Structural Studies on Phenylalanine Hydroxylase and Implications Toward Understanding and Treating Phenylketonuria

Home , Tryptophan hydroxylase, Tyrosine hydroxylase

Heidi Erlandsen, DrSci; Marianne G. Patch, PhD; Alejandra Gamez, PhD; Mary Straub; and Raymond C. Stevens, PhD

ABSTRACT. Mutations in the gene encoding for phe- discusses some of the structural effects of the cur- nylalanine hydroxylase (PAH) result in phenylketonuria rently known mutations in the PAH gene, including (PKU) or hyperphenylalaninemia (HPA). Several 3-di- some of the BH4-responsive PKU/HPA mutations mensional structures of truncated forms of PAH have (PKU database at www.pahdb.mcgill.ca and PAH been determined in our laboratory and by others, using Mutation Analysis Consortium Newsletter, Decem- x-ray crystallographic techniques. These structures have allowed for a detailed mapping of the >250 missense ber 2001). mutations known to cause PKU or HPA found through- Human (liver) PAH (EC. 1.14.16.1) exists in a pH- out the 3 domains of PAH. This structural information dependent equilibrium of homotetramers and ho- has helped formulate rules that might aid in predicting modimers,7 and, like the 2 other aromatic amino acid the likely effects of unclassified or newly discovered hydroxylases tyrosine hydroxylase (EC 1.14.16.2) PAH mutations. Also, with the aid of recent crystal struc- and tryptophan hydroxylase (EC 1.14.16.4), consists ture determinations of co-factor and substrate analogs of 3 domains: an N-terminal regulatory domain (res- bound at the PAH active site, the recently discovered idues 1–142), a catalytic domain (residues 143–410), tetrahydrobiopterin-responsive PKU/HPA genotypes can be mapped onto the PAH structure, providing a molecu- and a C-terminal tetramerization domain (residues lar basis for this tetrahydrobiopterin response. Pediatrics 411- 452; Fig 1). Because of the difficulty of crystal- 2003;112:1557–1565; phenylalanine hydroxylase, 3-dimen- lizing full-length PAH, no full-length tetrameric sional structure, structural basis, phenylketonuria, BH4- structures exist for PAH. However, several truncated responsive hyperphenylalaninemia, co-factor therapy. forms of PAH have been structurally characterized, including a dimeric form containing the regulatory 3 ABBREVIATIONS. PAH, phenylalanine hydroxylase; l-Phe, l- and catalytic domains and a tetrameric form con- 2 phenylalanine; BH4, tetrahydrobiopterin; PKU, phenylketonuria; taining the catalytic and tetramerization domains. HPA, hyperphenylalaninemia; ARS, autoregulatory sequence; On the basis of these structures and a higher-resolu- CBR, co-factor–binding region. tion dimeric double-truncated form of PAH,1 a composite full-length structural model was constructed uman phenylalanine hydroxylase (PAH) by superimposing the respective catalytic domain converts the essential amino acid l-phenyl- regions (Figs 2–4).8 alanine (l-Phe) into l-tyrosine using the The regulatory domain of PAH contains an ␣-␤ H sandwich with an interlocking double ␤␣␤ motif co-factor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin ␤␣␤␤␣␤ 3 (BH4) and molecular oxygen. Catalysis by this iron- ( topology) (Fig 4). The N-terminal autoreg- dependent enzyme is the major pathway for cata- ulatory sequence (ARS; residues 19–33) extends over bolic degradation of dietary l-Phe and accounts for the active site in the catalytic domain. approximately 75% of the l-Phe disposal. The auto- An N-terminal truncated form of PAH that in- somal recessive disorder phenylketonuria (PKU) is cludes the catalytic and tetramerization domains the result of a deficiency of PAH enzymatic activity (residues 116–452) crystallized as a tetramer (dimer or loss of enzyme expression as a result of mutations of dimers).2 The tetramerization domain contains 2 in the PAH gene. Because of extensive newborn ␤-strands, forming a ␤-ribbon, and a 40-Å-long ␣-he- screening for PKU and genotyping of the PAH al- lix.2,9 The 4 ␣-helices (1 from each monomer) pack leles, Ͼ400 mutations in the PAH gene are known to into a tight antiparallel coiled-coil motif in the center cause PKU or the milder form referred to as hyper- of the tetramer structure (Fig 4). phenylalaninemia (HPA). The recently solved crystal Recombinant double-truncated human PAH 1–6 ⌬ ⌬ structures of PAH provide a structural scaffold to ( NH102- COOH428, hPAHCat) represents a fully explain the effects of PAH mutations. This review activated form of the enzyme, without any cooper- covers the structural work done so far on PAH and ativity of l-Phe binding.10 Thus, this structure represents the activated, or R state, of PAH. The catalytic From the Department of Molecular Biology, Scripps Research Institute, La domain region of the PAH structure (hPAHCat) has a Jolla, California. basket-like arrangement, with a total of 13 ␣-helices Reprint requests (R.C.S.) to Department of Molecular Biology, Scripps Re- and 8 ␤-strands. Because part of the C-terminal tet- search Institute, 10550 North Torrey Pines Rd, La Jolla, CA 92037. E-mail: ramerization domain is missing, this double-trun- [email protected] PEDIATRICS (ISSN 0031 4005). Copyright © 2003 by the American Acad- cated PAH construct forms a dimer in solution, as emy of Pediatrics. well as in the crystal.

