Purification, Cloning, and Functional Expression of Dihydroneopterin Triphosphate 2'-Epimerase from Escherichia coli*

(Received for publication, February 28, 1997, and in revised form, April 2, 1997)

Chiyoung Ahn , Jonghoe Byun and Jeongbin Yim Dagger

From the Department of Microbiology, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Dihydroneopterin triphosphate (H2NTP) 2'-epimerase from Escherichia coli catalyzes the epimerization of H2NTP to dihydromonapterin triphosphate (H2MTP). The enzyme was purified 954-fold to apparent homogeneity by a combination of ammonium sulfate fractionation and column chromatography of Cibacron blue 3GA dye ligand, phenyl-Sepharose CL-4B, methotrexate-agarose, and Superdex 200 HR 10/30 FPLC column. The molecular mass of the epimerase determined on a Superdex column was 82.6 kDa, while the subunit molecular mass determined on SDS-polyacrylamide gel electrophoresis was 13.7 kDa. This implies that the epimerase most probably exists as homohexamer. The 20-amino acid sequence from the N terminus was determined (AQPAAIIRIKNLRLRTFIGI). Based on this sequence, the gene encoding the epimerase was cloned using a simple polymerase chain reaction approach. Translation of the nucleotide sequence of the cloned gene revealed the presence of an open reading frame containing 120 amino acids with a predicted molecular mass of 13,993 Da. The epimerase gene located in a 2.3-kilobase BamHI-EcoRI fragment from Kohara's clone 406 was overexpressed 300-fold, which was confirmed by the prominent increase in the 14-kDa protein band on SDS-polyacrylamide electrophoresis gels. It showed no homology with the sequences of isomerases or other enzymes in GenBank/EMBL data bases.


INTRODUCTION

When GTP is incubated with a crude extract of Escherichia coli, both erythro- and threo-neopterin1 are formed (1). Since neopterin contains two chiral centers on its side chain, four possible stereoisomers can exist: D-erythro-neopterin, L-erythro-neopterin, D-threo-monapterin, and L-threo-monapterin.

D-erythro-Neopterin (1'S,2'R) or D-neopterin is a conventional neopterin. It is one of the major pterins found in human and many other species. An elevated level of D-neopterin in serum is a sensitive marker for an increased activity of cellular immunity and is useful for biochemical monitoring of infectious diseases, including AIDS (2). L-erythro-Neopterin (1'R,2'S) or L-neopterin, termed bufochrome, is an enantiomer of neopterin and its occurrence has been reported in toad skins (3). D-threo-Neopterin (1'R,2'R) or D-monapterin, tentatively termed as umanopterin (3), has been found in human urine and normally takes up 4-15% of D-neopterin. It is also present in Tetrahymena pyriformis (4). L-threo-Neopterin (1'S,2'S) or L-monapterin appears to be the major pterin in E. coli (5). This compound has also been found in substantial quantities in Pseudomonas species (6), where its tetrahydro form functions as a coenzyme in the enzymatic hydroxylation of phenylalanine to tyrosine. It can also act as an inhibitor for human 6-pyruvoyl tetrahydropterin synthase (EC 4.6.1.10) (7).

The cyclic monophosphate of L-threo-neopterin has been identified in Methylococcus capsulatus and its suggested function was a cofactor for alcohol dehydrogenase (8). Also in Dictyostelium discoideum a distinct chemotactic activity toward L-monapterin was observed during the developmental phase after starvation and its implication in cell sorting has been suggested (9).

E. coli excrete L-monapterin during their logarithmic growth phase. At the switch from the logarithmic to the stationary phase, there is a burst increase in excretion of L-monapterin and its role as a marker for cell proliferation has been suggested (10). However, little is known about the biological function of L-monapterin in this organism.

In E. coli, L-monapterin is made from H2MTP after successive dephosphorylation and oxidation. H2MTP is formed by an epimerase (11) acting on C2' carbon of H2NTP (Fig. 1), which is made from GTP by GTP cyclohydrolase I (EC 3.5.4.16) (12). H2NTP is a key intermediate for the biosynthesis of many pteridine compounds of biological significance. These include tetrahydrobiopterin, methanopterin, molybdopterin, drosopterin, sepiapterin, limipterin, and folates (13-18).


