(Received for publication, February 28, 1997, and in revised form, April 2, 1997)
From the Department of Microbiology, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea
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.
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
(1S,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).
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.
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. [-32P]UTP and [
-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
DH5 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 Extract500 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 FractionationTo 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 ChromatographyA 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 ColumnTo 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 ColumnActive 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 ColumnThe 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
(
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).
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.
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 EpimeraseThe 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.
|
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 SequencingThe 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.
PCR Cloning of N Terminus, 5
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 (
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.
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 DH5 cells
carrying pEPIFL. When the lysates of DH5
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.
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.
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.
We again examined an enhancement in the activity of the epimerase in
pMPS-transformed DH5 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.
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.
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U47639[GenBank].
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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