Downloaded from www.aappublications.org/news PEDIATRICSby guest on September Vol. 11225, 2021 No. 6 December 2003 1557 Fig 1. Scheme showing the secondary structure assignment of human PAH sequence (SWISS-PROT P00439). Sec- ondary structure was assigned using the program DSSP56 on the composite PAH model.8 Residues that have PKU mutations associated with them are marked with grey dots. Secondary structural elements of the 3 regulatory domain are indicated as arrows for ␤-strands and coils for ␣-helical regions. Reprinted with permission from Erlandsen and Stevens.49

Fig 2. Bar graph showing the trunca- tions made in PAH to form crystals. The figure contains information about the oligomeric state of the protein along with the resolution of the crystal structures.

The active site, located in the center of the catalytic on the “floor” of the active site, at the intersection of domain, consists of a 13-Å-deep and 10-Å-wide the channel and the active site pocket. The Fe(III) pocket (Fig 3).1 Adjacent to the active site is a 16-Å- atom is coordinated to His285, His290, and 1 oxygen long and 8-Å-wide channel that may provide sub- atom in Glu330. Both His285 and His290 have been strate access to the active site. The majority of the 34 shown by site-directed mutagenesis to be required amino acids lining the active site are hydrophobic, for iron binding.12 Well-defined electron density is but 3 charged glutamates, 2 histidines, and 1 tyrosine also observed for 3 water molecules coordinated to are also located in this region. Covering the entrance the iron. The iron ligands are arranged in an octahe- to the active site is a short loop (residues 378–381) dral geometry, making the iron 6-coordinate as pre- that contains some of the highest B-factors in the viously suggested from spectroscopic studies.13 structure (60–80 Å2), indicative of a flexible, or dis- The PAH hydroxylation reaction requires the ordered, loop region. binding of BH4, dioxygen, and Fe(II) before hydroxy- As isolated, the PAH protein contains an active site lation of l-Phe can occur. A stretch of 27 PAH amino Fe(III) atom.1,11 In the crystal structures, the iron acids (His263 to His289), which is highly conserved atom is located 10 Å below the surface of the protein in all 3 aromatic amino acid hydroxylases, was

