From the Department of Chemistry, University of
California and Physical Biosciences Division, Lawrence Berkeley
National Laboratory, Berkeley, California 94720, ¶ Protein
Crystallography Group, Chemistry Department, University of Tromsø,
N-9037 Tromsø, Norway, and
Department of Biochemistry and
Molecular Biology, University of Bergen, Årstadveien 19, N-5009
Bergen, Norway
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ABSTRACT |
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Phenylalanine hydroxylase (PheOH) catalyzes the conversion of L-phenylalanine to L-tyrosine, the rate-limiting step in the oxidative degradation of phenylalanine. Mutations in the human PheOH gene cause phenylketonuria, a common autosomal recessive metabolic disorder that in untreated patients often results in varying degrees of mental retardation. We have determined the crystal structure of human PheOH (residues 118-452). The enzyme crystallizes as a tetramer with each monomer consisting of a catalytic and a tetramerization domain. The tetramerization domain is characterized by the presence of a domain swapping arm that interacts with the other monomers forming an antiparallel coiled-coil. The structure is the first report of a tetrameric PheOH and displays an overall architecture similar to that of the functionally related tyrosine hydroxylase. In contrast to the tyrosine hydroxylase tetramer structure, a very pronounced asymmetry is observed in the phenylalanine hydroxylase, caused by the occurrence of two alternate conformations in the hinge region that leads to the coiled-coil helix. Examination of the mutations causing PKU shows that some of the most frequent mutations are located at the interface of the catalytic and tetramerization domains. Their effects on the structural and cellular stability of the enzyme are discussed.
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INTRODUCTION |
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Mammalian phenylalanine hydroxylase (PheOH,1 phenylalanine 4-monooxygenase, EC 1.14.16.1) is an iron- and tetrahydropterin-dependent enzyme that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine. The reaction is the rate-limiting step in the catabolic pathway of phenylalanine resulting in the complete degradation of the amino acid. PheOH activity is tightly regulated by reversible phosphorylation and substrate activation. Mutations in the human PheOH gene result in the metabolic disorder known as phenylketonuria (PKU), a relatively common autosomal recessive disease that, if not diagnosed and properly treated from birth, ultimately leads to severe mental retardation. Approximately 280 mutant alleles of the human PheOH gene have been identified in patients with PKU and non-PKU hyperphenylalaninemia (1). The mutations lead to a variety of clinical and biochemical phenotypes with different degrees of severity (2, 3).
PheOH shares many biochemical and physical properties with tyrosine hydroxylase (TyrOH) and tryptophan hydroxylase (TrpOH) suggesting that they evolved from a common ancestor and might constitute a family of structurally related proteins (for review, see Ref. 4). PheOH, TyrOH, and TrpOH catalyze similar hydroxylation reactions using the same nonheme iron atom but differ in their substrate specificity. All three hydroxylases are organized into three domains: a 12-19-kDa N-terminal domain involved in regulation of the activity followed by a segment of about 38 kDa, which consist of a catalytic domain and a 5-kDa tetramerization domain. The crystal structure of a dimeric form of PheOH (5) containing only the catalytic domain shows a remarkable similarity to the previously reported structure of TyrOH (6). The tetramerization domain, located at the C terminus of TyrOH, is characterized by the presence of a long helix that promotes oligomerization through a coiled-coil motif (6). The sequence homology among the aromatic amino acid hydroxylases is very high, particularly in the catalytic and tetramerization domains (6-8). Proteolysis experiments on rat PheOH and TyrOH have shown that deletion of the N-terminal domain results in active truncated proteins with reduced substrate binding specificity. The attachment of either N-terminal regulatory domain (as chimeric proteins) enhances the substrate specificity displayed by the catalytic domain (9).
Since the discovery of the relationship between PKU and mutations in PheOH, the enzyme has been the subject of intense biochemical and genetic studies but for many years it has been difficult to gain detailed structural information on PheOH. Crystallization and diffraction to 2.7 Å resolution of a phosphorylated tetrameric form of full-length PheOH was reported several years ago (10), but no structure determination was completed from those studies. More recent crystallization experiments have shown that highly diffracting crystals can be obtained only for dimeric forms of PheOH missing the C-terminal tetramerization domain (11, 12).
Here we report the first crystallization and structure determination of a truncated form of human tetrameric PheOH (residues 118-452) to which we will refer as PheOHCterm. The crystal structure of PheOH reveals that within one monomer the catalytic and tetramerization domains can adopt two different orientations, thus forming a tetramer similar to that in TyrOH (6) but with an unexpected asymmetry in the packing of the four subunits. Because some of the most common PKU mutations map at the junction between catalytic and tetramerization domain, this structure of PheOH can provide a template for analyzing the molecular basis of PheOH deficiency.
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EXPERIMENTAL PROCEDURES |
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Protein Purification and Crystallization-- The gene encoding the catalytic and the tetramerization domain of human PheOH (residues 118-452 of human phenylalanine hydroxylase, PheOHCterm), cloned into vector pET-3a-d (Novagen), was overexpressed in Escherichia coli BL21. For protein purification, frozen cell paste was resuspended in 50 mM Tris-HCl buffer, pH 8.0, containing 20 mM NaCl, 5% glycerol, and 3 mM methionine, 0.5 mM phenylmethylsulfonyl fluoride. Cell lysis was started by adding lysozyme and completed by French press treatment. All purification steps were performed at 4 °C. The soluble fraction of the protein extract was diluted with one volume of 50 mM Tris-HCl buffer, pH 8.0, 5% glycerol, 3 mM methionine and applied onto a DEAE Fast Flow column (Amersham Pharmacia Biotech). The fractions containing PheOHCterm were pooled, and the protein was further purified by ammonium sulfate precipitation at 30-50% saturation. The precipitate was resuspended in 50 mM MES buffer, pH 5.8, 2.5% glycerol, 3 mM methionine and loaded onto a Perseptive Biosystem HS column and eluted with a NaCl gradient. PheOHCterm fractions were pooled, concentrated and further purified by size-exclusion chromatography on a Superdex 200 Pharmacia column eluted with 50 mM Bis-Tris-propane buffer, pH 7.0, 200 mM NaCl, 2.5% glycerol, 3 mM methionine. After adding 2 mM dithiothreitol, the protein was concentrated to about 10 mg/ml in an Amicon pressure cell. The purified PheOHCterm appeared homogeneous (tetramer) as assessed by size-exclusion chromatography and silver-stained SDS-polyacrylamide gel electrophoresis.
Initial crystallization conditions were identified by using the sparse matrix approach (14). PheOHCterm was crystallized at room temperature by hanging drop vapor diffusion from 0.6 M MgSO4, 0.2 M sodium, potassium tartrate, 10% polyethylene glycol 4000, 100 mM Bis-Tris-propane buffer, pH 7.0. Crystals grew within 1-2 days as triangular plates with dimension 0.6 × 0.6 × 0.2-mm3 in space group P3112 (a = b = 119.5 Å, c = 126.0 Å,Data Collection and Structure Determination-- X-ray diffraction data to 3.1 Å were collected at Beam Line 7-1 of the Stanford Synchrotron Radiation Laboratory (SSRL) on flash frozen crystals. A solution of 30% glycerol in 25% mother liquor was used as a cryoprotectant. The programs DENZO and SCALEPACK were used for data processing and reduction (15). A total of 120,922 measurements were recorded for 18,446 unique reflections. The data set is 99% complete between 20 and 3.1 Å with an Rmerge value of 7.5%.
The PheOHCterm structure was solved by molecular replacement. The search model was based on the atomic coordinates of the crystal structure of tyrosine hydroxylase (6), because the crystal structure of PheOH (5) was not yet available when the present study was started. The amino acid sequence identity between the catalytic domains of PheOH and TyrOH is approximately 80%. The best search model included only the catalytic domain of TyrOH in which all the residues that were not identical in the two proteins were replaced with alanine. Molecular replacement was performed using the program AMoRe (16, 17). In the cross-rotation function all data between 8 and 4.0 Å were used and model Patterson vectors were selected within a radius of 30 Å. The translation function and a subsequent step of rigid body refinement were carried out with all data between 8 and 4.0 Å. This procedure allowed a clear identification of the two molecules in the asymmetric unit. The search for monomer A gave a correlation coefficient of 33% and an R-factor of 50%, whereas the search for monomer B gave a correlation coefficient of 22% and an R-factor of 53%. Orientation and position of the dimer obtained by molecular replacement were improved by rigid body refinement using the program X-PLOR (18). This lowered the R-factor to 41% and provided a more accurate definition of the noncrystallographic two-fold operator. The ![]() |
RESULTS |
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A truncated form of PheOH including the catalytic and
tetramerization domains, residues 118-452, was expressed in E. coli, purified and crystallized. This fragment of PheOH
(PheOHCterm) retains full catalytic properties but is
more soluble than the wild-type enzyme. Dynamic light scattering
analysis and size-exclusion chromatography revealed that the
recombinant purified protein is 99% monodisperse in solution with a
calculated molecular mass around 160 kDa, corresponding to a tetramer
(data not shown). PheOHCterm crystallizes in space group
P3112 with two molecules in the asymmetric unit forming a
tetramer (dimer of dimers) through the crystallographic two-fold
symmetry (Fig. 1). The structure was
solved by molecular replacement using a model derived from the crystal
structure of TyrOH. The PheOHCterm catalytic domain contains 13 -helices and 8
-strands and is identical, within coordinate error, to the structure of a dimeric form of PheOH recently
determined at 2.0 Å resolution, which we will refer to as
PheOHCat (5). The PheOHCterm catalytic
domain in the tetramer structure can be superimposed on the
corresponding domain of the TyrOHCterm structure
with a root mean square deviation of 0.64 Å for all the C
atoms.
The active site of human PheOHCterm is located in a
deep cleft in the core of each monomer and is very similar to that of
TyrOHCterm. Although iron was not added during the
purification and crystallization procedures, electron density compatible with that of a metal ion is present in the catalytic center
in the same site as seen in the structure of
TyrOHCterm (6) and PheOHCat (5).
His-285, His-290, and Glu-330 coordinate the iron in agreement with
site-directed mutagenesis studies (23-25) and with the structure of
PheOHCat. Glu-286, proposed to be involved in pterin
binding in PheOH but not in TyrOH (26), occupies nearly identical positions in both structures. The absence of significant structural differences in the active site of PheOHCterm and
TyrOHCterm is not surprising because a similar
low substrate binding specificity is observed for the catalytic domains
in steady-state kinetic analyses (9).
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The architecture of the PheOHCterm matches the overall fold
and organization found in the structure of
TyrOHCterm (6). The assembly of PheOH and
TyrOH might be described by a domain swapping mechanism in which
secondary structural elements mutually switch their position to promote
oligomerization (27). The tetramerization domain is formed by a
C-terminal "arm" consisting of two -strands, forming a
-ribbon, and a 40 Å long
-helix. The C-terminal arm extends over
a neighboring monomer bringing the four helices (one from each monomer)
into a tightly packed anti-parallel coiled-coil motif in the center of
the structure (Fig. 1). In the PheOH structure the tetramer is formed
by two conformationally different dimers resulting in a distortion of
the 222 symmetry. A superposition of the two monomers shows that the
catalytic domains and tetramerization domains, if taken separately, are
identical but they adopt different relative orientations (Fig.
2A). The distortion of the
tetramer symmetry is also evident from a surface calculation: in the
PheOHCterm the subunits interact with a buried surface area
of about 1200 Å2 and 1700 Å2, across the
crystallographic 2-fold dimerization interfaces AC and BD, respectively
(Fig. 1). The buried surface calculated for the
TyrOHCterm structure is about 1600 Å2
for both dimerization interfaces AC and BD. The discrepancy results from a change in backbone conformation at the hinge region that connects the
-ribbon to the coiled-coil helix accompanied by another
rotation in the coiled-coil helix.
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Using the program DYNDOM (28) it was possible to identify two rotation
points in the tetramerization domain. The first hinge axis is found at
residue Thr 427 leading to a 22° movement of the catalytic domain
with respect to the tetramerization domain. All the residues in the
hinge (425-429) are nicely defined in electron density, except for
Thr-427, which is very disordered indicating flexibility in this part
of the polypeptide chain. The second change in backbone conformation is
found at residue Gly-442 that generates a kink in the coiled-coil helix
and a rotation of about 20° (Fig. 2B). A conformational
change in the helix could be necessary to preserve the coiled-coil
interactions in response to the change in relative orientation of the
subunits. In the TyrOH sequence Thr-427 and Gly-442 are replaced
by a proline and a histidine residue, repectively (Fig. 2C).
It has recently been proposed that the presence of a proline in the
exchanging arm induces conformational constraints on the peptide
backbone that favor the optimal conformation for oligomerization (29).
As shown in Fig. 2C and Fig.
3, both PheOH and TyrOH sequences
contain a total of three proline residues in the tetramerization
domain. In PheOHCterm two prolines (Pro-407 and Pro-409)
define the sharp turn that links the tetramerization domain to the
catalytic domain. Another proline is also found at the C-terminal end
of -strand 7 (residue 416), but no proline residue is located at the
last hinge region connecting to the coiled-coil where the
conformational change is detected. The lack of a proline residue at
this position may allow more flexibility at this linker region and
modulate the arrangement of the quaternary structure in PheOH. In
TyrOH, a proline residue is present in each of the hinge regions
of the tetramerization domain, probably stabilizing its domain-swapped conformation.
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DISCUSSION |
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Full-length PheOH has been described to exist as both a tetramer
and dimer in a pH-dependent equilibrium and possibly as
some monomer (4, 30). Lowering the pH, as well as binding of
L-phenylalanine, shifts the equilibrium toward the
tetrameric form of PheOH (31, 32). By contrast, TyrOH and TrpOH
are always reported to be a tetramer in solution (4). Truncated forms
of PheOH and TyrOH where the C-terminal domain is left intact
have so far only been isolated as tetramers. Removal of the last 34 residues at the C terminus results in dimeric (PheOH) or monomeric
(TyrOH) active hydroxlases. The three-dimensional structure of
the PheOHCterm tetramer matches the overall fold and
organization found in the structure of tetrameric
TyrOHCterm (6). Despite the high degree of sequence
and structural identity, superposition of TyrOHCterm and PheOHCterm shows that the relative orientation of the
subunits forming the tetramer is different in the two enzymes. The
difference is caused by a distortion of the 222-fold symmetry in the
PheOH tetramer, whereas in the TyrOH tetramer the four subunits
are related by crystallographic symmetry and form a dimer of dimers with only two unique subunit-subunit interfaces (6). With the evidence
of a conformational change at hand it is tempting to hypothesize
whether this structural difference is physiologically meaningful. PheOH
and TyrOH share a common catalytic mechanism in which reduced
pterin cofactor, molecular oxygen, and iron are required to hydroxylate
the aromatic amino acid, but they are regulated through different
mechanisms. PheOH activation is believed to be achieved by specific
phosphorylation on the regulatory domain and/or by substrate
activation. During the past few years the mechanism of activation has
been the subject of intense biochemical and spectroscopic studies,
which have showed that activation of PheOH by the substrate is highly
cooperative and always accompanied by alterations in the tertiary
structure, but the nature of the conformational change has not been
ascertained yet. Substrate activation has been proposed to involve
cooperativity among all four subunits of the tetramer (33, 34) and
radiation target analysis has shown that activation of PheOH results in
modification of the monomer structure and promotes stronger association
at the dimer interface (35). These results are further supported by
evidence of increase in volume of the tetrameric protein (30), and by a
shift of the dimer tetramer equilibrium toward the tetrameric form
(31) upon binding of L-phenylalanine. Recent infrared
spectroscopic studies (36) led to the same conclusion and have also
shown that the alterations measured upon PheOH activation are not
accompanied by any measurable changes in the secondary structure. The
asymmetric structure of the PheOHCterm tetramer could
represent an intermediate state and could suggest that high flexibility
in the tetramerization domain is required to optimally orient the
domains for catalysis. Further experiments are required to understand
if and how the observed asymmetry of the tetramer is related to
cooperative substrate activation.
A possible contribution to the conformational heterogeneity in the
crystal structure of PheOHCterm could be a chemical
modification. PheOHCterm crystals lose their diffraction
power after a few days and mass spectroscopy analysis shows a
time-dependent heterogeneity of the protein in solution.
Two-dimensional electrophoresis of full-length recombinant wild-type
human PheOH (as isolated from E. coli) has shown a
microheterogeneity in terms of isoelectric point (five components),
which has been proposed (37) and recently proven2 to be a result of
progressive deamidation of rather labile amide groups. Studies on short
peptides have demonstrated that the rate of nonenzymatic deamidation of
asparagine residues primarily depends on the amino acid at positions
n 1 and n + 1, with half-lives from a few
hours, when the residue in the n + 1 position is a glycine,
to several days (38). Polar residues preceding Asn and neighboring Ser
and Thr increase deamidation rates, whereas bulky, nonhydrophobic
residues preceding Asn correlate with low deamidation rates (38).
Deamidation may result in marked conformational changes as first shown
for bovine heart cytochrome c (39). The sequence positions
of the labile amide groups in PheOH are, however, not yet identified.
Two putative targets for nonenzymatic deamidation are both highly
conserved within the mammalian PheOH, and map on the tetramerization
domain, Asn-426 (Asp-Asn-Thr), in the hinge loop that seems to confer
high flexibility to the tetramerization domain and Asn-438
(Ile-Asn-Ser) in the middle of the coiled coil. Asn-426 and Asn-438 are
well defined by the electron density, but Thr-427 is very weakly
outlined. Mutation of these residues may confirm if a nonenzymatic
deamidation mechanism is responsible for the heterogeneity of
PheOHCterm and for the difficulties encountered in the
crystallization of this enzyme in its tetrameric form.
In discussing the implications of the asymmetric organization of PheOHCterm we should also consider that the observed arrangement could be the result of crystal packing interactions. This possibility can be excluded because the catalytic domains are nearly identical in both the dimeric and tetrameric form of PheOH as well as in the TyrOH structure, even though they crystallize in different space groups.
Phenylketonuria-- PKU is a common recessive genetic disease caused by mutations in the human PheOH gene. PKU was the first inborn error of metabolism shown to affect the central nervous system and the first form of mental retardation related to a specific enzymatic defect (40). Approximately 280 mutant alleles have been identified so far and they have been associated with different degrees of PKU severity (for review see Refs. 2 and 3 and PKU data base: http://www.mcgill.ca/pahdb). The majority of PKU mutations map onto the catalytic domain, but 12 mutations occur in residues at the interface of the catalytic and tetramerization domain or within the tetramerization domain. Of particular interest in this study are the mutations that have been shown by expression studies to severely affect the stability of the enzyme.
The splicing mutation IVS12ntg ![]() |
ACKNOWLEDGEMENTS |
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We thank Christelle Sabatier and Andy-Mark Thunnissen for fruitful discussions and critical reading of the manuscript and also Steven Hayward for making the program DYNDOM available prior to distribution. We also thank Randi S. Svebak and Ali S. Muñoz for expert technical assistance.
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FOOTNOTES |
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* This work received support from the Physical Biosciences Division of Lawrence Berkeley National Laboratory (to R. C. S.), the Research Council of Norway (to H. E. and T. F.), Rebergs Legat (to T. F.), the Novo Nordisk Foundation (to T. F.), and the European Commission (to T. F.).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 atomic coordinates and structure factors (code 2PAH) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
§ Present address: Laboratory of Biophysical Chemistry, Dept. of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
** To whom correspondence should be addressed. Tel.: 510-643-8285; Fax: 510-643-9290; E-mail: stevens{at}adrenaline.berkeley.edu.
1 The abbreviations used are: PheOH, phenylalanine hydroxylase; PKU, phenylketonuria; TyrOH, tyrosine hydroxylase; TrpOH, tryptophan hydroxylase; MES, (2-(N-morpholino)ethanesulfonic acid); Bis-Tris-propane,2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
2 T. Solstad and T. Flatmark, unpublished data.
3 P. M. Knappskog, E. Bjørgo, and T. Flatmark, unpublished data.
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REFERENCES |
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