From the Department of Organic Chemistry and Biochemistry, Technische Universität München, Lichtenbergstraße 4, D-85747 Garching, Federal Republic of Germany
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ABSTRACT |
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An open reading frame located at 69.0 kilobases on the Escherichia coli chromosome was shown to code for dihydroneopterin aldolase, catalyzing the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin in the biosynthetic pathway of tetrahydrofolate. The gene was subsequently designated folB. The FolB protein shows 30% identity to the paralogous dihydroneopterin-triphosphate epimerase, which is specified by the folX gene located at 2427 kilobases on the E. coli chromosome. The folX and folB gene products were both expressed to high yield in recombinant E. coli strains, and the recombinant proteins were purified to homogeneity. Both enzymes form homo-octamers. Aldolase can use L-threo-dihydroneopterin and D-erythro-dihydroneopterin as substrates for the formation of 6-hydroxymethyldihydropterin, but it can also catalyze the epimerization of carbon 2' of dihydroneopterin and dihydromonapterin at appreciable velocity. Epimerase catalyzes the epimerization of carbon 2' in the triphosphates of dihydroneopterin and dihydromonapterin. However, the enzyme can also catalyze the cleavage of the position 6 side chain of several pteridine derivatives at a slow rate. Steady-state kinetic parameters are reported for the various enzyme-catalyzed reactions. We propose that the polarization of the 2'-hydroxy group of the substrate could serve as the initial reaction step for the aldolase as well as for the epimerase activity. A deletion mutant obtained by targeting the folX gene of E. coli has normal growth properties on complete medium as well as on minimal medium. Thus, the physiological role of the E. coli epimerase remains unknown. The open reading frame ygiG of Hemophilus influenzae specifies a protein with the catalytic properties of an aldolase. However, the genome of H. influenzae does not specify a dihydroneopterin-triphosphate epimerase.
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INTRODUCTION |
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The pathway of tetrahydrofolate biosynthesis is an important drug target in bacteria and protozoal parasites (1-3). Thus, the sulfonamides inhibiting dihydropteroate synthase were the first synthetic agents that could be used with high efficiency against a wide variety of microbial and protozoal parasites. Even after more than 60 years, they remain important therapeutic agents (1, 4). More recently, trimethoprim was found to act as an inhibitor of bacterial dihydrofolate reductase (5). The combined application of trimethoprim and a sulfonamide is an important protocol for the treatment of bacterial infections (5-8).
Tetrahydrofolate is biosynthesized by plants and many microorganisms. The first committed step catalyzed by GTP cyclohydrolase I is a mechanistically complex ring expansion reaction conducive to the formation of dihydroneopterin triphosphate (Compound 1) (Fig. 1) (9). A pyrophosphatase and a phosphatase have been proposed to convert Compound 1 to dihydroneopterin (Compound 3) in two consecutive steps (10), but firm evidence for the involvement of specific enzymes in the conversion of Compound 1 to Compound 3 has not been obtained. Recently, it has been proposed that the release of pyrophosphate from Compound 1 can be catalyzed of two-valent ions such as Mg2+ and Ca2+ (11).
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The conversion of Compound 3 to 6-hydroxymethyl-7,8-dihydropterin (Compound 4) is catalyzed by dihydroneopterin aldolase (12). Compound 4 is converted to dihydrofolate by the subsequent condensation with 4-aminobenzoate and glutamate by the catalytic action of dihydropteroate synthase and dihydrofolate synthetase, respectively (13).
An enzyme catalyzing the epimerization of the folate precursor, dihydroneopterin triphosphate, under formation of 7,8-dihydromonapterin (Compound 2) had been observed in Escherichia coli by Heine and Brown (14). Recently, the primary structure of this enzyme has been determined and has been found to show similarity to the dihydroneopterin aldolase domain of a multifunctional folate biosynthetic enzyme from Pneumocystis carinii (15). On the other hand, the epimerase is similar to a reading frame at 69.0 kb1 of the E. coli chromosome designated ygiG (EMBL accession number L12966) in the close vicinity of the bacitracin resistance gene. This reading frame specifies the dihydroneopterin aldolase of E. coli, which is a potential target for the development of enzyme inhibitors with antibacterial and/or antiprotozoal activity.
This paper reports on structural and mechanistic similarities of dihydroneopterin-triphosphate epimerase and dihydroneopterin aldolase. It also shows that the single Hemophilus influenzae homolog of these proteins (specified by the unannotated open reading frame ygiG_haein) is an aldolase-type enzyme.