1558 STUDIES ON PAHDownloaded AND UNDERSTANDING from www.aappublications.org/news AND TREATING by guest PKUon September 25, 2021 with Phe254, and the pterin ring and dihydroxypropyl side-chain are positioned by a Tyr325 hydrophobic interaction. The BH2 dihydroxypropyl side chain O2Ј atom hydrogen bonds to the carbonyl oxygen of Ala322, and Glu286 hydrogen bonds to 1 of the water molecules coordinated to the iron and also hydrogen bonds to a water molecule that is hydrogen bonded to the N3 position of the pterin ring. In addition, a major conformational change occurs in the active site upon pterin binding; residues 245 to 250 move in the direction of the iron, allowing several protein hydrogen bonds to the pterin ring to be formed. Bound pterin co-factor is in an ideal location for dioxygen binding in a bridging position between the iron and the pterin, as is presumed to happen during the l-Phe hydroxylation reaction. Fig 3. Structure of a monomer of human PAH full-length composite model. The regulatory domain (residues 19–142), the cata- Although several alternative co-factor analogs are lytic domain (residues 143–410), and the tetramerization domain capable of being used in the PAH hydroxylation comprise the full-length monomer. The active site iron is shown as reaction, only the natural co-factor (BH4) inhibits the a sphere. Reprinted with permission from Erlandsen and Stevens.8 l-Phe induced activation of the enzyme13; the molecular mechanism of this inhibition is not yet under- 3,13,16 thought to be responsible for tetrahydrobiopterin stood. BH2 inhibits the activation by l-Phe less 14,15 17 binding before any structures were known. In the than the inhibition observed with BH4. Hydrogen PAH structures, 10 residues (Phe263, Cys265, bonding between the pterin dihydroxypropyl side Thr266, Thr278, Pro279, Glu280, Pro281, His285, chain and the carbonyl oxygen of Ala322, in combi- Glu286, and Gly289) in this co-factor–binding motif nation with the PAH loop 245 to 250 conformational are present in the active site. changes observed on co-factor binding, could pro- Based on co-crystal structures of double-truncated vide a specific regulatory function of the pterin upon Fe(III)-containing human PAH with oxidized co- binding at the active site. It is interesting that when ⅐ ⅐ factor (hPAHCat 7,8-BH2) or Fe(II)-containing hu- the binary complex hPAHCat 7,8-BH2 structure is man PAH with reduced (natural) co-factor superimposed onto the structure of the ligand-free ⅐ 5 (hPAHCat BH4), structural details of co-factor bind- C-terminal truncated rat PAH (rPAHReg containing 3 ing have been determined. The pterin binds close to the regulatory and catalytic domains ), BH2 interacts the catalytic iron and forms hydrogen bonding inter- with the N-terminal ARS region. actions with 2 of the water molecules coordinated to This interaction of pterin with the ARS gives a the iron, as well as to the main chain carbonyl oxy- plausible structural explanation for the inhibitory gens of Ala322, Gly247, and Leu249; the main chain effect of BH4 on the rate of phosphorylation of Ser16 amide of Leu249; and the O␥ atom of Ser251.4,5 The in rPAH by the cAMP-dependent protein kinase, pterin ring forms an aromatic ␲-stacking interaction which is specific for the R-isomer of the natural

Fig 4. Three views of the tetrameric form of the composite model of PAH. The active site iron is shown as a sphere. A, Front view. B, Side view, seen in the plane of the paper along the x axis as compared with A. C, Side view, seen in the plane of the paper along the y axis as compared with A. Reprinted with permission from Erlandsen and Stevens.49

Downloaded from www.aappublications.org/news by guest on September 25, 2021 SUPPLEMENT 1559 18 co-factor (BH4). Furthermore, phosphorylation of and/or stability that has been observed. The first Ser16 in the mobile ARS region may facilitate access category involves mutations that affect both PAH of l-Phe to the active site. These conclusions are kinetics and stability. A second class describes struc- consistent with the finding that the phosphorylated turally stable mutations with altered kinetic proper- form of rPAH requires less l-Phe to be activated (S0.5 ties, whereas a third class encompasses PAH muta- ϭ ␮ ϭ 29 M) than the nonphosphorylated form (S0.5 tions displaying normal kinetics but with reduced ␮ 18 21 51 M). Thus, the regulatory properties of BH4 in stability in vitro and in vivo. In addition, on the full-length PAH does not require an additional bind- basis of the 3-dimensional structure of PAH, muta- ing site of the co-factor in the regulatory domain as tions that cause PKU/HPA affect residues in 5 dif- previously postulated.3 ferent categories: 1) residues located at the active site, 2) structural residues, 3) residues involving interdo- STRUCTURAL BASIS FOR HPA AND PKU main interactions in a monomer, 4) residues that Currently, 269 missense point mutations are interact with the N-terminal ARS, and 5) residues at known for the PAH gene (PAH Mutation Analysis the dimer or tetramer interface regions of the struc- Consortium Newsletter, December 2001 issue; PKU ture. The most prevalent single point mutations in database at www.pahdb.mcgill.ca), in addition to 23 the PAH gene are discussed according to these 5 nonsense mutations and 10 silent mutations. Most of different structural groups. the point mutations map onto the exon 5 (PAH residue 148) to exon 12 (PAH residue 438) region of the PAH Active Site Mutations sequence. As shown in Figs 1 and 5, 57 PAH muta- In total, 31 point mutations can be found in the tions are located in the regulatory domain sequence residues lining the active site. Seven of these can be (residues 1–142), whereas 231 PAH mutations are found in the putative pterin-binding motif, consist- located in the catalytic domain (residues 143–410) ing of residues 264 to 29015 (PKU database at www. and 14 are located in the tetramerization domain pahdb.mcgill.ca). sequence (residues 411–452). Previously, a summary The D143G mutation is associated with a severe of genotype/phenotype/structural interpretations PKU phenotype.22 Asp143 is located at the entrance for these mutations was published.8 to the active site and may be involved in controlling In vitro expression analyses have been used to substrate/co-factor access to the active site. Another characterize PAH gene mutations. In general, the PKU/HPA mutation is Thr278, which is also located PAH mutations that lead to PKU or HPA genotypes at the entrance to the active site, forming a hydrogen result in reduced enzyme activity and stability to bond with Glu280.23,24 The mutations T278I and varying extents, and some mutations also have been T278A substitute a polar amino acid into different found to alter the oligomeric state of the protein.19,20 hydrophobic amino acids, losing an important hy- At least 3 groups of HPA/PKU mutations have been drogen bond that would perturb the structure categorized, dependent on the kinetic behavior around the entrance to the active site. The more

