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Computational study of missense mutations in phenylalanine hydroxylase

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Abstract

Hyperphenylalaninemia (HPA) is one of the most common metabolic disorders. HPA, which is transmitted by an autosomal recessive mode of inheritance, is caused by mutations of the phenylalanine hydroxylase gene. Most mutations are missense and lead to reduced protein stability and/or impaired catalytic function. The impact of such mutations varies, ranging from classical phenylketonuria (PKU), mild PKU, to non-PKU HPA phenotypes. Despite the fact that HPA is a monogenic disease, clinical data show that one PKU genotype can be associated with more in vivo phenotypes, which indicates the role of other (still unknown) factors. To better understand the phenotype–genotype relationships, we analyzed computationally the impact of missense mutations in homozygotes stored in the BIOPKU database. A total of 34 selected homozygous genotypes was divided into two main groups according to their phenotypes: (A) genotypes leading to non-PKU HPA or combined phenotype non-PKU HPA/mild PKU and (B) genotypes leading to classical PKU, mild PKU or combined phenotype mild PKU/classical PKU. Combining in silico analysis and molecular dynamics simulations (in total 3 μs) we described the structural impact of the mutations, which allowed us to separate 32 out of 34 mutations between groups A and B. Testing the simulation conditions revealed that the outcome of mutant simulations can be modulated by the ionic strength. We also employed programs SNPs3D, Polyphen-2, and SIFT but based on the predictions performed we were not able to discriminate mutations with mild and severe PKU phenotypes.

The structural impact of the missense mutations with mild and severe phenotypes in the tetrameric structure of the phenylalanine hydroxylase was analyzed using in silico analysis and molecular dynamics simulations.

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References

  1. Kaufman S (1987) Enzymology of the phenylalanine-hydroxylating system. Enzyme 38:286–295

    CAS  Google Scholar 

  2. Stenson PD, Mort M, Ball EV, Shaw K, Phillips AD, Cooper DN (2014) The Human Gene Mutation Database: building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine. Hum Genet 133:1–9

    Article  CAS  Google Scholar 

  3. Gjetting T, Petersen M, Guldberg P, Guttler F (2001) In vitro expression of 34 naturally occurring mutant variants of phenylalanine hydroxylase: Correlation with metabolic phenotypes and susceptibility toward protein aggregation. Mol Genet Metab 72:132–143

    Article  CAS  Google Scholar 

  4. Pey AL, Desviat LR, Gamez A, Ugarte M, Perez B (2003) Phenylketonuria: genotype-phenotype correlations based on expression analysis of structural and functional mutations in PAH. Hum Mutat 21:370–378

    Article  CAS  Google Scholar 

  5. Gersting SW, Kemter KF, Staudigl M, Messing DD, Danecka MK, Lagler FB, Sommerhoff CP, Roscher AA, Muntau AC (2008) Loss of function in phenylketonuria is caused by impaired molecular motions and conformational instability. Am J Hum Genet 83:5–17

    Article  CAS  Google Scholar 

  6. Gregersen N, Bross P, Andresen BS, Pedersen CB, Corydon TJ, Bolund L (2001) The role of chaperone-assisted folding and quality control in inborn errors of metabolism: protein folding disorders. J Inherit Metab Dis 24:189–212

    Article  CAS  Google Scholar 

  7. Guldberg P, Rey F, Zschocke J, Romano V, Francois B, Michiels L, Ullrich K, Hoffmann GF, Burgard P, Schmidt H, Meli C, Riva E, Dianzani I, Ponzone A, Rey J, Guttler F (1998) A European multicenter study of phenylalanine hydroxylase deficiency: classification of 105 mutations and a general system for genotype-based prediction of metabolic phenotype. Am J Hum Genet 63:71–79

    Article  CAS  Google Scholar 

  8. Desviat LR, Perez B, Gamez A, Sanchez A, Garcia MJ, Martinez-Pardo M, Marchante C, Boveda D, Baldellou A, Arena J, Sanjurjo P, Fernandez A, Cabello ML, Ugarte M (1999) Genetic and phenotypic aspects of phenylalanine hydroxylase deficiency in Spain: molecular survey by regions. Eur J Hum Genet 7:386–392

    Article  CAS  Google Scholar 

  9. Desviat LR, Perez B, Garcia MJ, MartinezPardo M, Baldellou A, Arena J, Sanjurjo P, Campistol J, Couce ML, Fernandez A, Cardesa J, Ugarte M (1997) Relationship between mutation genotype and biochemical phenotype in a heterogeneous Spanish phenylketonuria population Eur. J Hum Genet 5:196–202

    CAS  Google Scholar 

  10. Zhu TW, Ye J, Han LS, Qiu WJ, Zhang HW, Liang LL, Gu XF (2013) Variations in genotype-phenotype correlations in phenylalanine hydroxylase deficiency in Chinese Han population. Gene 529:80–87