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EXPERIMENTAL PROCEDURES |
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Materials-- 6-Hydroxymethylpterin, D-dihydroneopterin, L-dihydromonapterin, D-dihydromonapterin, L-neopterin, L-monapterin, D-dihydroneopterin monophosphate, D-neopterin, and dihydrobiopterin were purchased from Dr. B. Schircks (Jona, Switzerland). Synthetic oligonucleotides were purchased from MWG (Ebersberg, Germany). Restriction enzymes and T4 DNA ligase were obtained from Pharmacia (Freiburg, Germany), Life Technologies, Inc. (Eggenstein, Germany), and New England Biolabs Inc. (Beverly, MA). Goldstar DNA polymerase was obtained from Eurogentec (Seraing, Belgium). GTP cyclohydrolase I was purified from a recombinant E. coli strain as described earlier (9).
Microorganisms and Plasmids-- The bacterial strains and plasmids used in this study are summarized in Table I.
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Assay of Epimerase Activity-- The epimerase substrate, dihydroneopterin triphosphate, was produced enzymatically from GTP. Reaction mixtures containing 100 mM Tris-HCl, pH 7.5, 0.1 M KCl, 50 mM GTP, 5 mM dithiothreitol, and 0.05 mg/ml recombinant E. coli GTP cyclohydrolase I were incubated for 4 h at 37 °C. Protein was then removed by ultrafiltration using a YM-30 membrane (Amicom Inc.).
An aliquot of this reaction mixture (20 µl) was added to 80 µl of a solution containing 80 mM PIPES, pH 6.2, and 8 mM MgCl2. The epimerase reaction was started by the addition of protein solution. The reaction mixture was incubated at 55 °C for 10 min. The reaction was terminated by the addition of a solution (30 µl) containing 1% I2 and 2% (w/v) KI in 1 M HCl. The samples were incubated at room temperature for 5 min. Excess iodine was reduced by the addition of 2% ascorbic acid (10 µl, w/v). A solution (210 µl) containing 1.5 M Tris-HCl, pH 8.5, 4.8 mM ZnCl2, 4.5 mM EDTA, and 0.5 units of alkaline phosphatase (Boehringer Mannheim, grade II) was added. The mixture was incubated at 37 °C for 90 min. Trichloroacetic acid (50 µl, 40%, w/v) was added. Aliquots of 10 µl were analyzed by HPLC using a reversed-phase column (4 × 250 mm) of Nucleosil RP18 and an eluent containing 0.5% (w/v) H3BO3, pH 4.7. The effluent was monitored fluorometrically (excitation, 365 nm; emission, 446 nm). The flow rate was 2 ml/min. The retention volume was 7 ml for D-erythro-neopterin and 9 ml for L-threo-neopterin (monapterin).Assay of Dihydroneopterin Aldolase-- The assay is based on a method described earlier (12). Assay mixtures contained 100 mM Tris-HCl, pH 8.0, 5 mM dithiothreitol, 70 µM dihydroneopterin, and protein. The mixture was incubated at 70 °C for 10 min. The reaction was terminated by the addition of 30 µl of 1 M HCl containing 1% I2 and 2% (w/v) KI. The samples were incubated at room temperature for 5 min. Excess iodine was reduced by the addition of 2% ascorbic acid (10 µl, w/v). The samples were analyzed by reversed-phase HPLC using a column (4 × 250 mm) of Nucleosil RP18 and an eluent containing 30 mM HCOOH and 7% MeOH. 6-Hydroxymethylpterin was detected fluorometrically (excitation, 365 nm; emission, 446 nm). The retention volume was 6.5 ml.