Fig 5. C␣-trace of 1 monomer of the full-length model of PAH. The trace is shaded for residues associated with PKU mutations, and the active site iron is shown for reference as a sphere. Note that sites of PKU mutations are located through- out the 3 domains of PAH. Reprinted with permission from Erlandsen and Stevens.8

1560 STUDIES ON PAHDownloaded AND UNDERSTANDING from www.aappublications.org/news AND TREATING by guest PKUon September 25, 2021 electrostatically conserved mutation T278N would location and electrostatics of the residues that act as most likely result in a changed hydrogen bonding iron ligands, thus destabilizing the active site struc- pattern that would have the same effect as the 2 other ture. One additional residue that is important in mutations, although presumably slightly smaller in holding the catalytic iron in place is Ser349, which magnitude. A frequent mutation among black Amer- hydrogen bonds to His285, one of the iron ligands. icans is the L255S mutation, which causes severe Two PKU mutations have been reported for this PKU.25 Leu255 is most likely involved in controlling residue, one that results in classical PKU (S349P35). the separation of helices C␣6 and C␣9, and in this This substitution into a proline so close to the cata- active site mutation, a nonpolar group is substituted lytic iron will change the shape of the active site. As with a smaller polar side chain that would be ex- expected, expression of S349P hPAH in E coli or COS pected to result in significant structural perturbation. cells results in only Ͻ0.2 to Ͻ1% residual activity. Another mutation in the active site is the E280K The second mutation of Ser349, S349L,36 also has mutation, found within the pterin-binding motif15 minimal activity in E coli or COS cells. Both muta- and associated with mild to severe PKU pheno- tions will alter the hydrogen bond to His285, having types.26 Glu280 hydrogen bonds to His146 and also severe detriment on the PAH catalytic activity. forms a salt bridge to Arg158. Also, in the active site of PAH, there are only 2 free, charged groups (both PAH Mutations in Structural Residues glutamic acids), and substitution of Glu280 into a The G46S mutation in the regulatory domain is lysine represents a dramatic change in the electro- associated with classical PKU and is one of the most static potential of the active site. As expected, expres- frequent mutations found clinically. Gly46 is located sion of E280K human PAH in Escherichia coli results on the surface of the regulatory domain in a loop in an enzyme with only approximately 1% of the preceding helix R␣1. Substitution into a serine would specific activity of wild-type PAH.27 The R158Q mu- generate potential side-chain hydrogen bonding to 1 tation is a frequent mutation in patients with PKU. or more residues in close proximity to Gly46, result- As mentioned above, Arg158 forms a salt bridge to ing in distortions of the regulatory domain second- Glu280, but also forms a hydrogen bond to Tyr268. ary structure. This G46S mutation thus potentially Both of these interactions are important for conserv- results in the formation of inactive PAH aggregates, ing the shape of the active site, and substitution into as previously reported.37 Another PAH mutation a glutamine or the larger aromatic residue trypto- found in the regulatory domain is I65T. This muta- phan (R158W) will alter the active site architecture tion is associated with non-PKU HPA to variant and lower enzymatic activity.28 Another active site PKU.38 Ile65 is located in the hydrophobic core of the residue that is involved in BH4 co-factor binding is regulatory domain, and substitution into a polar Phe254, which ␲-stacks onto the pterin ring in threonine (or asparagine as in the I65N mutant) ⅐ 4 hPAHCat 7,8-BH2. Phe254 is located 5.9 Å away would result in a significant structural perturbation from the active site iron, and substitution into an of the regulatory domain core. A frequent PAH mu- isoleucine, as in PKU mutant F254I, would interfere tation found in patients with PKU is A104D. This with the proper binding of the pterin and possibly mutation is associated with variant PKU, and Ala104 the Phe substrate. is