Fig. 1. Reaction catalyzed by H2NTP 2'-epimerase. Inversion of hydroxyl occurs at 2' carbon of side chain. P3 denotes triphosphate residues.
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Despite its long history of existence in E. coli, H2NTP 2'-epimerase has not previously been purified to homogeneity. Some characteristics of the epimerase have been studied with only partially purified enzymes (7, 11). The aims of the previous studies were not to get a highly purified enzyme, but rather to learn as much as possible about how L-monapterin is made. The present work was initiated to elucidate the biological function of L-monapterin in E. coli as well as to further investigate the physico-chemical properties of the epimerase with completely purified enzymes. We report here the purification, partial amino acid sequence, cloning, and overexpression of the epimerase from E. coli.


EXPERIMENTAL PROCEDURES

Materials

All chemicals used were of reagent grade. D-7,8-Dihydroneopterin, D-neopterin, and L-monapterin were purchased from Dr. B. Schircks Laboratories (Switzerland). The frozen E. coli cells (ATCC 11303), harvested in late log phase from Kornberg medium, were obtained from General Biochemicals. GTP, alkaline phosphatase, calibration proteins for gel filtration (MW-GF-20), Sephadex G-10, Cibacron blue 3GA dye resin, phenyl-Sepharose CL-4B, methotrexate-agarose, and Superdex 200 HR 10/30 gel were purchased from Sigma. Calibration proteins for SDS-PAGE were purchased from Boehringer Mannheim GmbH.

The restriction enzymes and DNA modifying enzymes were purchased from New England Biolabs, Promega Corp., Postech Biotechnology, and Boehringer Mannheim. Dynazyme for polymerase chain reaction (PCR) were purchased from Finnzymes Oy (Finland). Nylon membranes were purchased from ICN. [alpha -32P]UTP and [alpha -35S]dATP were purchased from Amersham Nuclear Corp. Sequenase version 2.0 sequencing kit was purchased from U. S. Biochemical Corp. The primers were synthesized at DNA International. E. coli Gene Mapping Membrane was purchased from TaKaRa Shuzo. E. coli genomic library was from CLONTECH Laboratories. Other chemicals were purchased from Sigma.

Strains, Plasmids, and Phage

DH5alpha E. coli strain was used as a host cell for plasmids, while Gln358 was used for phage propagation. pBluescript II KS (Stratagene) and pBSTA (pBluescript II KS backbone; a plasmid designed for direct-cloning of PCR product) were used for cloning of general DNA fragments and for PCR products, respectively. Phage 406 (9D2) is a clone of the Kohara's miniset (19). Luria-Bertani (LB) and NZCYM media were used for E. coli and phages, respectively (20).

Preparation of H2NTP

H2NTP was made enzymatically from GTP as described previously (12). Since H2NTP is very unstable and prone to photooxidation, many aliquots were made and stored frozen at -70 °C in the darkness until used.

Epimerase Assay

The enzyme reaction mixture (25 µl) contained 40 mM PIPES (pH 6.2), 4 mM MgCl2, 40 mM H2NTP, and the epimerase preparation. The reaction was carried out by incubating the mixture for 10 min at 50 °C in the darkness and terminated by heat for 1 min in boiling water. To dephosphorylate the remaining substrate (H2NTP) and the product (H2MTP), the mixture was treated with 2.5 units of alkaline phosphatase for 30 min at 37 °C, followed by treatments of 2.5 µl of 30% trichloroacetic acid and 5 µl of 1% I2, 2% KI for 15 min at 4 °C for the oxidation of reactants. After proteins were removed by centrifugation (10,000 × g for 15 min), a 30-µl aliquot of the supernatant was transferred to a fresh tube and neutralized by 2 µl of 2 N NaOH. Excess iodine was reduced with 4 µl of 2% ascorbic acid. A 2-µl aliquot of the final assay mixture was subjected to reversed-phase HPLC (Waters, Model 510) equipped with Partisil ODS C18 column (Whatman, 0.39 × 30 cm). The column was pumped using water as a mobile phase at a flow rate of 1.0 ml/min and the eluent was monitored with a fluorescence detector (Shimadzu RF-540). Enzyme activity was determined by integrating the peak area of L-monapterin.