Construction of Expression Plasmids for the folX and folB Genes-- The expression plasmids for the folX and folB genes were constructed as described by Richter et al. (16). The folX and folB genes of E. coli were amplified by PCR with chromosomal DNA from E. coli XL1-Blue as template. The folB gene of H. influenzae was amplified by PCR using the plasmid GHIFL0I as template (Table I). The folX gene was amplified with the oligonucleotides EpiExsense as forward primer and EpiExanti as reverse primer (Table II). The folB gene of E. coli was amplified with ECAL1 as forward primer and ECAL2 as reverse primer (Table II), and the folB gene of H. influenzae was amplified with HAEALD1 as forward primer and HAEALD2 as reverse primer (Table II). Each amplification product served as template for a second PCR with the universal forward primer kEcoRI (Tables II and Table III). The reverse primer EpiExanti was used for the folX gene. The reverse primer ECAL2 was used for the folB gene of E. coli, and the reverse primer HAEALD2 for the folB gene of H. influenzae. The PCR products (folB of H. influenzae and folX of E. coli) were cleaved with EcoRI and BamHI and ligated in the expression vector pNCO113, which had been digested with EcoRI and BamHI, whereas the folB gene of E. coli was cleaved with PstI and EcoRI and then ligated into the expression vector pNCO113, which had been digested with PstI and EcoRI. The expression plasmids designated pEPI (containing the folX gene of E. coli), pEAL (containing the folB gene of E. coli), and pHIAL containing the folB gene of H. influenzae) (Table I) were transformed into E. coli M15(pREP4). Kanamycin (20 µg/ml) and ampicillin (180 µg/ml) were added for the maintenance of the plasmids in the host strain.
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Construction of the Vector Used for the Deletion of the folX Gene-- The folX gene and the adjacent regions were amplified by PCR with EpiKpn as forward primer and EpiPst as reverse primer (Table II) using chromosomal E. coli XL1 DNA as template. The amplification product was cleaved with KpnI and PstI and then ligated into the vector pMAK705, which had previously been digested with KpnI and PstI. The vector contains the temperature-sensitive pSC101 origin of replication and a chloramphenicol resistance gene. The resulting plasmid, pMAK705E, was digested with EcoRV. The folX gene contains two internal EcoRV sites, and digestion results in the excision of a DNA fragment of 105 bp. A kanamycin resistance cassette was amplified by PCR using the forward primer kan-Rbs-1 and the reverse primer Kana4 (Table II) with the plasmid pREP4 (Table I) as template. The EcoRV-digested plasmid pMAK705E was T-tailed with Taq polymerase, and the product was ligated with the kanamycin resistance cassette, yielding the plasmid pMAK705EK (Table I and Fig. 2).
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Replacement of the Chromosomal folX Gene with Its Inactivated Variant-- The replacement of the chromosomal folX gene is a modification of the methods described by Hamilton et al. (17), Russel and Model (18), and Fermér and Swedberg (19). The plasmid pMAK705EK was transformed into the recA+ E. coli strain SK6600. The plasmid replicates at 30 °C, but not at 44 °C. Growth at 44 °C on plates that contain chloramphenicol selects clones with the plasmid integrated into the chromosome by homologous recombination. This results in a tandem arrangement of the intact folX gene adjacent to the inactivated folX gene with the kanamycin insert. PCR screening was performed using two pairs of primers in order to check whether the integration of the inactivated folX gene had occurred by homologous recombination. The forward primer Epi01, complementary to the E. coli chromosome in the direct vicinity of the 5'-end of the folX insert, in combination with the reverse primer kan02, complementary to the folX insert, should result in a fragment of ~1.2 kb. The forward primer kan01, complementary to the kanamycin gene, and the reverse primer Epi02 (Table II), complementary to the E. coli chromosome in the direct vicinity of the 3'-end of the folX insert, should result in the amplification of a 1.4-kb fragment. One of 12 clones screened with this method showed the correct pattern of amplificates. The resolution of this cointegrate was achieved by overnight incubation of the bacterial strain in LB liquid medium containing 20 mg/liter chloramphenicol at 30 °C. Cells were then diluted 105-fold and grown on plates containing chloramphenicol at 30 °C. Plasmid isolation of the resulting colonies showed that ~50% of the colonies now contained an intact folX gene on the plasmid and therefore the defective folX gene on the chromosome. Curing of the clones was achieved by growing them at 44 °C on LB plates. The resulting clones were sensitive to chloramphenicol. Epimerase activity could not be detected in cell extracts of the mutants. PCR amplification using the forward primer Episense and the reverse primer Epianti (Table II) resulted in amplificates of ~1400 bp, whereas the intact folX gene yields amplificates of ~300 bp. For the comparison of growth rates, SK6600 cells with intact or defective folX genes were grown on LB medium as well as on M9 minimal medium supplemented with vitamin-free casamino acids (Gibco BRL).
Purification of Recombinant Dihydroneopterin
Aldolase--
E. coli strain M15(pREP4) carrying the
expression plasmid pEAL was grown to a density of 0.6 A600 nm units in shaking flasks with LB medium
containing 20 µg/ml kanamycin and 180 µg/ml ampicillin at 37 °C.