located in a loop between R␣2 and R␤4inthe One of the most frequent PAH mutations found in regulatory domain. Substitution into a larger and southeastern Europe is the P281L mutation that is charged residue may destabilize this loop structure. associated with HPA to severe PKU phenotypes.29 Consistent with this hypothesis, in vitro expression Pro281 helps to define the shape of the active site led to increased PAH aggregation and marginal ac- very close to the iron. Therefore, substitution to a less tivity.39 Cys203 is involved in a disulfide bond with rigid leucine will change the conformation of the Cys334 in the rat PAH structure,3 but this disulfide active site by removing the conformational con- link is not observed in any of the human PAH struc- straints imposed by the proline. As expected, in vitro tures.1,2 Both cysteine residues are associated with expression of P281L PAH in E coli or African green PKU mutations. Arg243 is located at the end of C␤1, monkey Cercopithecus aethiops kidney (COS) cells re- forming a salt bridge to Asp129 in the C␣1 helix. In sults in complete loss of PAH activity.30,31 vitro expression of either of 2 PKU mutants (R243Q Three residues in the active site that are located or R243X) in COS cells resulted in Ͻ10% residual near the (putative) location of substrate binding or in activity40 or Ͻ1% residual activity.26 the region near the catalytic iron are Phe331, Ala345, Arg252 forms a salt bridge to Asp315 and hydro- and Gly346. Phe331 ␲-stacks onto Trp326, a residue gen-bonds to the carbonyl oxygen of Ala313 as well that may be involved in determining substrate spec- as the side chain of Asp27 in the ARS. There are 3 ificity in the aromatic amino acid hydroxylases.32 PKU mutations associated with this residue: R252Q, Two PKU mutations have been reported for Phe331, R252G, and R252W. The R252W mutation results in F331C and F331L,33,34 and both of these mutations classical PKU, and in vitro expression of the R252G will remove important ␲-stacking interactions of the and R252W mutations results in Ͻ1% residual activ- active site. Both Ala345 and Gly346 are located close ity.25,27,28 Recombinant expression of the R252Q mu- to the triad of residues that bind the iron (His285, tation, however, results in somewhat larger residual His290, and Glu330). Ala345 has 2 reported PKU activity (16% and 3%).25 Thus, any substitution at mutations, A345S and A345T, and Gly346 has 1 mu- Arg252 results in disruption of stabilizing interac- tation, G346R. All 3 of these mutations into larger tions in the catalytic domain. and more polar residues would interfere with the Ala259 is buried in a hydrophobic pocket approx-

Downloaded from www.aappublications.org/news by guest on September 25, 2021 SUPPLEMENT 1561 imately 4 Å away from Leu311 and Leu308, which gen-bonds to Arg71 as well as Glu422 in a second both hydrogen-bond to Arg408. These residues hold monomer. Arg297 has 2 PKU mutations, R297H and the tetramerization arm close to the catalytic domain, R297C,37 and both substitutions will result in dis- and thus substitution of Ala 259 into a larger or polar rupting the dimer-stabilizing hydrogen bonds, re- residue, as in A259T and A259V, cannot be accom- sulting in unstable PAH. Gln304 in ␣-helix C␣8is modated. Both A259T and A259V hPAH when ex- also involved in dimer/tetramer interactions, form- pressed in vitro result in 0.2% to 0.3% residual activ- ing hydrogen bonds to Tyr417 in a second monomer, ity.27 One of the most frequent mutations in the PKU and Arg261. Gln304 has 2 PKU mutations, a silent database is R261Q. This mutation results in pheno- mutation (Q304Q)43 and Q304R; substitution of a types varying from variant PKU to classical PKU. polar glutamine to a charged arginine would alter Arg261 is located in a loop between helix C␣6 and both hydrogen bonds and destabilize PAH. Last, C␤2 in the catalytic domain, forming hydrogen both Pro362 and Pro366 position Glu368, which me- bonds to Gln304 and Thr238, and substituting an diate dimer contacts. Thus, the PKU mutations arginine for proline (R261P) would destabilize the P362T44 and P366H45 will most likely prevent proper catalytic domain structure. dimer formation.

