Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 513-518
doi:10.1111/j.1745-7270.2008.00422.x
Role of Phe-99 and Trp-196 of
sepiapterin reductase from Chlorobium tepidum in the production of L-threo-tetrahydrobiopterin
Supangat1#,
Sun Ok Park4#, Kyung Hye Seo1,2,3, Sang Yeol Lee1,2,3,
Young Shik Park4*, and Kon Ho Lee1,2,3*
1 Division of Applied Life Science (BK21
Program), Gyeongsang National University, Jinju 660-701, Korea
2 Plant Molecular Biology and Biotechnology
Research Center, Gyeongsang National University, Jinju 660-701, Korea
3 Environmental Biotechnology National Core
Research Center, Gyeongsang National University, Jinju 660-701, Korea
4 Mitochondrial Research Group, School of
Biotechnology and Biomedical Science, Inje University, Kimhae 621-749, Korea
Received: February
13, 2008
Accepted: April 8,
2008
This work was
supported by the grants from the BK21 Program (Supangat, KHS), grant
R15-2003-002-01001-0 to the EB-NCRC (KHL), and the Inje Academic Research Fund
2006 (YSP)
#
These
authors contributed equally to this work
*Corresponding
authors:
Kon Ho Lee: Tel,
82-55-751-6257; Fax, 82-55-759-9363; E-mail, [email protected]
Young
Shik Park: Tel: 82-55-320-3263; Fax, 82-55-336-7706; Email, [email protected]
Sepiapterin
reductase from Chlorobium tepidum (cSR) catalyzes the synthesis of a distinct
tetrahydrobiopterin (BH4), L-threo-BH4, different from the mammalian
enzyme product. The 3-D crystal structure of cSR has revealed that the product
configuration is determined solely by the substrate binding mode within the
well-conserved catalytic triads. In cSR, the sepiapterin is stacked between two
aromatic side chains of Phe-99 and Trp-196 and rotated approximately 180 around
the active site from the position in mouse sepiapterin reductase. To confirm
their roles in substrate binding, we mutated Phe-99 and/or Trp-196 to alanine
(F99A, W196A) by site-directed mutagenesis and comparatively examined substrate
binding of the purified proteins by kinetics analysis and differential scanning
calorimetry. These mutants had higher Km values than
the wild type. Remarkably, the W196A mutation resulted in a higher Km increase compared with the F99A mutation. Consistent with
the results, the melting temperature (Tm) in the
presence of sepiapterin was lower in the mutant proteins and the worst was
W196A. These findings indicate that the two residues are indispensable for
substrate binding in cSR, and Trp-196 is more important than Phe-99 for
different stereoisomer production.
Keywords Chlorobium tepidum; tetrahydrobiopterin; sepiapterin
reductase; site-directed mutagenesis; enzyme kinetics; differential scanning
calorimetry
Sepiapterin reductase (SR; EC 1.1.1.153) is an essential enzyme
catalyzing the last step in tetrahydrobiopterin (BH4) biosynthesis [1]. BH4 is
found ubiquitously in higher animals and is a well-known essential co-factor
for aromatic amino acid hydroxylases [2] and nitric oxide synthases [3]. The de
novo biosynthesis of BH4 from guanosine triphosphate (GTP) is catalyzed by
at least three enzymes, GTP cyclohydrolase I (EC 3.5.4.16),
6-pyruvoyltetrahydropterin synthase (EC 4.2.3.12), and SR [1]. GTP is first
converted to dihydroneopterin triphosphate by GTP cyclohydrolase I, then to
6-pyruvoyltetrahydropterin (PPH4) by 6-pyruvoyltetrahydropterin synthase. SR
finally catalyzes NADPH-dependent reduction of the diketo groups in the side
chain of PPH4 to BH4 [4]. BH4 has also been found in some bacteria such as Cyanobacteria
and Chlorobium spp., even though its function is still unknown [5–9]. BH4
synthesis in bacteria follows the same biochemical steps in higher organisms
[10,11].As BH4 has two chiral carbons in the C6 side chain, BH4 could be
produced in four different stereoisomeric forms: L-erythro-BH4 [6R-(1’S,
2’S)], D-threo-BH4 [6R-(1’S, 2’S)], L-threo-BH4 [6R-(1’S, 2’S)],
and D-erythro-BH4 [6R-(1’S, 2’S)]-5,6,7,8-BH4. In nature, L-erythro-BH4
is commonly found in higher animals, whereas the D-threo-BH4 form
(dictyopterin) was isolated from Dictyostelium [5], and L-threo-stereoisomer
was found in Chlorobium tepidum as a glycoside (tepidopterin, L-threo-BH4-N-acetylglucosamine)
[8]. The D-erythro-BH4 stereoisomer has not been reported yet. SR from C. tepidum (cSR) is able to catalyze the conversion
of sepiapterin into L-threo-H2-biopterin as well as PPH4 into L-threo-BH4
[1]. cSR and animal SRs share 43% amino acid sequence similarity, suggesting
that those enzymes diverged from the same ancestral protein. Therefore, it is
reasonable to question how these similar enzymes catalyze the synthesis of
different isomeric forms of BH4 from the same substrate. Recent X-ray
crystallographic analysis of cSR has revealed that these enzymes share almost
identical overall protein folding and well-conserved catalytic triad residues
within their 3-D structures [12–14]. The structures revealed that the specific
product configuration is determined solely by the binding mode of the substrate
to the enzyme within the well-conserved architecture consisting of the hydride
donor (NADPH) and the proton donor (Tyr residue). In cSR, the pterin ring in
sepiapterin is rotated approximately 180 around the active site from the
position observed in mouse SR and stacked between two aromatic side chains of
Trp-196 and Phe-99 (Fig. 1). To confirm the roles of these two aromatic
side chains in substrate binding, we mutated either/both of these residues into
alanines by site-directed mutagenesis and examined substrate binding by kinetic
analysis and differential scanning calorimetry (DSC). Here, we report the results indicating the two residues are crucial
for substrate binding and thus responsible for different stereoisomer
production.