    Article  CAS  Google Scholar 

  11. Trunzo R, Santacroce R, D’Andrea G, Longo V, De Girolamo G, Dimatteo C, Leccese A, Lillo V, Papadia F, Margaglione M (2013) Mutation analysis in hyperphenylalaninemia patients from South Italy. Clin Biochem 46:1896–1898

    Article  CAS  Google Scholar 

  12. Song F, Qu YJ, Zhang T, Jin YW, Wang H, Zheng XY (2005) Phenylketonuria mutations in Northern China. Mol Genet Metab 86:S107–S118

    Article  CAS  Google Scholar 

  13. Blau N, Belanger-Quintana A, Demirkol M, Feillet F, Giovannini M, MacDonald A, Trefz FK, van Spronsen F, Centers EP (2010) Management of phenylketonuria in Europe: survey results from 19 countries. Mol Genet Metab 99:109–115

    Article  CAS  Google Scholar 

  14. Leandro J, Nascimento C, de Almeida IT, Leandro P (2006) Co-expression of different subunits of human phenylalanine hydroxylase: evidence of negative interallelic complementation. Biochim Biophys Acta 1762:544–550

    Article  CAS  Google Scholar 

  15. Leandro J, Leandro P, Flatmark T (2011) Heterotetrameric forms of human phenylalanine hydroxylase: Co-expression of wild-type and mutant forms in a bicistronic system. Biochim Biophys Acta 1812:602–612

    Article  CAS  Google Scholar 

  16. Thony B, Auerbach G, Blau N (2000) Tetrahydrobiopterin biosynthesis, regeneration and functions. Biochem J 347:1–16

    Article  CAS  Google Scholar 

  17. Pontoglio M, Barra J, Hadchouel M, Doyen A, Kress C, Bach JP, Babinet C, Yaniv M (1996) Hepatocyte nuclear factor 1 inactivation results in hepatic dysfunction, phenylketonuria, and renal Fanconi syndrome. Cell 84:575–585

    Article  CAS  Google Scholar 

  18. Thony B, Neuheiser F, Kierat L, Blaskovics M, Arn PH, Ferreira P, Rebrin I, Ayling J, Blau N (1998) Hyperphenylalaninemia with high levels of 7-biopterin is associated with mutations in the PCBD gene encoding the bifunctional protein pterin-4a-carbinolamine dehydratase and transcriptional coactivator (DCoH). Am J Hum Genet 62:1302–1311

    Article  CAS  Google Scholar 

  19. Citron BA, Davis MD, Milstien S, Gutierrez J, Mendel DB, Crabtree GR, Kaufman S (1992) Identity of 4a-carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, and Dcoh, a transregulator of homeodomain proteins. Proc Natl Acad Sci USA 89:11891–11894

    Article  CAS  Google Scholar 

  20. Erlandsen H, Stevens RC (1999) The structural basis of phenylketonuria. Mol Genet Metab 68:103–125

    Article  CAS  Google Scholar 

  21. Erlandsen H, Fusetti F, Martinez A, Hough E, Flatmark T, Stevens RC (1997) Crystal structure of the catalytic domain of human phenylalanine hydroxylase reveals the structural basis for phenylketonuria. Nat Struct Biol 4:995–1000

    Article  CAS  Google Scholar 

  22. Fusetti F, Erlandsen H, Flatmark T, Stevens RC (1998) Structure of tetrameric human phenylalanine hydroxylase and its implications for phenylketonuria. J Biol Chem 273:16962–16967

    Article  CAS  Google Scholar 

  23. Kobe B, Jennings IG, House CM, Michell BJ, Goodwill KE, Santarsiero BD, Stevens RC, Cotton RG, Kemp BE (1999) Structural basis of autoregulation of phenylalanine hydroxylase. Nat Struct Biol 6:442–448

    Article  CAS  Google Scholar 

  24. Pey AL, Stricher F, Serrano L, Martinez A (2007) Predicted effects of missense mutations on native-state stability account for phenotypic outcome in phenylketonuria, a paradigm of misfolding diseases. Am J Hum Genet 81:1006–1024

    Article  CAS  Google Scholar 

  25. Khan S, Vihinen M (2010) Performance of protein stability predictors. Hum Mutat 31:675–684

    Article  CAS  Google Scholar 

  26. Flück CE, Mullis PE, Pandey AV (2009) Modeling of human P450 oxidoreductase structure by in silico mutagenesis and MD simulation. Mol Cell Endocrinol 313:17–22

    Article  Google Scholar 

  27. Thusberg J, Vihinen M (2009) Pathogenic or not? And if so, then how? Studying the effects of missense mutations using bioinformatics method. Hum Mutat 30:703–714