PAH Mutations Involved in Interdomain Interactions POTENTIAL PKU/HPA THERAPEUTIC in a Monomer APPROACHES The most important residues at the interface be- In light of the difficulty of PKU/HPA patients in tween the catalytic and tetramerization domains are adhering to a restrictive diet for life,46,47 new and less Leu311, Leu308, and Arg408. Arg408, located in the restrictive therapies for PKU/HPA are needed. Be- loop between C␣12 and T␤1, forms hydrogen bonds cause gene therapy solutions are many years off, to the carbonyl oxygens of Leu308 (at the end of C␣8) alternative solutions such as enzyme replacement and Leu311 (in the loop between C␣8 and C␣9). therapy using recombinantly produced PAH (in a R408W is the single most frequent mutation in the truncated, monomeric, and constitutively active PKU database, resulting in a severe PKU phenotype, form) or the plant enzyme phenylalinine ammonia with a low (Ͻ1% to Ͻ2.7%) recombinant PAH activ- lyase may provide some relief to PKU/HPA Phe ity.41,42 Another mutation, R408Q, associated with an load. For example, phenylalanine has been shown to HPA phenotype, reportedly shows 55% residual lower l-Phe levels, as well as reduce the symptoms PAH activity.42 Substitution of Arg408 into a larger of HPA, in mice engineered with a model for HPA.48 and bulkier tryptophan would alter the hydrogen bonding network at the interface of the tetrameriza- BH4-RESPONSIVE HPA/PKU MUTATIONS tion and catalytic domains, interfering with the cor- Recently, a number of patients harboring a subset rect positioning of the ␤-ribbon (T␤1 and T␤2). of PAH mutations showed a marked lowering (nor- malization) of their blood l-Phe levels upon oral PAH Mutations in Residues That Interact With the administration of the PAH co-factor BH4 (10–20 mg N-Terminal ARS 49,51 BH4/kg body weight). It is believed that this Tyr377 is 1 of the residues in the catalytic domain subset of mutations results in expressed mutant en- 3 that is in contact with the N-terminal ARS and hy- zymes that are Km variants of PAH with an altered drogen-bonds to Ser23 in the regulatory domain, binding affinity for BH4. More and more PAH muta- along with stacking on top of Trp326 (a residue tions are continuing to be reported as being respon- 50,51 involved in determining substrate specificity for the sive to BH4, and characterization of the biochem- aromatic amino acid hydroxylases). The proximity of ical, molecular, and physiologic bases for their BH4 Tyr377 to the ARS and the putative substrate binding responsiveness is required for a detailed understand- site may regulate access to the active site. In the PAH ing of this phenomenon. PKU mutant Y377C, the hydrogen bond to Ser23 Most of the BH4-responsive PKU mutations are in would be lost, altering access to the active site. the PAH catalytic domain, either located in co-factor–binding regions (CBR 1 [residues 245–266], CBR PAH Mutation in Residues at the Dimer or Tetramer 2 [residues 280–283], CBR 3 [residues 322–326], and Interfaces CBR 4 [residues 377–379]) or in locations that directly Residues Ser67, Glu76, Arg297, Gln304, and interact with the CBR regions involved with co-factor Glu422 can be found at the dimer interface and have binding. The following PAH genotypes were found 1–3 PKU mutations associated with them. Ser67 is lo- to be responsive to BH4: P407S/R252W, IVS4–1g- cated in ␤-strand R␤2, forming a hydrogen bond to Ͼa/A373T, R413P/R241C,52 IVS10nt-11g-Ͼa/ Tyr216 in another monomer; the PKU mutation S67P E390G,53 A313T/1099insC, V190A/R243X, A300S/ would alter the PAH structure at this interface. A403V, and R241C/A403V.54 These mutations are Glu76 is located in ␤-strand R␤3 on the surface of the found in distinct regions of the PAH primary se- regulatory domain, hydrogen-bonding to His208 in quence (Fig 1) and tertiary structure of the composite another monomer, as well as Asn73 in the same model of hPAH (Fig 6). monomer. The PKU mutations E76A and E76G are Neither residue in the R252W/P407S genotype in- substitutions of a charged residue into surface-ex- teracts directly with the co-factor, but Arg252 follows posed hydrophobic residues, resulting in destabili- Ser251 and is located in CBR 1 (Figs 1 and 6). Ser251 zation of the secondary structure in the regulatory is presumed to position BH4 by hydrogen bonding to domain. Arg297 is located in ␣-helix C␣8 and hydro- the dihydroxypropyl side chain. As previously men-

