Structure of Tetrameric Human Phenylalanine Hydroxylase and Its Implications for Phenylketonuria

Abstract

Phenylalanine hydroxylase (PheOH) catalyzes the conversion of L-phenylalanine to L-tyrosine, the ratelimiting 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 coiledcoil. 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.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 pr...

PAHdb 2003: What a locus-specific knowledgebase can do

“…X-ray crystallography has been used previously to determine the 3D structures of truncated forms of phenylalanine hydroxylase [Erlandsen et al, 1997;Fusetti et al, 1998;Kobe et al, 1999], but because it is difficult to crystallize the full-length PAH subunit, no primary full-length tetrameric PAH structure has yet been analyzed. However, two truncated forms have been characterized.…”

“…However, two truncated forms have been characterized. These include a dimeric form containing regulatory and catalytic domains [Kobe et al, 1999] and a tetrameric form containing catalytic and tetramerization domains [Fusetti et al, 1998]. From these two structures, and from a higherresolution dimeric double-truncated form of the PAH enzyme [Erlandsen et al, 1997], it has been possible to derive a composite full-length structural model [Erlandsen and Stevens, 1999] (Fig.…”

“…The tetramerization domain contains two b-strands forming a b-ribbon and a 40 Å long a-helix. The four a-helices (one from each monomer) pack into a tight anti-parallel coiledcoil motif in the center of the tetramer structure [Fusetti et al, 1998]. The region containing the catalytic domain has a basket-like arrangement with a total of 13 a-helices and 8 b-strands.…”

Structural interpretation of mutations in phenylalanine hydroxylase protein aids in identifying genotype–phenotype correlations in phenylketonuria

“…We have recently determined the crystal structure of a truncation mutant of rat PAH (lacking 24 C-terminal residues) with catalytic and regulatory properties of the wild-type protein. 11 Previously, the crystal structures of smaller fragments, lacking the regulatory domain, of human PAH 12,13 and a homologous enzyme, rat tyrosine hydroxylases (TyrOH) 14 have also been determined. Jointly, this structural information now allows a structural interpretation of any PAH mutation resulting in PAH deficiency.…”

“…The tetramerisation domain is a 40Å-long α-helix, and the tetramer forms through the association of four helices into an antiparallel coiled coil. 13 Site-directed mutagenesis in conjunction with a threedimensional structure of a protein has emerged as one of the most powerful methods in understanding protein function. Here, we use the database of naturally occurring mutations in PAH jointly with the three-dimensional structure in an analogous way to elucidate their effects on protein function, which ultimately defines the metabolic phenotype of PKU.…”