  28. Thusberg J, Vihinen M (2006) Bioinformatic analysis of protein structure-function relationships: case study of leukocyte elastase (ELA2) missense mutations. Hum Mutat 27:1230–1243

    Article  CAS  Google Scholar 

  29. Cozzi R, Nuccitelli A, D’Onofrio M, Necchi F, Rosini R, Zerbini F, Biagini M, Norais N, Beier C, Telford JL, Grandi G, Assfalg M, Zacharias M, Maione D, Rinaudo CD (2012) New insights into the role of the glutamic acid of the E-box motif in group B Streptococcus pilus 2a assembly. Faseb J 26:2008–2018

    Article  CAS  Google Scholar 

  30. Skalova D, Zidkova J, Vohanka S, Mazanec R, Musova Z, Vondracek P, Mrazova L, Kraus J, Reblova K, Fajkusova L (2013) CLCN1 mutations in Czech patients with myotonia congenita. In silico analysis of novel and known mutations in the human dimeric skeletal muscle chloride channel. PLoS ONE 8:e82549

    Article  Google Scholar 

  31. Carluccio C, Fraternali F, Salvatore F, Fornili A, Zagari A (2013) Structural features of the regulatory ACT domain of phenylalanine hydroxylase PLoS ONE 8:

  32. Banerjee S, Wu Q, Yu P, Qi M, Li C (2014) In silico analysis of all point mutations on the 2B domain of K5/K14 causing epidermolysis bullosa simplex: a genotype-phenotype correlation. Mol Biosyst 10:2567–2577

    Article  CAS  Google Scholar 

  33. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR (2010) A method and server for predicting damaging missense mutations. Nat Methods 7:248–249

    Article  CAS  Google Scholar 

  34. Ng PC, Henikoff S (2001) Predicting deleterious amino acid substitutions. Genome Res 11:863–874

    Article  CAS  Google Scholar 

  35. Yue P, Melamud E, Moult J (2006) SNPs3D: candidate gene and SNP selection for association studies. BMC Bioinforma 7:166

    Article  Google Scholar 

  36. Reblova K, Hruba Z, Prochazkova D, Pazdirkova R, Pouchla S, Fajkusova L (2013) Hyperphenylalaninemia in the Czech Republic: Genotype-phenotype correlations and in silico analysis of novel missense mutations. Clin Chim Acta 419:1–10

    Article  CAS  Google Scholar 

  37. Sali A, Blundell TL (1993) Comparative protein modeling by satisfaction of spatial restraints. J Mol Biol 234:779–815

    Article  CAS  Google Scholar 

  38. Pearlman DA, Case DA, Caldwell JW, Ross WS, Cheatham TE, Debolt S, Ferguson D, Seibel G, Kollman P (1995) Amber, a package of computer-programs for applying molecular mechanics, normal-mode analysis, molecular-dynamics and free-energy calculations to simulate the structural and energetic properties of molecules. Comput Phys Commun 91:1–41

    Article  CAS  Google Scholar 

  39. Brocchieri L, Karlin S (1994) Geometry of interplanar residue contacts in protein structures. Proc Natl Acad Sci USA 91:9297–9301

    Article  CAS  Google Scholar 

  40. Humphrey W, Dalke A, Schulten K (1996) VMD—visual molecular dynamics. J Mol Graph Model 14:33–38

    Article  CAS  Google Scholar 

  41. Frishman D, Argos P (1995) Knowledge-based protein secondary structure assignment. Proteins 23:566–579

    Article  CAS  Google Scholar 

  42. Chothia C (1976) The nature of the accessible and buried surfaces in proteins. J Mol Biol 105:1–12

    Article  CAS  Google Scholar 

  43. Grantham R (1974) Amino acid difference formula to help explain protein evolution. Science 185:862–864

    Article  CAS  Google Scholar 

  44. Andersen OA, Stokka AJ, Flatmark T, Hough E (2003) 2.0 angstrom resolution crystal structures of the ternary complexes of human phenylalanine hydroxylase catalytic domain with tetrahydrobiopterin and 3-(2-thienyl)-l-alanine or l-norleucine: substrate specificity and molecular motions related to substrate binding. J Mol Biol 333:747–757

    Article  CAS  Google Scholar 

  45. Case DA, Darden TA, Cheatham I, T.E., Simmerling CL, Wang J, Duke RE, Luo R, Walker RC, Zhang W, Merz KM, Roberts B, Hayik S, Roitberg A, Seabra G, Swails J, Götz AW, Kolossváry I, Wong KF, Paesani F, Vanicek J, Wolf RM, Liu J, Wu X, Brozell SR, Steinbrecher T, Gohlke H, Cai Q, Ye X, Wang J, Hsieh MJ, Cui G, Roe DR, Mathews DH, Seetin MG, Salomon-Ferrer R, Sagui C, Babin V, Luchko T, Gusarov S, Kovalenko A, and Kollman PA, AMBER 12. 2012: University of California, San Francisco.

