdUMP, fluoro-dUMP. Back


   ACKNOWLEDGMENTS
 
We thank Pal Stenmark, Stockholm University, for helpful discussions on the structure of FDTS and Heidi Wood for reading the manuscript.



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 ABSTRACT
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 DISCUSSION
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