To avoid photooxidation of pteridines, all operations were conducted in dim light. All assays were performed in duplicate. During the routine assay of the epimerase activity, the percent conversion of D-neopterin to L-monapterin ((area of D-monapterin × 100)/(area of D-neopterin + L-monapterin)) was monitored. One unit of enzyme activity was defined as the amount of enzyme which catalyzes the formation of 1 nmol of H2MTP/min. The volume of the epimerase preparation in the assay mixture was chosen so that the ratio of L-monapterin/D-neopterin never exceeded 0.3 at the end of the assay. Product formation was shown to be linear with enzyme concentration and incubation time under standard reactions.

Purification

All enzyme purification steps were performed at 4 °C.

Step 1: Preparation of Crude Extract

500 g of frozen cells were resuspended in 0.1 M Tris-HCl (pH 8.0) and disrupted by sonication (Ultrasonic Processor XL, Misonix, Inc.). Total process time was 10 min with 5 s of pulse on time and 10 s of pulse off time. Cell debris were removed by centrifugation (5,500 × g for 30 min).

Step 2: Ammonium Sulfate Fractionation

To the crude extract was added enough ammonium sulfate to give a 50% saturated solution. The resulting precipitated proteins were discarded and additional ammonium sulfate was added to the supernatant with slow stirring to 75% saturation. The precipitates were recovered by centrifugation (13,000 × g for 30 min), dissolved in minimal amount of 20 mM Tris-HCl (pH 7.8) (buffer S), and subjected to dialysis three times against 5 liters of buffer S for 4 h. This dialyzed fraction was again centrifuged (13,000 × g for 20 min) to obtain clear supernatant.

Step 3: Cibacron Blue 3GA Column Chromatography

A 50-75% ammonium sulfate fraction was applied to a column (5 × 16 cm) of Cibacron blue 3GA dye resin that had been equilibrated with buffer S. The column was washed with buffer S containing 0.12 M NaCl until the absorbance (280 nm) dropped to the baseline. Then the column was eluted with the buffer S containing 0.7 M NaCl. Fractions of 10 ml were collected at a rate of 30 ml/h.

Step 4: Phenyl-Sepharose CL-4B Column

To the active fractions pooled from the Cibacron blue 3GA dye resin were added ammonium sulfate with slow stirring to give a 1.0 M solution of ammonium sulfate. This was loaded onto a column (3 × 7 cm) of phenyl-Sepharose CL-4B, pre-equilibrated with buffer S containing 1.0 M ammonium sulfate. The column was developed with 150 ml of a linear gradient (1.0-0 M) of ammonium sulfate dissolved in buffer S. The column was further eluted with buffer S. Fractions of 5 ml were collected at a rate of 30 ml/h.

Step 5: Methotrexate-agarose Column

Active fractions from the phenyl-Sepharose CL-4B column were combined and applied to a column (1.5 × 15 cm) of methotrexate-agarose pre-equilibrated with buffer S. The column was developed with 60 ml of a linear gradient (0-0.7 M) of NaCl dissolved in buffer S. Then the column was further eluted with buffer S containing 0.7 M NaCl until the absorbance (280 nm) dropped to the baseline. Fractions of 4 ml were collected at a rate of 30 ml/h. The active fractions were pooled and concentrated in Centriprep-10 (Green Base: Mr cut-off = 10,000; Amicon) down to 1 ml, which was further concentrated to 200 µl by Centricon-10 filter (Amicon).

To reduce the possible irreversible binding of proteins, methotrexate-agarose was subjected to pre-use treatment as follows: liver concentrate (5 g) was dissolved in 50 ml of 50 mM potassium phosphate buffer (pH 8.5) and centrifuged to obtain clear supernatant. Then 20 ml of gel cake was slurried in the clarified liver extract and mixed gently by shaking for 20 min. The top layer was decanted and the gel cake washed with 100 ml of folic acid solution (1 mg/ml in 50 mM potassium phosphate buffer, pH 8.5) followed by 100 ml of 0.5 N NaCl.

Step 6: Superdex 200 HR 10/30 FPLC Column

The methotrexate-purified enzyme concentrate was subjected to Superdex 200 HR 10/30 FPLC column chromatography. The column was equilibrated with 50 mM Tris-HCl (pH 8.0) buffer containing 0.15 M NaCl at a flow rate of 0.4 ml/min and calibrated with blue dextran 2000 (for void volume), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), and lysozyme (14.3 kDa). Fractions of 0.5 ml were collected.