  46. Hornak V, Abel R, Okur A, Strockbine B, Roitberg A, Simmerling C (2006) Comparison of multiple amber force fields and development of improved protein backbone parameters. Proteins 65:712–725

    Article  CAS  Google Scholar 

  47. Dang LX, Kollman PA (1995) Free energy of association of the K+: 18-crown-6 complex in water: a new molecular dynamics study. J Phys Chem 99:55–58

    Article  CAS  Google Scholar 

  48. Gresh N, Sponer JE, Spackova N, Leszczynski J, Sponer J (2003) Theoretical study of binding of hydrated Zn(II) and Mg(II) cations to 5′-guanosine monophosphate. Toward polarizable molecular mechanics for DNA and RNA. J Phys Chem B 107:8669–8681

    Article  CAS  Google Scholar 

  49. Berendsen HJC, Postma JPM, Vangunsteren WF, Dinola A, Haak JR (1984) Molecular-dynamics with coupling to an external bath. J Chem Phys 81:3684–3690

    Article  CAS  Google Scholar 

  50. Fitzpatrick PF (2012) Allosteric regulation of phenylalanine hydroxylase. Arch Biochem Biophys 519:194–201

    Article  CAS  Google Scholar 

  51. McLaughlin RN, Poelwijk FJ, Raman A, Gosal WS, Ranganathan R (2012) The spatial architecture of protein function and adaptation. Nature 491:138–U163

    Article  CAS  Google Scholar 

  52. Molina-Vila MA, Nabau-Moreto N, Tornador C, Sabnis AJ, Rosell R, Estivill X, Bivona TG, Marino-Buslje C (2014) Activating mutations cluster in the “molecular brake” regions of protein kinases and do not associate with conserved or catalytic residues. Hum Mutat 35:318–28

    Article  CAS  Google Scholar 

  53. Bardelli T, Donati MA, Gasperini S, Ciani F, Belli F, Blau N, Morrone A, Zammarchi E (2002) Two novel genetic lesions and a common BH4-responsive mutation of the PAH gene in Italian patients with hyperphenylalaninemia. Mol Genet Metab 77:260–266

    Article  CAS  Google Scholar 

  54. Balmer C, Pandey AV, Rufenacht V, Nuoffer JM, Fang P, Wong LJ, Haberle J (2014) Mutations and polymorphisms in the human argininosuccinate lyase (ASL). Gene Hum Mutat 35:27–35

    Article  CAS  Google Scholar 

  55. Lesueur F, Bouadjar B, Lefevre C, Jobard F, Audebert S, Lakhdar H, Martin L, Tadini G, Karaduman A, Emre S, Saker S, Lathrop M, Fischer J (2007) Novel mutations in ALOX12B in patients with autosomal recessive congenital ichthyosis and evidence for genetic heterogeneity on chromosome 17p13. J Investig Dermatol 127:829–834

    Article  CAS  Google Scholar 

  56. Yang HQ, Liu L, Shin HD, Li JH, Du GC, Chen J (2013) Structure-guided systems-level engineering of oxidation-prone methionine residues in catalytic domain of an alkaline alpha-amylase from Alkalimonas amylolytica for significant improvement of both oxidative stability and catalytic efficiency. PLoS ONE 8:e57403

    Article  CAS  Google Scholar 

  57. Lindorff-Larsen K, Piana S, Dror RO, Shaw DE (2011) How fast-folding proteins fold. Science 334:517–520

    Article  CAS  Google Scholar 

  58. Eldar A, Rozenberg H, Diskin-Posner Y, Rohs R, Shakked Z (2013) Structural studies of p53 inactivation by DNA-contact mutations and its rescue by suppressor mutations via alternative protein-DNA interactions. Nucleic Acids Res 41:8748–8759

    Article  CAS  Google Scholar 

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Acknowledgments

This study was supported by the project CEITEC (CZ.1.05/1.1.00/02.0068) and the project SuPReMMe (CZ.1.07/2.3.00/20.0045). Access to the MetaCentrum computing facilities provided under the program “Projects of Large Infrastructure for Research, Development, and Innovations” LM2010005 funded by the Ministry of Education, Youth, and Sports of the Czech Republic is acknowledged. Access to the CERIT-SC computing and storage facilities provided under the programme Center CERIT Scientific Cloud, part of the Operational Program Research and Development for Innovations, reg. no. CZ. 1.05/3.2.00/08.0144 is acknowledged.

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  1. Central European Institute of Technology (CEITEC), Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic

    Kamila Réblová, