Structural Studies on Phenylalanine Hydroxylase and Implications Toward Understanding and Treating Phenylketonuria 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 com- posite 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 repre- sents 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 re- gions. 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 dihydroxypro- pyl side-chain are positioned by a Tyr325 hydropho- bic 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 hydro- gen 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 com- posite 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 molec- ular 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.