Amino Acid Sequencing

A 5-µg aliquot of purified epimerase was subjected to 15% SDS-PAGE and electroblotted onto polyvinylidene difluoride membrane (Immobilon-P, Millipore) as described previously (21). Protein was visualized by Coomassie Blue staining. The N-terminal amino acid sequence was analyzed using a protein sequencer (Applied Biosystems, Inc., Model 476A) at the Korea Basic Science Institute (Daejon, Korea). The sequence was verified independently using another protein sequencer (Milligen 6600B).

Design of Oligonucleotide Primers for PCR

Two degenerate primers were synthesized based on the N-terminal amino acid sequence of the epimerase: EP1, 5'-GCICARCCIGCIGCIATYATYCGIAT-3'; EP2, 5'-ATNCCRATRAANGTRCG-3' (I = inosine; R = A + G; Y = C + T; N = A + C + G + T). Other specific primers used are as follows: EP3, 5'-ATAAAGAACCTTCGTTTG-3'; EP4, 5'-CGCAAACGAAGGTTCTTT-3'; EP5, 5'-CTGCTAAAAGCACAACTC-3'; EP6, 5'-AGAACGGAACTGGCTTTCTG-3'; GT11R (lambda gt11 reverse), 5'-CACCAGACCAACTGGTAATG-3'.

Polymerase Chain Reaction/DNA Sequencing

Polymerase chain reaction was performed as described (20). DNA sequencing was performed by the dideoxy chain termination method (22) using the Sequenase version 2.0 DNA sequencing kit, according to the supplier's protocol.

Preparation of RNA Probe

Uniformly radiolabeled RNA probe of antisense strand for Southern blot analysis was prepared by in vitro transcription as recommended in Protocols and Applications Guide from Promega Corp.

Southern Blot Analysis

Southern analysis was performed as described (20) with some modifications. The digested phage DNA run on the gel was transferred onto a nylon membrane by capillary action (23) and cross-linked by UV irradiation (StratalinkerTM 1800). The blot was hybridized at 50 °C in hybridization buffer (150 mM sodium phosphate, 250 mM NaCl, 50% formamide, 10% polyethylene glycol, 1% SDS, 1 mM EDTA, 2 × Denhardts) and the membrane was washed with the washing solution (0.1 × SSC, 5 mM sodium phosphate, 1% SDS, 0.02% sodium pyrophosphate). E. coli Gene Mapping Membrane was hybridized in the same way as above.

Other Procedures

Protein was determined by the method of Bradford (24) using bovine serum albumin as a standard. Protein of column chromatography fractions was monitored by measuring absorbance at 280 nm.

SDS-polyacrylamide gel electrophoresis was done as described by Hames (25). Protein sequence homology comparison was performed by the Blast program (via the NCBI BLAST E-mail server, National Institutes of Health).


RESULTS

Separation of L-Monapterin from D-Neopterin

A new procedure for the epimerase assay has been developed by the use of a reversed-phase HPLC column equipped with a fluorescence detection system. After epimerization reaction, the substrate (H2NTP) and the product (H2MTP) are oxidized and dephosphorylated to give D-neopterin and L-monapterin, respectively. Complete baseline separation of L-monapterin from D-neopterin was achieved in a Whatmann Partisil 5 ODS-3 column with water as a mobile phase (Fig. 2). D-Neopterin eluted at 7.2 min and L-monapterin about 3 min later at a flow rate of 1.0 ml/min. Slight enhancement in separation was achieved by incorporating boric acid (0.5% (w/v), pH 4.7) or cupric sulfate (4 mM)/D-phenylalanine (8 mM) in the mobile phase (4). However, standard assay was routinely performed without these additives.


Fig. 2. Separation of D-neopterin and L-monapterin by reversed phase HPLC. The injected amounts of D-neopterin and L-monapterin were 100 and 50 nmol, respectively. The column (Whatmann Partisil 5 ODS-3, 0.39 × 30 cm) was pumped at 1.0 ml/min with water (pH 5.6). The eluent was monitored by fluorescence (excitation at 350 nm, emission at 450 nm). NEOPT, D-neopterin; MONAPT, L-monapterin.
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Since fluctuations were observed in the retention times, comparison of the retention time with that of standards alone was not accurate for identifying the compound. Therefore a spiking test was performed with authentic compound. The standard D-neopterin or L-monapterin, indeed, coeluted with the corresponding D-neopterin or L-monapterin from the reaction mixture, respectively (data not shown).

