Structure of Tetrameric Human Phenylalanine Hydroxylase and Its Implications for Phenylketonuria*

Fabrizia FusettiDagger §, Heidi Erlandsen, Torgeir Flatmarkparallel , and Raymond C. StevensDagger **

From the Dagger  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 parallel  Department of Biochemistry and Molecular Biology, University of Bergen, Årstadveien 19, N-5009 Bergen, Norway

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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, alpha  = beta  = 90°, gamma  = 120°). The asymmetric unit contains two PheOHCterm monomers, each consisting of a catalytic and a tetramerization domain. Two asymmetric units form one tetramer by a crystallographic two-fold symmetry axis. The solvent content is about 60%. To obtain good diffraction it was essential to harvest and flash freeze the crystals immediately after growth was completed (1-2 days).

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 sigma A weighted |Fo| - |Fc| maps (19) calculated at this stage showed electron density for the helices forming the coiled-coil domain that were not included in the search model. Simulated annealing omit maps (20) were calculated for manual model building with the program "O" (21). Strict noncrystallographic symmetry was initially imposed during the refinement. After addition of the tetramerization domain the restrain option was used because the relative positions of the core and the tetramerization domains within the tetramer are described by different noncrystallographic two-fold operators. The refinement was performed imposing tight noncrystallographic symmetry restraints (weight of 300) for the core of the catalytic domain (residues 118-393) but relaxed restraints for the tetramerization domain (weight of 10). Alternating refinement cycles with X-PLOR (18) and model building produced the final structure. In the final model all residues are defined in the electron density with the exception of residues 138-144. The final model of the two subunits in the asymmetric unit, comprising 654 residues, has an R-factor of 22.7% for all measured data between 20 and 3.1 Å and an R-free of 33.7% (22). The final structure has a root mean square deviation in bond length and angles of 0.006 Å and 1.7°, respectively.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha -helices and 8 beta -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 Calpha 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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Fig. 1.   Ribbon diagram representations of the PheOHCterm and TyrOHCterm tetramers. The four subunits are shown in different colors. In the PheOH tetramer the 2-fold crystallographic axis relates subunits A (green) and B (blue) to C (yellow) and D (orange), the noncrystallographic 2-fold relates the dimer AC to BD. In the TyrOH tetramer the four subunits are related by a crystallographic 222 symmetry. The coiled-coil motif is visible in the center of each tetramer. The iron bound in the active sites are shown as red spheres. The figure was generated using the programs MOLSCRIPT (48) and RASTER3D (49).

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 beta -strands, forming a beta -ribbon, and a 40 Å long alpha -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 beta -ribbon to the coiled-coil helix accompanied by another rotation in the coiled-coil helix.


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Fig. 2.   A, stereo view of the superposition of the PheOHCterm showing that in PheOHCterm the relative orientation of the coiled-coil helix in subunit A (green) is different than in B (blue). B, stereo view of the PheOHCterm tetramerization domain. The superposition of the coiled-coil helices shows that the conformational change in the tetramerization domain causes a different orientation of the beta -ribbon that leads to the catalytic domain. The positions at which the conformational changes are detected are highlighted in red. Color code is as shown in A. For clarity, only the catalytic domain of B is shown in gray. C, sequence alignment of the PheOH and TyrOH tetramerization domains. Proline residues located at the hinge regions are shown in red. Secondary structure elements are represented as boxes (alpha -helices) and arrows (beta -strands). An arrow indicates the positions at which the conformational changes are detected. Regions of identity are indicated with a vertical bar. The figure was generated using the program MOLSCRIPT (48).

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 beta -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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Fig. 3.   A stereo view of the tetramerization domain of PheOHCterm, (subunit A). PKU mutations sites are shown in white as ball-and-stick representations (see text for details). The hydrogen-bond interactions of Arg-252 and Arg-408 with the main chain carbonyl groups of Ala-313, Leu-308, Leu-309, and Leu-311, are shown as dashed lines. The proline residues in the tetramerization domain are shown in green as ball-and-stick representations. The figure was generated using the program MOLSCRIPT (48).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 left-right-arrow  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 IVS12ntgright-arrowa in intron 12 of the human PheOH gene is the most prevalent PKU allele among Caucasians (41). The clinical and metabolic phenotype of homozygotes with the mutation is a severe ("classical") form of PKU. The mutation leads to the synthesis of a truncated form lacking the last 52 C-terminal amino acids, which include the tetramerization domain. Expression studies in eukaryotic systems indicated that the deletion abolished PheOH activity in the cell as a result of protein instability. Another frequent allele is caused by a CGG-to-TGG transition in exon 12, resulting in an amino acid substitution Arg/Trp at position 408. This mutation also results in undetectable levels of PheOH in vivo and in vitro and a severe metabolic PKU phenotype (42). Analysis of the TyrOH and PheOH structures shows that the highly conserved Arg-408 is located on the hinge loop that connects the tetramerization arm to the core of the PheOH monomer, flanked by the above mentioned Pro-407 and Pro-409 (Fig. 3). The arginine side chain is buried and forms a hydrogen bond with the main chain carbonyl of Leu-308, Leu-309, and Leu-311 at the C-terminal end of helix 8. Arg-408 could be involved in stabilizing domain swapping and promoting oligomerization by anchoring the tetramerization arm onto the catalytic domain in a proper orientation. Correct alignment of arm exchange in domain swapping is believed to be necessary for the association process (27). This suggestion is consistent with the analysis of mutation R408Q that causes a non-PKU hyperphenylalaninemia in homozygotes (43). Expression of R408Q mutants in E. coli results in the synthesis of a tetrameric form of the enzyme with high residual activity.3 Mutations R252W, A259L/T, F299C, and L311P (43-45) also result in extremely reduced levels of PheOH and are associated with severe PKU phenotype. As described above, the carbonyl atom of Leu-311 forms a hydrogen bond with Arg-408. Residue Ala-259 is buried in a hydrophobic pocket about 4 Å from Leu-311 and Leu-308; a larger or polar residue cannot be accommodated without disturbing the surrounding environment. Phe-299 is also involved in hydrophobic interactions that stabilize the helix where Leu-311 is located and Arg-252 forms a hydrogen bond with the carbonyl atom of Ala-313. As shown in Fig. 3, these mutations map in the same region as Arg-408, and they could dramatically alter the hydrogen bond network or produce steric hindrance, interfering with the correct positioning of the beta -ribbon. It is likely that their effect will have consequences for the arrangement of the catalytic and tetramerization domains and thus for the normal oligomerization process. Mutation Y414C has also been reported, and the expressed enzyme retains approximately 50% of normal activity (46). The mild effect of this mutation can be observed also at structural level: a substitution Tyr right-arrow Cys in this position should not have drastic consequences on the interactions with the neighboring residues (Fig. 3). One of the most frequent alleles in the oriental population is the missense mutation R413P in exon 12, which causes a severe metabolic form of PKU. This mutation results in protein instability with almost undetectable levels of PheOH when expressed in eukaryotic cells (47). Arg-413 is located in the middle of the first strand (beta 7) of the beta -ribbon in the tetramerization domain. A proline residue in this position would disrupt the main chain to main chain hydrogen bond network that stabilizes the beta -ribbon. The PKU mutation R413P most certainly leads to extreme disorder of the tetramerization arm thus compromising the normal oligomeric state and stability of PheOH.

The analysis of these mutations suggest that changes in the beta -ribbon have major effects on the stability of the enzyme. The possibility that the beta -ribbon represents a mutation "hot spot" points to the structural importance of this element.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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