Isopropyl--D-thiogalactopyranoside was added to a
concentration of 2.0 mM, and incubation was continued for a
period of 6 h. Cells were harvested by centrifugation; washed with
50 mM Tris-HCl, pH 8.0; and stored at
20 °C.
Purification of Recombinant Epimerase--
E. coli
strain M15(pREP4) harboring the expression plasmid pEpi was grown in LB
medium containing 150 mg/liter ampicillin and 20 mg/liter kanamycin.
The culture was incubated at 37 °C with shaking. At a cell density
of 0.6 A600 nm units,
isopropyl--D-thiogalactopyranoside was added to a final
concentration of 2 mM, and incubation was continued
overnight. The cells were harvested by centrifugation and stored at
20 °C.
Determination of Kinetic Parameters-- Steady-state kinetic experiments were performed at 37 °C. Km and Vmax were determined by least-square fit according to the Michaelis-Menten equation using the programs Origin (Microcal) and Excel (Microsoft). The equilibrium constant for epimerization of dihydroneopterin triphosphate and dihydromonapterin triphosphate catalyzed by dihydroneopterin-triphosphate epimerase was estimated from the composition of reaction mixtures that had reached a state of apparent equilibrium.
Analytical Ultracentrifugation--
Experiments were performed
with an analytical ultracentrifuge (Optima XL-I, Beckman Instruments)
equipped with absorbance and interference optics. Experiments were
performed in double sector cells with aluminum centerpieces and
sapphire windows. Fluorochemical FC43 (10 µl; Beckman Instruments)
was added to the sample compartment for sedimentation equilibrium
experiments. The partial specific volume was estimated on the basis of
amino acid compositions (20). Protein (~2 mg/ml) dissolved in 100 mM sodium phosphate, pH 7.0 ( = 1.0228 g
cm
3), was centrifuged at 4 °C and 7000 rpm for 72 h. Protein concentration was monitored by laser interferometry.
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RESULTS |
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Recombinant Expression of Dihydroneopterin Aldolases-- We have recently reported the sequence of dihydroneopterin-triphosphate epimerase from E. coli (15). A data base search indicated that an open reading frame in the neighborhood of the bacA gene located at 69.0 kb on the E. coli chromosome specifies a similar protein (30% identical amino acid residues). This open reading frame, which was subsequently designated folB, also shows similarity to putative dihydroneopterin aldolases from a variety of microorganisms (Fig. 3) (2, 21-26, 28).2
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Recombinant Expression of Epimerase-- The folX gene of E. coli specifying dihydroneopterin-triphosphate epimerase was expressed under the control of the lac promoter and T5 operator. The recombinant protein was purified by a sequence of ion-exchange chromatography, heat treatment, and gel permeation chromatography (Table V). The purified protein migrated as a single band of 14 kDa on SDS-polyacrylamide gels.
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Quaternary Structure-- The molecular masses of the proteins under study were determined by sedimentation equilibrium analysis. The molecular masses are summarized in Table VI and suggest that the three proteins under study form homo-octamers.
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Kinetic Properties of Aldolases-- In light of the sequence similarity between epimerase and aldolase, it was in order to perform a detailed comparison of their catalytic and kinetic properties. Initial enzyme experiments were performed at the temperature optimum of the enzymes studied (70 °C for dihydroneopterin aldolase and 55 °C for dihydroneopterin-triphosphate epimerase) (Tables IV and V). The more detailed kinetic analyses reported in Table VII were performed at 37 °C.
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Kinetic Properties of Epimerase-- As shown in Table VII and Fig. 4, dihydroneopterin-triphosphate epimerase uses the triphosphates of dihydroneopterin and dihydromonapterin efficiently as substrates for epimerization. A value of 0.77 was estimated for the equilibrium constant on the basis of the steady-state kinetic parameters. Dihydroneopterin and dihydromonapterin can also serve as substrates for epimerization, but the reaction velocities of the unphosphorylated substrates are slower by more than a factor 700. However, the unphosphorylated substrates are also slowly converted to hydroxymethyldihydropterin. The velocity of the aldolase-type reaction catalyzed by epimerase is <1% as compared with the cleavage of dihydroneopterin by the FolB protein. The cleavage reactions cannot be attributed to a contamination of recombinant dihydroneopterin aldolase with dihydroneopterin-triphosphate epimerase specified by the chromosomal folX gene since aldolase and epimerase are efficiently separated by the anion-exchange chromatography step (epimerase elutes at a sodium chloride concentration of ~150 mM, whereas aldolase elutes at ~600 mM NaCl).