Purification of the Epimerase

The purification scheme included ammonium sulfate fractionation of crude extract followed by chromatography on Cibacron blue 3GA, phenyl-Sepharose CL-4B, methotrexate-agarose, and Superdex 200 HR 10/30 column. The most effective step was methotrexate-agarose column chromatography which eliminated most of the contaminating proteins (Fig. 3). Final purification was achieved by gel filtration on Superdex 200 HR 10/30 column, which gave one peak of the epimerase activity coincident with a major protein peak (data not shown). This purification scheme resulted in the isolation of an essentially homogeneous enzyme as judged by SDS-polyacrylamide gel electrophoresis (Fig. 3). A summary of the purification is shown in Table I. Overall, the epimerase was purified 954-fold over the crude extract with an activity yield of 5.4% to a final specific activity of 505 unit/mg.


Fig. 3. SDS-PAGE analysis of proteins at various stages of purification. M, size marker; CE, crude extract (20 µg); AS, ammonium sulfate (10 µg); CB, Cibacron blue 3GA (5 µg); PS, phenyl-Sepharose CL-4B (2 µg); MA, methotrexate-agarose (2 µg); SD, Superdex 200 HR fractions (numbers 27-31). Gels were stained with Coomassie Blue G-250.
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Table I. Purification of epimerase


Purification step Protein Activity Specific activity Recovery Purification

mg nmol H2MTP/min unit/mg % -fold
Crude extract 35,217 18,647 0.530 100 1
Ammonium sulfate (50-75%) 3,940 4,110 1.043 22.0 1.97
Cibacron blue 3GA-agarose 913 2,936 3.215 15.7 6.07
Phenyl-Sepharose CL-4B 60 2,265 37.73 12.1 71.3
Methotrexate-agarose 3.4 1,660 484 8.90 914
Superdex 200 HR 2.0 1,010 505 5.42 954

Molecular Mass of the Epimerase

The molecular mass of the epimerase was determined on Superdex 200 HR 10/30 FPLC column with standard calibrator proteins including lysozyme (14.3 kDa), ovalbumin (43 kDa), bovine serum albumin (66 kDa), and alcohol dehydrogenase (150 kDa). Epimerase activity eluted at a position consistent with a molecular mass of 82.6 kDa (data not shown). This was confirmed using another gel filtration in HPLC (Protein Pak 125, Waters). This compares favorably with earlier determinations of 87-89 (11) and 88 (7) kDa. However, the minimum subunit molecular mass of the epimerase was 13.7 kDa (Fig. 3). This value is in close agreement with the molecular mass (13,993 Da) deduced from the open reading frame of the cloned epimerase gene.

N-terminal Amino Acid Sequencing

The N-terminal sequence of the epimerase subunit was determined by microsequencing. A 20-amino acid sequence from the N terminus is presented in Fig. 4. It showed no homology with the sequences of isomerase or any other enzymes in GenBank/EMBL data bases, indicating that it is a novel sequence.


Fig. 4. Amino acid sequence of the N-terminal region of the epimerase. The sequences are given in single-letter code. The arrows labeled EP1 and EP2 indicate the amino acid residues used to design specific degenerate primers for PCR cloning.
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PCR Cloning of N Terminus, 5'-Upstream, and 3'-Downstream DNA

The gene encoding the epimerase was cloned by the PCR approach as follows. Since there was no other information available on the epimerase gene except the N-terminal amino acid sequence, we first cloned the N terminus DNA of the epimerase gene by PCR amplification with two degenerate primers (EP1 and EP2) designed based on the N-terminal amino acid sequence (Fig. 4). The PCR performed on E. coli chromosomal DNA produced a 59-bp band corresponding to the size of 20 amino acids. This fragment was cloned into pBSTA and the sequence was determined. The 59-bp DNA indeed contained amino acid sequences identical with the N-terminal amino acid sequence of the epimerase. Based on this DNA sequence, two gene-specific primers, sense primer EP3 and antisense primer EP4, were designed for cloning 5'-upstream and 3'-downstream DNA.

To obtain the 5'-upstream portion, the first PCR was performed with EP2 and GT11R primers using the DNA prepared from E. coli genomic library (lambda gt11) as a template. The amplified product was then subjected to the second PCR using the sequence-specific primer (EP4 and GT11R primer) to obtain a DNA fragment with increased specificity. A prominent DNA band of about 200 bp was obtained and cloned into pBSTA for sequencing. Analysis of the sequence revealed that this 200-bp DNA contained 1-9 residues of the N-terminal amino acid sequence as well as an initiation codon (ATG) and a presumed ribosomal binding site.

To obtain the 3'-downstream portion, the same PCR process was employed, except that the degenerate primer (EP1) and sequence-specific primer (EP3) were used. Three prominent DNA bands were obtained and each fragment was cloned into pBSTA for DNA sequencing. Analyses of both terminal regions of the three fragments revealed that only the 1.2-kb DNA fragment contained the amino acid sequence corresponding to 15-20 residues of the N-terminal amino acid sequence of the epimerase and open reading frame that stretched to 339 bp downstream.

Next, we linked the 5'-upstream and 3'-downstream DNA sequences and found an open reading frame of 360 bp encoding a polypeptide of 120 amino acid residues. The deduced molecular mass was 13,993 Da in close agreement with the 13.7 kDa determined by SDS-PAGE of the purified epimerase. These results indicated that the entire nucleotide sequence for the epimerase gene was obtained.

PCR Cloning of the Entire DNA and Expression of the Epimerase

To obtain the DNA fragment covering the entire epimerase gene, the specific sense primer (EP5) and antisense primer (EP6) were designed, based on the linked DNA sequence. The PCR with EP5 and EP6 produced the DNA fragment of 0.6 kb, which was cloned into pBSTA to generate pEPIFL.

Then we examined the specific activity of the epimerase in DH5alpha cells carrying pEPIFL. When the lysates of DH5alpha cells were assayed for the epimerase activity, there was 2.8-fold more epimerase activity in cells transformed with pEPIFL than those transformed with pBluescript control (Fig. 7). This indicates we successfully cloned the epimerase gene.


Fig. 7. Activities of the epimerase in DH5alpha cells carrying pEPIFL and pMPS. pEPIFL and pMPS carry the gene cloned by PCR approach and the gene isolated from the library (Kohara's clone), respectively. The cells carrying plasmids were cultured overnight in LB broth containing ampicillin. The cells were harvested, resuspended in 0.1 M Tris-HCl (pH 8.0), and disrupted. The lysates were collected after removing the cell debris by centrifugation and subjected to enzyme assay. The shaded boxes indicate the open reading frame encoding the epimerase. Numbers, -98 and -130, indicate 5'-upstream ends from initiation codon. F, EcoRV; M, MluI.
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Cloning of the Epimerase Gene from the Genomic Library and Its Overexpression

Three factors led us to clone the epimerase gene from the Kohara's genomic library of E. coli: (i) to obtain DNA containing more of distal 5' and 3' regions besides open reading frame; (ii) to compare the DNA sequence obtained from PCR with that from the chromosomal DNA, as there is frequently PCR errors; and (iii) to establish the location of the gene on E. coli genome. First, when the radiolabeled RNA probe prepared from the 0.6-kb insert of a plasmid pEPIFL was hybridized to E. coli gene mapping membrane, numbers 405 and 406 were found to be the positive clones. The DNA of Kohara's phage clone 406 was subjected to Southern hybridization analysis. The epimerase gene was located within the 2.3-kb BamHI-EcoRI segment of phage clone 406 DNA (Fig. 5). To further map the location and determine the direction of transcription, the 2.3-kb BamHI-EcoRI fragment was subcloned into pBluescript II KS, yielding pMPS. It was then analyzed for restriction sites, EcoRV and MluI, and the location of the gene is marked by the arrow in Fig. 5.


Fig. 5. Location of the epimerase gene on map of Kohara phage 406 DNA. The arrow indicates the position of the epimerase gene and transcriptional direction. E, EcoRI; B, BamHI; H, HindIII; F, EcoRV; M, MluI.
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Next, we determined the entire nucleotide sequence of the epimerase gene in pMPS using T7 primer and the primers used for the PCR cloning. Fig. 6 shows the entire nucleotide sequence of the gene that encodes the epimerase. There was no difference between the sequence of pMPS and that obtained from PCR.