Materials and Methods
Site-directed mutagenesis
Site-directed mutations were introduced into the cSR coding regions of
the expression vector pET-28b using the QuickChange site-directed mutagenesis
kit (Stratagene, La Jolla, USA) following the manufacturer’s protocol with two
complementary oligonucleotide primers containing the desired mutations from
Trp-196 and Phe-99 to Ala (GCC) (W196A and F99A, respectively), shown
underlined: W196A, 5-TATACGCCGATGGCCGGTAAAGTTGAC-3 (forward) and
5-GTCAACTTTACCGGCCATCGGCCTATA-3 (backward); and F99A, 5-GGGGTTGGCCGTGCCGGAGCACTGAGC-3
(forward) and 5-GCTCAGTGCTCCGGCAGCGCCAACCCC-3 (backward).The plasmids containing the cSR DNA fragments with single- and
double-site mutations were generated by polymerase chain reaction using the
primers, digested with DpnI and transformed into XL1-Blue bacteria
cells. All the mutated DNA sequences were confirmed by sequencing. Plasmids
without unintended missense mutations were then transformed into BL21(DE3)
bacteria cells for protein expression.
Protein expression and
purification
Mutant cSR enzymes were expressed in Escherichia coli strain
BL21(DE3) and purified in the same way used for wild-type protein (WT) as
described previously [10,12]. Briefly, the transformed cells were grown at 37 ?C
in Luria-Bertani/kanamycin broth with vigorous shaking, then induced by 0.4 mM
isopropyl-D-thiogalactoside when OD600
reached approximately 0.6. After 3 h induction, the cells were harvested by
centrifugation. The cells were washed, resuspended in lysis buffer [50 mM
sodium phosphate (pH 8.0), 300 mM NaCl, and 5 mM imidazole] and disrupted by
sonication. After centrifugation, the clear supernatant was applied onto a
column of Ni-NTA beads (Qiagen, Hilden, Germany). The column was washed with 20
column volumes of lysis buffer then with 20 column volumes of washing buffer
[50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, and 30 mM imidazole]. The soluble mutant
proteins were eluted with elution buffer [50 mM Tris-HCl (pH 8.0), 100 mM NaCl,
and 300 mM imidazole]. Fractions containing recombinant proteins were pooled,
concentrated, and exchanged to 50 mM Tris-HCl (pH 8.0) by ultrafiltration
(Centriprep YM-30; Millipore, Bedford, USA). The recombinant proteins were
further purified by anion-exchange chromatography (MonoQ; GE Healthcare,
Buckinghamshire, UK), then by gel filtration chromatography (Superdex 200; GE
Healthcare) in 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl. The proteins were
concentrated to 10 mg/ml in 20 mM Tris-HCl (pH 8.0) by ultrafiltration
(Millipore). The purity of the proteins was monitored by SDS-PAGE. Protein
concentration was determined by Bradford assay using bovine serum albumin as
standard.Mutant cSR enzymes were expressed in Escherichia coli strain
BL21(DE3) and purified in the same way used for wild-type protein (WT) as
described previously [10,12]. Briefly, the transformed cells were grown at 37 ?C
in Luria-Bertani/kanamycin broth with vigorous shaking, then induced by 0.4 mM
isopropyl-D-thiogalactoside when OD600
reached approximately 0.6. After 3 h induction, the cells were harvested by
centrifugation. The cells were washed, resuspended in lysis buffer [50 mM
sodium phosphate (pH 8.0), 300 mM NaCl, and 5 mM imidazole] and disrupted by
sonication. After centrifugation, the clear supernatant was applied onto a
column of Ni-NTA beads (Qiagen, Hilden, Germany). The column was washed with 20
column volumes of lysis buffer then with 20 column volumes of washing buffer
[50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, and 30 mM imidazole]. The soluble mutant
proteins were eluted with elution buffer [50 mM Tris-HCl (pH 8.0), 100 mM NaCl,
and 300 mM imidazole]. Fractions containing recombinant proteins were pooled,
concentrated, and exchanged to 50 mM Tris-HCl (pH 8.0) by ultrafiltration
(Centriprep YM-30; Millipore, Bedford, USA). The recombinant proteins were
further purified by anion-exchange chromatography (MonoQ; GE Healthcare,
Buckinghamshire, UK), then by gel filtration chromatography (Superdex 200; GE
Healthcare) in 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl. The proteins were
concentrated to 10 mg/ml in 20 mM Tris-HCl (pH 8.0) by ultrafiltration
(Millipore). The purity of the proteins was monitored by SDS-PAGE. Protein
concentration was determined by Bradford assay using bovine serum albumin as
standard.