The physiological role of the FolB protein in the pathway of tetrahydrofolate biosynthesis is well understood. However, the role of the epimerase occurring in E. coli, but not in H. influenzae, is not clear. We therefore constructed a folX deletion mutant by gene targeting in E. coli using the method described by Kushner and co-workers (17). The construction of the required plasmids and the gene deletion are described under "Experimental Procedures." PCR analysis of chromosomal DNA confirmed that the chromosomal gene of the mutant carries an insertion of 807 bp after bp 149 of the folX gene. Cell extracts of the mutant are devoid of epimerase activity. Since the enzyme assay used is very sensitive, any residual activity below the detection limit would have been <0.5% as compared with the E. coli wild-type strain. The folX mutant showed normal growth properties on minimal medium as well as on complete medium. Thus, the biological role of epimerase remains unknown. ![]() |
DISCUSSION |
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The enzymes specified by the folX and folB genes of E. coli are similar in terms of their primary structure and quaternary structure. The sequence similarity extends to dihydroneopterin aldolases of several other microorganisms (Fig. 3). Some of these proteins are large multidomain proteins catalyzing several steps in the biosynthesis of tetrahydrofolate.
It appears safe to assume that the folB gene is involved in the pathway of tetrahydrofolate biosynthesis, where it is responsible for the shortening of the position 6 side chain of the pteridine precursor. On the other hand, the role of the dihydroneopterin-triphosphate epimerase is still unknown. Growth tests of E. coli with a defective folX gene showed no apparent phenotype.
Pteridines with the threo configuration of the trihydroxypropyl side chain that is generated by the epimerization reaction have been found in a variety of organisms (29, 30), but their metabolic role is unknown. Even if these stereoisomers have a specific metabolic function, a specific epimerase may not be required for their formation since, at least in E. coli and H. influenzae, they can be formed by the aldolase specified by the folB gene.
H. influenzae has a folB gene (i.e. the unannotated reading frame ygiG_haein) specifying a protein whose catalytic properties are very similar to those of the FolB protein of E. coli. However, the genomes of H. influenzae and Bacillus subtilis appear to be devoid of folX genes specifying a pteridine epimerase, in line with the hypothesis that the epimerase is not an essential protein in bacteria.
A hypothetical reaction mechanism is shown in Fig. 5. We propose tentatively that both reaction types can be initiated by protonation of N-5 followed by deprotonation at the acidic C-1' of dihydroneopterin- or dihydromonapterin-type substrates. Epimerase- as well as aldolase-type reactions can be catalyzed by both the FolB and FolX proteins. This raises the question of a common transition state for both types of reaction. A retroaldol cleavage of the C-C bond between C-1' and C-2' is proposed to be the crucial reaction step for both enzyme reactions. Epimerization at C-2 would result from reversal of the cleavage reaction without stereochemical control. The different product ratios of FolB- and FolX-type proteins could result from differences in compartmentalization between the different exit pathways originating from the common transition state.
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Epimerization could occur if the reaction products resulting from
cleavage of the ,
-carbon bond, instead of being released from the
enzyme, react under regeneration of a pteridine, albeit without stereo
control. Partitioning between the reaction pathways for cleavage and
isomerization could then depend on the compartmentalization of the
cleavage fragments at the active site. This would imply that in the
case of epimerase, the release of the cleavage products from the enzyme
could be hindered by the protein conformation, and release would occur
only after religation of the cleavage fragments.
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ACKNOWLEDGEMENTS |
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We thank Angelika Werner for expert help with the preparation of this manuscript, Dr. A. P. G. van Loon (Hoffmann-La Roche AG) for bacterial strains and plasmids, and Dr. T. Mayer for useful discussions.
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FOOTNOTES |
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* This work was supported in part by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie and by Grant ERBCHRXCT930243 from the European Community.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.
Dedicated to Prof. Wolfhart Rüdiger on the occasion of his 65th birthday.
To whom correspondence should be addressed. Tel.:
49-89-289-13360; Fax: 49-89-289-13363; E-mail:
Bacher{at}oc3gra.org.chemie.tu-muenchen.de.
1 The abbreviations used are: kb, kilobase(s); PIPES, 1,4-piperazinediethanesulfonic acid; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; bp, base pair(s).
2 Sen-Gupta, M., Beinhauer, J. D., Fiedler, T. A., and Hegemann, J. H. (1996) EMBL/GenBankTM/DDBJ accession number 96722.
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REFERENCES |
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