Fig. 6. The entire nucleotide sequence of the gene encoding H2NTP 2'-epimerase. The deduced amino acid sequence is also shown. The stop codon is indicated by the asterisks (***). Numbers at the left and right are for amino acids and nucleotides, respectively. Location of the sequences used to design the primers, EP5 and EP6, are marked by the arrows.
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We again examined an enhancement in the activity of the epimerase in pMPS-transformed DH5alpha cells. Surprisingly, the cell had 300-fold more activity than the cells transformed with pBluescript control (Fig. 7). Also, there was a prominent increase in 14-kDa protein band in SDS-PAGE (Fig. 8). These results again confirm the successful cloning and overexpression of the epimerase.


Fig. 8. Overexpression of the epimerase in DH5alpha cells carrying pMPS. An aliquot (40 µg) of the lysates of cells carrying pBluescript (control) and pMPS was subjected to 15% SDS-PAGE. The 14-kDa protein band is indicated by the arrow. Lane M, marker protein; pBS, the lysate of the cells carrying pBluescript; pMPS, the lysate of the cells carrying pMPS.
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Although pMPS and pEPIFL are identical in coding regions, pMPS has some additional 5' and 3' sequences which may account for the higher level of the epimerase activity (Fig. 7). To narrow down the positive regulatory elements, extra 5' region (-130 to -102) of pMPS has been deleted and the epimerase activity was compared with that from pMPS. There was no significant difference in the activity between the 5'-deleted pMPS and intact pMPS. This suggests strongly a positive regulatory role of the 3' end downstream sequence on the overexpression of the epimerase gene.


DISCUSSION

Despite the ubiquitous distribution of polyhydroxypropyl-pterins in nature, much of the exact stereoisomerism and their implicated roles have not been elucidated completely. Monapterin has been gaining wide attention regarding its nature and its biological role in various organisms. It has been found in human, bovine, and rat retina (26) as well as in human urine. In patients with retinitis pigmentosa, which is a progressive dystrophic disease of rod-photoreceptor cells of the retina, monapterin was significantly decreased in lymphocytes and erythrocytes (27). So far the function of monapterin in humans is unknown. Its role as a protective pigment against light damage has been suggested (28).

Recently, the exact stereochemistry of natural monapterins has been determined by chiral HPLC as well as by measurements of their circular dichroism spectra (3, 4). Those present in human urine were found to be D-monapterin, while those present in E. coli and Pseudomonas are L-monapterin. Although the function of L-monapterin has been suggested as a cofactor for phenylalanine hydroxylase in Pseudomonas (6), L-monapterin has no known function in E. coli.

To further understand the function and the biosynthesis of L-monapterin in E. coli, the preparation of highly purified epimerase that is enzymatically fully active is required. However, preparations of the epimerase have suffered from limited purity and low yields until now. The epimerase, completely purified here, is the first to be purified and microsequenced among the many enzymes involved in the biosynthesis of pteridines in E. coli other than those committed toward folic acid biosynthesis. Overall, 954-fold purification was achieved with 5.4% recovery. This compared well with earlier partial purifications: Heine and Brown (11), purification of 7.5-fold, recovery of 6.2%; Blau et al. (7), purification of 53-fold, recovery of 0.35%.

One of the unique findings made from this complete purification was the multimeric nature of the epimerase. Taken with the native molecular mass, 82.6 kDa, estimated from gel filtration chromatography, the value of 13.7 kDa for the denatured enzyme and 13,993 Da for predicted molecular mass suggest that the epimerase is a hexamer of identical polypeptides. It is interesting to note that 6-pyruvoyl tetrahydropterin synthase, a key enzyme in the biosynthetic pathway of L-tetrahydrobiopterin, is a hexamer of identical subunits (31). 6-Pyruvoyl tetrahydropterin synthase (EC 4.6.1.10) also acts on H2NTP like the epimerase.

Based on the N-terminal amino acid sequences presented here (Fig. 4), the gene encoding the H2NTP epimerase was cloned and functionally expressed. The cloned gene contained one open reading frame encoding 120 amino acid residues and its expression increased the epimerase activity as well as the amount of protein corresponding to the deduced molecular mass (13,993 Da) in SDS-PAGE. The predicted N-terminal amino acid sequence matched perfectly with that determined by microsequencing of the N terminus of the purified epimerase.

