Journal of Inherited Metabolic Disease

, Volume 24, Issue 2, pp 213–230 | Cite as

A structural hypothesis for BH4 responsiveness in patients with mild forms of hyperphenylalaninaemia and phenylketonuria

  • H. Erlandsen
  • R. C. Stevens


Deficiencies in the human enzyme phenylalanine hydroxylase (PAH) due to mutations in the PAH gene (PAH) result in the inborn error of metabolism phenylketonuria (PKU). The clinical symptom of this disease is an elevated concentration of L-phenylalanine (L-Phe) in blood serum. To prevent mental retardation due to the buildup of neurotoxic metabolites of L-Phe, patients with severe PKU must be treated with a low-L-Phe diet starting early in their life. Owing to extensive newborn screening programmes and genotyping efforts, more than 400 different mutations have been identified in the PAH gene. Recently, there have been several reports of PKU patients showing a normalization of their L-Phe concentrations upon oral administration of the natural cofactor to PAH, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4). In an attempt to correlate the clinical responsiveness to BH4 administration with PKU genotype, we propose specific structural consequences for this subset of PAH mutations. Based on the location and proximity of this subset of mutations to the cofactor-binding site in the three-dimensional structure of PAH, a hypothesis for BH4 responsiveness in PKU patients is presented. It is believed that some of these mutations result in expressed mutant enzymes that are Km variants (with a lower binding affinity for BH4) of the standard PAH enzyme phenotype. Oral administration of excess BH4 thus makes it possible for these mutant enzymes to suppress their low binding affinity for BH4, enabling this subset of PAH mutations to perform the L-Phe hydroxylation reaction. Most of the BH4-responsive PAH mutations map to the catalytic domain of PAH in either of two categories. Residues are located in cofactor-binding regions or in regions that interact with the secondary structural elements involved in cofactor binding. Based on the series of known mutations that have been found to be responsive to BH4, we propose that other subsets of PAH mutations will have a high likelihood of being responsive to oral BH4 administration.