1562 STUDIES ON PAHDownloaded AND UNDERSTANDING from www.aappublications.org/news AND TREATING by guest PKUon September 25, 2021 Fig 6. Backbone structure of a monomer of the composite model of PAH. Side chains of the residues involved in BH4 responsiveness are shown.49 The active site iron is shown as a sphere. The co-factor BH4 is positioned into the structure based on the electron density for the co-factor analog 7,8-dihydro-l-biopterin co-complex crystal structure with human double-truncated PAH.4 The CBRs are shown as darkened loops (CBR 1 [residues 245–266], CBR 2 [residues 280–283], CBR 3 [residues 322–326], and CBR 4 [residues 377–379]). Re- printed with permission from Erlandsen and Stevens.49

tioned, Arg252 hydrogen bonds to residues Ala313, responsive to BH4 oral loading, having an IVS10nt- Asp315, and Asp27 in the ARS. The second allele 11g-Ͼa/E390G genotype. The latter mutation, residue in the R252W/P407S genotype, Pro407, pre- E390G, must be responsible for the BH4 responsive- cedes the start of the tetramerization domain. Muta- ness, because the other mutation leads to a splicing tion into a less rigid serine residue might inhibit defect in intron 10 of the PAH gene, with no func- PAH tetramerization; however, dimeric or mono- tional protein expressed. The E390G PAH mutation meric P407S mutant PAH might still retain co-factor shows 70% residual activity when expressed in COS binding affinity and activity. Kure et al52 also re- cells (PKU database). In the structure of tetrameric ported a non–BH4-responsive patient, having a PAH, Glu390 is located on the surface in the catalytic P407S/R111X genotype. The R111X mutation results domain, following 1 of the CBRs (CBR 4)2 (Figs 1 and in a truncated PAH lacking enzymatic activity. 6). The Glu390 side chain points toward the catalytic Therefore, enzymatic activity for this second geno- domain of a second monomer and also toward the type must be attributable to the P407S PAH. On the tetramerization interface. Because Glu390 is located basis of these observations, BH4 responsiveness is on the surface, mutation into a flexible glycine may dependent on the specific PAH genotype. destabilize the PAH tetramer and reduce enzymatic The R413P/R241C genotype was also responsive activity somewhat. The E390G mutant PAH most to co-factor loading.52 Arg241 is located on the sur- likely retains co-factor–binding ability but with an face of the catalytic domain preceding 1 of the CBRs, increased Km; however, this hypothesis must still be at the start of C␤1. In the tetrameric structure of experimentally confirmed. PAH, Arg241 is hydrogen-bonded to Gln419 of the Another BH4-responsive genotype reported by tetramerization domain, participating in important Kure et al52 is IVS4–1g-Ͼa/A373T, with a mild HPA intradomain interactions.2 Arg413 is located in the phenotype. The first mutation affects intron 4, result- tetramerization domain and hydrogen-bonds to ing in nonfunctional protein expression. Ala373 Glu422, forming 1 of a few hydrogen bonds that hold forms hydrophobic interactions with helix C␣9 con- the ␤-ribbon formed by T␤1 and T␤2 together. This taining Tyr325, Trp326, and Ala322 (located in CBR interaction ensures proper tetramer formation by po- 3), which hydrogen-bonds to the dihydroxypropyl sitioning the 40-Å-long tetramerization domain ␣-he- side chain of the co-factor. An A373T substitution lix for intermonomer coiled-coil interactions. Three might be relatively easily accommodated in this re- PKU mutations have been found for Arg413: R413S, gion of the structure and might result in minimal R413C, and R413P. The R241C mutation, as a result structural perturbation of the C␣9 helix and the ac- of the mild effect of this mutation on the PAH struc- tive site. Thus, this A373T PAH retains some binding ture, as compared with the R413P mutation, must be affinity for BH4, providing for the observed BH4 responsible for the BH4 responsiveness of the responsiveness. R413P/R241C genotype. The variant HPA genotype A313T/1099insC was 53 54 Trefz et al reported a patient with PKU who was also found to be responsive to BH4 loading. The

Downloaded from www.aappublications.org/news by guest on September 25, 2021 SUPPLEMENT 1563 A313T allele is most likely BH4 responsive, because CONCLUSIONS the 1099insC mutation results in a frameshift after Crystal structures of different truncated forms of Leu367, but this frameshift mutation has all of the human and rat PAH have allowed for the construc- residues needed for catalysis and so might retain tion of a composite structural model for full-length, some enzymatic activity. Unfortunately, no expres- tetrameric PAH and provided a structural basis for sion information for either of these mutants exists. the numerous mutations resulting in deficient PAH Ala313 hydrogen-bonds to Arg252 (in CBR 1) and is activity. In addition, PAH crystal structures with located in a loop between helices C␣8 and C␣9, at the substrate analog, inhibitor, or co-factor bound at the interface with the regulatory domain (close to active site have provided details of ligand binding. Pro119). The effect of the A313T substitution on co- This structural information has helped formulate factor binding might not be too large because the rules that may aid in predicting the likely effects of residue preceding Ala313 is a glycine, which might unclassified or newly discovered PAH mutations. compensate for a distortion in the backbone imposed Also, with the aid of recent crystal structure deter- by a threonine mutation at Ala313. minations, recently discovered BH4-responsive Genotype V190A/R243X also displays variant PKU/HPA genotypes can be mapped onto PAH HPA, and the BH -responsive mutation must be 4 structure, providing a molecular basis for this BH4- V190A, because the R243X mutation results in a trun- dependent response. However, more structural work cated enzyme missing all of the catalytically required is needed, such as determination of the structure of residues. Val190 is located in helix C␣3, behind full-length PAH, as well as more site-directed mu- His285, and close to Cys284 and Arg270. Substitution tagenesis studies of PAH, to understand completely into an alanine at position 190 would create space the catalytic mechanism and substrate specificity of where the valine side chain was originally located. PAH and tyrosine hydroxylase/tryptophan hydrox- The mutant enzyme might have Cys284 (located at ylase, the other members of the aromatic amino acid the end of CBR 2 [residues 280–283]) and His285 hydroxylases. rearranged into the empty space created by the V190A substitution. These alterations to the co-factor ACKNOWLEDGMENTS binding site might require higher concentrations of This study was funded by National Institutes of Health grant co-factor for productive catalysis, consistent with the no. R01-HD38718 from the National Institute of Child Health and BH4 responsiveness observed. Human Development (Bethesda, MD). Variant HPA genotypes A300S/A403V and R241C/A403V both were found responsive to BH4 REFERENCES loading. Both genotypes contain the A403V allele. 1. Erlandsen H, Fusetti F, Martinez A, Hough E, Flatmark T, Stevens RC. Ala403 is located at the end of helix C␣12, close to Crystal structure of the catalytic domain of human phenylalanine hy- Ala309 in helix C␣8. Alanine or another smaller res- droxylase reveals the structural basis for phenylketonuria. Nat Struct idue might be necessary for close packing of helices Biol. 1997;4:995–1000 ␣ ␣ 2. Fusetti F, Erlandsen H, Flatmark T, Stevens RC. Structure of tetrameric C 8 and C 12. Thus, substitution into a larger valine human phenylalanine hydroxylase and its implications for phenylketo- as in A403V PAH might result in a less stable protein. nuria. J Biol Chem. 1998;273:16962–16967 The R241C/A403V genotype also shares 1 allele with 3. Kobe B, Jennings IG, House CM, et al. 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Downloaded from www.aappublications.org/news by guest on September 25, 2021 SUPPLEMENT 1565 Structural Studies on Phenylalanine Hydroxylase and Implications Toward Understanding and Treating Phenylketonuria Heidi Erlandsen, Marianne G. Patch, Alejandra Gamez, Mary Straub and Raymond C. Stevens Pediatrics 2003;112;1557

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