Estudos Estruturais e Funcionais da Fenilalanina Hidroxilase Humana

2014-02-01T11:19:02Z (GMT) by Fábio Madeira
<p>My master thesis on the "Structural and Functional Studies of Human Phenylalanine Hidroxilase", under the supervision of Prof. Maria João Romão (Crystallography group, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa) and Prof. Ana Paula Leandro (Metabolism and Genetics group (Faculdade de Farmácia, Universidade de Lisboa). Full text in Portuguese.</p> <p><strong>Abstract</strong></p> <p>Human phenylalanine hydroxylase (hPAH) is a non-heme iron dependent enzyme that catalyzes the hydroxylation of the essential aromatic amino acid L-phenylalanine (L- Phe) into L-tyrosine in the presence of the pterin co-factor tetrahydrobiopterin and molecular oxygen. This enzyme is part of the catabolic pathway of degradation of dietary L-Phe and accounts for 75% of the L-Phe disposal. Human PAH assumes particular importance in human health since deficient enzymatic activity or loss of enzyme expression as a consequence of mutations in the gene coding for hPAH, is responsible for the inherited metabolic disorder known as phenylketonuria (PKU), the most frequent disorder of amino acid metabolism, and which presents an average incidence in the Caucasian population of 1:10000. The treatment by dietary restriction of L-Phe is largely accepted and recommended, and must be followed for life soon after birth, to avoid severe mental retardation. However, and in light of the difficulty of PKU patients in adhering to the dietary treatment, new and less restrictive therapeutical approaches are under study, namely the enzyme substitution therapy.<br>Currently, only the crystal structures of truncated forms of both human and rat PAH, are known, which have significantly contributed to the elucidation of the catalytic pathway and to the identification of the molecular determinants of PKU. However, and due to the instability of the hPAH, none three-dimensional structure of the full-length PAH was solved yet.<br>In this work, with the aim of solving the structure of the full-length PAH, two variant proteins were expressed and purified, namely hPAH C29S and hPAH E360K, as they present a higher level of expression when compared to the wildtype form (hPAHwt), which suggests an increased protein stability. The existence of these stable forms of the protein has endorsed the starting material for crystallization trials and X-ray diffraction assays. Crystallization conditions were obtained for both mutants led to promising preliminary results, important to solve the three-dimensional structure of the full-length hPAH. The enzymatic characterization of the chimeric proteins was also carried out. Interestingly, only hPAH C29S showed an increase in the maximum reaction rate (Vmax) and in the half- denaturation temperature (Tm), when compared to the hPAHwt. Knowledge of the 3D structure will be fundamental to identify the determinants of PKU phenotypes and to a better understanding of the catalytic mechanism. Additionally, the chimeric protein C29S proved to be a strong candidate for the development and use in the treatment of PKU by enzyme substitution therapy.</p> <p> </p> <p><strong><br></strong></p> <p> </p>