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  1. Benit P,Rey F,Blandin-Savoja F,Munnich A,Abadie V,Rey J (1999) The mutant genotype is the main determinant of the metabolic phenotype in phenylalanine hydroxylase de¢ciency. Mol Genet Metab 68: 43–47.Google Scholar
  2. BjÖrgo E,Knappskog PM,Martinez A,Stevens RC,Flatmark T (1998) Partial characterization and three-dimensional-structural localization of eight mutations in exon 7 of the human phenylalanine hydroxylase gene associated with phenylketonuria. Eur J Biochem 257: 1–10.Google Scholar
  3. Durr E,Jelesarov I (2000) Thermodynamic analysis of cavity creating mutations in an engineered leucine zipper and energetics of glycerol-induced coiled coil stabilization. Bio-chemistry 39: 4472–4482.Google Scholar
  4. Erlandsen H,Stevens RC (1999) The structural basis of phenylketonuria. Mol Genet Metab 68: 103–125.Google Scholar
  5. Erlandsen H,Fusetti F,Martinez A,Hough E,Flatmark T,Stevens RC (1997a) Crystal struc-ture of the catalytic domain of human phenylalanine hydroxylase reveals the structural basis for phenylketonuria. Nature Struct Biol 4: 995–1000.Google Scholar
  6. Erlandsen H,Martinez A,Knappskog PM,Haavik J,Hough E,Flatmark T (1997b) Crystallization and preliminary di¡raction analysis of a truncated homodimer of human phenylalanine hydroxylase. FEBS Lett 406: 171–174.Google Scholar
  7. Erlandsen H,BjÖrgo E,Flatmark T,Stevens RC (2000) Crystal structure and site-speci¢c mutagenesis of pterin-bound human phenylalanine hydroxylase. Biochemistry 39: 2208–2217.Google Scholar
  8. Fusetti F,Erlandsen H,Flatmark T, C. SR (1998) Structure of tetrameric human phenylalanine hydroxylase and its implications for phenylketonuria. J Biol Chem 273: 16962–16967.Google Scholar
  9. Gamez A,Perez B,Ugarte M,Desviat LR (2000) Expression analysis of phenylketonuria mutations. Effect on folding and stability of the phenylalanine hydroxylase protein. J Biol Chem 275: 29737–29742.Google Scholar
  10. Ghosh S,Wolan D,Adak S, et al (1999) Mutational analysis of the tetrahydrobiopterin-binding site in inducible nitric-oxide synthase. J Biol Chem 274: 24100–24112.Google Scholar
  11. Harris TK,Wu G,Massiah MA,Mildvan AS (2000) Mutational, kinetic, andNMRstudies of the roles of conserved glutamate residues and of lysine-39 in the mechanism of the MutT pyrophosphohydrolase. Biochemistry 39: 1655–1674.Google Scholar
  12. Hubbard SJ,Argos P (1995) Evidence on close packing and cavities in proteins. Curr Opin Biotechnol 6: 375–381.Google Scholar
  13. Jennings IG,Cotton RG,Kobe B (2000) Structural interpretation of mutations in phenylalanine hydroxylase protein aids in identifying genotype-phenotype correlations in phenylketonuria. Eur J Hum Genet 8: 683–696.Google Scholar
  14. Kabsch W,Sander C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22: 2577–2637.Google Scholar
  15. Kayaalp E,Treacy E,Waters PJ,Byck S,Nowacki P,Scriver CR (1997) Human phenylalanine hydroxylase mutations and hyperphenylalaninemia phenotypes: a metanalysis of genotype-phenotype correlations. Am J Hum Genet 61: 1309–1317.Google Scholar
  16. Knappskog PM,Eiken HG,Martinez A,Bruland O,Apold J,Flatmark T (1996) PKU mutation (D143G) associated with an apparent high residual enzyme activity: expression of a kinetic variant form of phenylalanine hydroxylase in three di¡erent systems. Hum Mutat 8: 236–246.Google Scholar
  17. Kobe B,Jennings IG,House CM, et al (1999) Structural basis of autoregulation of phenylalanine hydroxylase. Nature Struct Biol 6: 442–448.Google Scholar
  18. Kraulis PJ (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Crystallogr 24: 946–950.Google Scholar
  19. Kure S,Hou DC,Ohura T, et al (1999) Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency. J Pediatr 135: 375–378.Google Scholar
  20. Martinez A,Knappskog PM,Olafdottir S, et al (1995) Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases. Biochem J 306: 589–597.Google Scholar
  21. Merritt EA,Bacon DJ (1997) Raster3D: photorealistic molecular graphics. Methods Enzymol 277: 505–524.Google Scholar
  22. Nagasaki Y,Matsubara Y,Takano H, et al (1999) Reversal of hypopigmentation in phenylketonuria mice by adenovirus-mediated gene transfer. Pediatr Res 45: 465–473.Google Scholar
  23. Okano Y,Eisensmith RC,Guttler F, et al (1991a) Molecular basis of phenotypic heterogen-eity in phenylketonuria. N Engl J Med 324: 1232–1238.Google Scholar
  24. Okano Y,Wang T,Eisensmith RC, et al (1991b) Phenylketonuria missense mutations in the Mediterranean. Genomics 9: 96–103.Google Scholar
  25. Okano Y,Hase Y,Shintaku H, et al (1994) Molecular characterization of phenylketonuric mutations in Japanese by analysis of phenylalanine hydroxylase mRNA from lymphoblasts. Hum Mol Genet 3: 659.Google Scholar
  26. Oue S,Okamoto A,Yano T,Kagamiyama H (1999) Redesigning the substrate speci¢city of an enzyme by cumulative e¡ects of the mutations of non-active site residues. J Biol Chem 274: 2344–2349.Google Scholar
  27. Sarkissian CN,Shao Z,Blain F, et al (1999) A different approach to treatment of phenylketonuria: phenylalanine degradation with recombinant phenylalanine ammonia lyase. Proc Natl Acad Sci USA 96: 2339–2344.Google Scholar
  28. Sarkissian CN,Boulais DM,McDonald JD,Scriver CR (2000) A heteroallelic mutant mouse model: a new orthologue for human hyperphenylalaninemia. Mol Genet Metab 69: 188–194.Google Scholar
  29. Scriver CR,Waters PJ,Sarkissian C, et al (2000) PAHdb: a locus-specific knowledgebase. Hum Mutat 15: 99–104.Google Scholar
  30. Spaapen LJM,Bakker JA,Velter C, et al (2000) Tetrahydrobiopterin-responsive hyper-phenylalaninemia (HPA) in dutch neonates. J Inherit Metab Dis 23:(supplement 1): 45.Google Scholar
  31. Trefz F,Blau N,Aulehla-Scholz C,Korall H,Frauendienst-Egger G (2000) Treatment of mild phenylketonuria (PKU) by tetrahydrobiopterin (BH4). J Inherit Metab Dis 23(supplement 1): 47.Google Scholar
  32. Wang T,Okano Y,Eisensmith RC, et al (1991) Founder e¡ect of a prevalent phenylketonuria mutation in the Oriental population. Proc Natl Acad Sci USA 88: 2146–2150.Google Scholar
  33. Waters PJ,Parniak MA,Nowacki P,Scriver CR (1998) In vitro expression analysis of mutations in phenylalanine hydroxylase: linking genotype to phenotype and structure to function. Hum Mutat 11: 4–17.Google Scholar
  34. Zaremba SM,Gregoret LM (1999) Context-dependence of amino acid residue pairing in antiparallel beta-sheets. J Mol Biol 291: 463–479.Google Scholar

Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • H. Erlandsen
    • 1
  • R. C. Stevens
    • 1
  1. 1.Department of Molecular Biology and Institute for Childhood and Neglected Diseases, La JollaThe Scripps Research InstituteUSA

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