BH4 responsiveness associated to a PKU mutation with decreased binding affinity for the cofactor Cristina Aguado a,c , Belen Pérez a,c , M. José García a,c , Amaya Bélanger-Quintana b,c , Mercedes Martínez-Pardo b,c , Magdalena Ugarte a,c, ⁎ , Lourdes R. Desviat a,c a Centro de Biología Molecular “Severo Ochoa” CSIC-UAM, Madrid, Spain b Unidad de Enfermedades Metabólicas, Servicio de Pediatría, Hospital Ramón y Cajal, Madrid, Spain c Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), Madrid, Spain Received 26 December 2006; received in revised form 13 February 2007; accepted 13 February 2007 Available online 22 February 2007 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. Method: 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. Conclusion: The D129G mutation which confers a BH4-responsive phenotype, has a decreased binding affinity for BH4. © 2007 Elsevier B.V. All rights reserved. Keywords: Tetrahydrobiopterin; Phenylketonuria; PKU mutation; Phenylalanine hydroxylase; BH4 responsiveness 1. Introduction Hyperphenylalaninemia is a result of a defect in the hydrox- ylation of phenylalanine (Phe) to tyrosine (Tyr), a reaction cat- alyzed by phenylalanine hydroxylase (PAH, EC.1.14.16.1) with the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH 4 ). In the majority of the cases, hyperphenylalaninemia is caused by mutations in the PAH gene (OMIM 261600), presenting with different phenotypes classified according to Phe tolerance: classical phenylketonuria (PKU), mild PKU and mild hyper- phenylalaninemia (MHP). More than 500 mutations have been described world-wide and the PAH enzyme has been fully characterized (http://www.pahdb.mcgill.ca). PAH has a three- domain structure consisting of an N-terminal regulatory domain, a catalytic domain which includes iron, substrate and BH4 binding sites and a C-terminal oligomerization domain [1–3]. Many reports have explored the genotype–phenotype correla- tions providing useful information on which to discuss ther- apeutic strategies. Dietary treatment is the choice for PKU patients while re- search is ongoing on gene and enzyme replacement therapies. However, after an initial report by Kure et al. [4], the in vivo response to BH4 supplementation resulting in a decrease in Phe levels has been confirmed to be a very common phenomenon in patients with PAH deficiency [5,6]. The positive response can be easily revealed by a standard BH4 loading test. About one third of PKU patients (mostly those with mild phenotypes) have been estimated to be potential candidates for BH4 treatment [6]. This has changed the therapeutic strategies for the disease, previously restricted to a life-long low-Phe diet. The diet avoids Clinica Chimica Acta 380 (2007) 8 – 12 www.elsevier.com/locate/clinchim ⁎ Corresponding author. Centro de Biología Molecular “Severo Ochoa” CSIC- UAM, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Tel.: +34 917347011; fax: +34 917347797. E-mail address: mugarte@cbm.uam.es (M. Ugarte). 0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2007.02.034