title: BH4 responsiveness associated to a PKU mutation with decreased binding affinity for the cofactor.
abstract: BACKGROUND
  Tetrahydrobiopterin (BH4), cofactor of phenylalanine hydroxylase, can be used to treat a subset of phenylketonuria (PKU) patients as it results in a reduction in blood phenylalanine levels. The molecular basis of the response appears to be multifactorial.
  
  
  METHODS
  A standard BH4 loading test (20 mg/kg) was performed. Genotyping was performed by DGGE and sequencing analysis. Expression analysis of the D129G mutation was performed in E. coli (expression as fusion protein MBP-PAH) and in a human hepatoma cell line with an N-terminal FLAG epitope.
  
  
  RESULTS
  We report the positive response and long-term treatment of a patient functionally hemizygous for the D129G mutation in the phenylalanine hydroxylase gene. Expression in the prokaryotic system revealed partial activity and a decreased binding affinity for BH4 of the mutant protein. In the eukaryotic system the mutant protein shows reduced stability.
  
  
  CONCLUSIONS
  The D129G mutation which confers a BH4-responsive phenotype, has a decreased binding affinity for BH4.
authors.affiliation: Centro de Biología Molecular Severo Ochoa CSIC-UAM, Madrid, Spin.
authors.forename: Cristina
authors.initials: C
authors.lastname: Aguado
authors.forename: Belen
authors.initials: B
authors.lastname: Pérez
authors.forename: M José
authors.initials: MJ
authors.lastname: García
authors.forename: Amaya
authors.initials: A
authors.lastname: Bélanger-Quintana
authors.forename: Mercedes
authors.initials: M
authors.lastname: Martínez-Pardo
authors.forename: Magdalena
authors.initials: M
authors.lastname: Ugarte
authors.forename: Lourdes R
authors.initials: LR
authors.lastname: Desviat
mesh_terms: D001708:Biopterins / Q000031:analogs & derivatives* / Q000494:pharmacology; D004252:DNA Mutational Analysis; D005838:Genotype; D006801:Humans; D007231:Infant, Newborn; D009154:Mutation / Q000235:genetics*; D015997:Neonatal Screening; D019001:Nitric Oxide Synthase / Q000378:metabolism; D010641:Phenotype; D010649:Phenylalanine; D010651:Phenylalanine Hydroxylase / Q000235:genetics* / Q000378:metabolism; D010661:Phenylketonurias / Q000175:diagnosis* / Q000235:genetics*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D001708:Biopterins; D010649:Phenylalanine; D019001:Nitric Oxide Synthase; D010651:Phenylalanine Hydroxylase; C003402:sapropterin