The cloning strategy employed here is unique in that only the 20-amino acid sequence from the N terminus was available for PCR and the entire gene sequence was deduced by linking the sequences of the 5' and 3' DNA fragments from two separate PCRs. This approach is likely to be very useful in cloning the genes that encode the proteins in a short time, provided that the N-terminal amino acid sequences are available. The problems that may arise from the PCR cloning can be solved as follows: (i) the PCR error can be reduced by using thermostable DNA polymerase with high fidelity and by determining and comparing the nucleotide sequences of multiple subclones. (ii) The absence of the native promoter may be circumvented by utilizing the exogenous promoter (e.g. T7 promoter) and E. coli strain that expresses T7 RNA polymerase (e.g. BL21(DE3)pLysS).

No significant sequence homology with other epimerase was found when GenBank/EMBL data bases were searched for homology with the epimerase gene. This suggests that it may be the first enzyme of its type to be cloned. Also, we could not find in the epimerase gene any potential pterin-binding domain which is conserved in aromatic acid hydroxylases and required for pterins to act as coenzymes (29, 30).

The similarity between L-tetrahydromonapterin from Pseudomonas and L-tetrahydrobiopterin from the human liver is that both act as a cofactor for the phenylalanine hydroxylase (EC 1.14.16.1) and neither is an efficient precursor of the pteridine moiety of folate (1). Since these compounds are not involved directly in the biosynthetic pathway of folic acid, their presence may be mainly for use in hydroxylation reactions. However, such oxygen-dependent hydroxylation of phenylalanine to tyrosine does not occur in E. coli and these two amino acids are made independently from the common precursor (prephenic acid) in this organism. Therefore the function of L-monapterin and its reduced forms in E. coli must be something other than being involved in the hydroxylation reactions.

In E. coli, the epimerase may function as a regulator system for folic acid biosynthesis. While D-erythro-dihydroneopterin, which is generated from H2NTP, is a good substrate for dihydroneopterin aldolase and greater portion of H2NTP may enter folate pathway, L-threo-dihydromonapterin which is made after the epimerization is still an efficient substrate (32, 33) and provide a shunt for H2NTP pool, thus regulating the level of folate. Also, monapterin or its derivative may play a role related to scavenging of toxic radicals generated during monooxygenase reactions in addition to the role in cell proliferation (10, 11). Once the mutant lacking this epimerase gene is obtained, the absolute requirements for L-monapterin or its derivatives can be determined and we will be a step closer to elucidating the biological function of L-monapterin in E. coli.


FOOTNOTES

*   This work was supported by a Genetic Engineering Research grant from the Korean Ministry of Education.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 GenBankTM/EMBL Data Bank with accession number(s) U47639[GenBank].


Dagger    To whom correspondence should be addressed: Dept. of Microbiology, Seoul National University, San 56-1, Shilimdong, Kwanakgu, Seoul 151-742, Korea. Tel.: 82-2-880-6702 or 82-2-880-6770; Fax: 82-2-871-4315; E-mail: jyim{at}plaza.snu.ac.kr.
1   The abbreviations and trivial names used are: pterin, 2-amino-4-hydroxypteridine; H2NTP (dihydroneopterin triphosphate), 6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin-3'-triphosphate; H2MTP (dihydromonapterin triphosphate), 6-(L-threo-1',2',3'-trihydroxypropyl)-7,8-dihydropterin-3'-triphosphate; D- or L-neopterin, 6-(D- or 6-(L-erythro-1',2',3'-trihydroxypropyl)pterin; D- or L-monapterin, 6-(D- or 6-(L-threo-1',2',3'-trihydroxypropyl)pterin; The epimerase, H2NTP 2'-epimerase; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s); HPLC, high performance liquid chromatography; PIPES, 1,4-piperazinediethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.

ACKNOWLEDGEMENTS

We thank Dr. Yuji Kohara for the supply of a phage clone, Dr. Byeongjae Lee for the gift of pBSTA, and Dr. Chin-ha Chung for providing the E. coli genomic library. We also thank to Dr. Sheldon Milstien for helpful discussions.


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