Enzyme assay and kinetic
analysis
The activity to convert sepiapterin to 7, 8-dihydrobiopterin was
measured by disappearance of the yellow color at 420 nm. Unless stated
otherwise, all reactions were carried out in the following conditions (standard
assay conditions). The reaction mixture contained 50 mM potassium phosphate (pH
6.5), 0.4 mM NADPH, 0.4 mM sepiapterin, and enzyme (0.1 mg for WT, 2 mg for F99A, 4 mg for W196A, and
16 mg
for the double mutant) in a total volume of 100 ml. The reaction was carried
out at 37 ?C for 20 min. A 1/10 volume of 20% cold trichloroacetic acid was
added to stop the reaction. After 10 min incubation on ice, the mixture was
centrifuged at 10,000 g for 10 min. The supernatant was diluted 5-fold
with water and measured for remaining sepiapterin. In order to normalize the
quenching effect of trichloroacetic acid on spectral absorption of sepiapterin
at 420 nm, every assay was accompanied by a blank reaction without enzyme. For
kinetic analysis, different quantities of sepiapterin were used in the standard
assay conditions. The concentration of sepiapterin was calculated from the
molar extinction coefficient of 10.4 mM–1?cm–1 at 420 nm [9,15].
Differential scanning
calorimetric analysis
DSC was carried out with a VP-DSC microcalorimeter (MicroCal,
Northampton, USA) with 0.52 ml cell volume. Before measurement, all protein
samples [1 mg/ml in 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl] were mixed with
NADP and sepiapterin to final concentrations of 1 mM and 0.3 mM, respectively,
and incubated for 3 h in ice. All samples were degassed for 5 min at 25 ?C and
filtered through a 0.2 mm Nanosep MF filter (Pall Life Sciences, East Hills, USA) before
loading into the instrument. The samples were kept under a constant pressure at
27 psi. Data were collected at a heating rate of 1 ?C/min and analyzed using
the MicroCal Origin DSC software package [16]. Structural modeling
Models of cSR F99A and W196A mutants were produced based on the
solved crystal structure of the cSR determined at 1.7 ? (PDB code 2BD0) [12]
using the Modeller 9v3 program [17] with alanine substitutions at residues 99
and 196. Ribbon diagrams of cSR WT and F99A and W196A mutants in complex with
sepiapterin were generated using PyMol [18].
Results
Construction of mutant cSR
enzymes
The 3-D structure of cSR in complex with sepiapterin and NADP [12]
(PDB code 2BD0) showed that sepiapterin is bound mainly by Phe-99 and Trp-196 (Fig.
1). In particular, the pterin ring of sepiapterin is stacked between two
aromatic side chains of Phe-99 and Trp-196. The residue Trp-196 in cSR is
unique compared with other known SRs (Fig. 2). Therefore, these two
aromatic amino acids seem to be essential for substrate binding and different
BH4 stereoisomer production in cSR. To confirm the roles of the two residues Phe-99 and Trp-196 in
substrate binding, single- and double-site mutations were introduced into the
aromatic amino acids. The mutant enzymes were expressed in E. coli using
the T7 expression system. The mutant enzymes were produced at the same level as
the WT enzyme and as stable as the WT. The cSR mutant proteins carrying single
mutation (W196A and F99A) or double mutation (F99A-W196A) were purified by
Ni-NTA affinity, anion exchange, and gel filtration chromatography in the same
way as used for the WT enzyme. Highly homogenous cSR mutant proteins could be
obtained after the three chromatographic steps (Fig. 3).
Catalytic properties of cSR
mutants
In order to compare catalytic properties of the purified WT and
mutant enzymes, kinetic parameters were determined for sepiapterin (Table 1).
The mutant enzymes manifested higher Km and
lower Vmax than WT and the W196A mutant did worse than
the F99A mutant. Km values were 3.9-fold higher
in F99A and 8.7-fold higher in W196A, and Vmax
values were 8.95% and 5.64% of WT in F99A and W196A, respectively. The activity
of the double mutant was so low that it could not be measured properly for
kinetic study. The kinetic data clearly suggest poor binding of sepiapterin in
the mutant enzymes, consequently leading to a decreased catalytic rate.
Therefore, the results show that both the Phe-99 and Trp-196 residues are
essential for enzyme activity and the latter is more important in substrate
binding.
Thermal denaturation of cSR WT
and mutant proteins
The thermal denaturation of the WT and mutant cSRs in the presence
of sepiapterin and NADP was analyzed by DSC and the melting temperatures (Tm) are shown in Fig. 4. The Tm value
for WT in the absence of sepiapterin was lower than that in the presence of
sepiapterin, suggesting that sepiapterin binding stabilized the protein. The Tm values for the mutants in the absence of sepiapterin were similar
to the value for WT without sepiapterin. In the presence of sepiapterin, the Tm value for WT was significantly higher compared with those for all
mutants. The Tm for the W196A mutant was much lower than
those of WT and the F99A mutant, consistent with the kinetic data, suggesting Trp-196
is a key residue for substrate binding. The Tm value
for the double mutant was almost the same as that for the F99A mutant. This
suggests that the effect of Trp-196 mutation is not that significant in
substrate binding when F99A is not present in the binding site.
Discussion
cSR has high amino acid sequence similarities with animal, insect,
fish, and other bacterial SR, carrying a well-conserved catalytic triad.
However, cSR produces a different stereoisomer, L-threo-BH4, and has an exceptionally
different substrate binding site formed mainly by Phe-99 and Trp-196, as shown
in the crystal structure [12]. In sequence alignment, cSR is the only exception
carrying a Trp at the 196 amino acid position instead of a well-conserved Gln (Fig.
2). In the cSR crystal structure, the loop containing Trp-196 was
disordered in the absence of substrate. On substrate binding, the loop was
fixed mainly by Trp-196 that stacked the pterin ring of the substrate together
with Phe-99 (Fig. 1). Specifically, the Trp-196 residue was positioned
above the pterin ring, covering the substrate at the entrance of the binding
site in the cSR-sepiapterin-NADP complex structure [12]. These observations
suggest that loss of any of the Phe-99 or Trp-196 residues might impair
substrate binding, leading to catalytic activity decrease. As expected, the
assay showed that both the F99A and W196A mutant enzymes had much less activity
than the WT. Notably, mutation of Trp-196 resulted in much pronounced changes
in the catalytic parameters compared with the mutation of Phe-99. The
significantly increased Km and decreased Vmax of the W196A mutant could be due to the different coordination of
the residue in substrate binding, as observed in the cSR complex structure
[12]. The mutation of Trp-196 would cause residue 196 to lose its interaction
with the pterin ring of substrate, so that the loop carrying residue 196 might
be disordered, as seen in the cSR structure with no substrate. The absence of
tryptophan, the residue with the largest side chain, might open up the binding
site, yielding a higher degree of freedom to the substrate around the binding
site. Therefore, this unavailability of the tryptophan indole ring at residue
196 might result in significant reduction in substrate binding and catalysis (Fig.
5). In the case of the F99A mutant, the Trp-196 residue alone, positioned
above the substrate, might be enough to confine the substrate to the active
site. Through interactions with Trp-196, the substrate, even in the absence of
Phe-99, might be positioned in the binding site (Fig. 5). The DSC data
was also consistent with the kinetic data, showing that the W196A mutant had a
lower Tm than that of the F99A mutant (Fig. 4).
Therefore, it appears that Trp-196 could contribute more to holding the
substrate in the binding site. So the loss of Trp-196 is more disruptive than
the loss of Phe-99 for the function of the enzyme. From the assays of mutant enzymes we conclude here that both the
Phe-99 and Trp-196 residues play key roles for stereospecific enzyme catalysis
by holding the substrate in the active site, and that Trp-196 is more important
in substrate binding, consistent with their structural roles as observed in the
cSR complex structure.
Acknowledgements
We thank
members of the Plant Molecular Biology and Biotechnology Research Center
(Jinju, Korea) and the Environmental Biotechnology National Core Research
Center (Jinju, Korea) for their assistance.
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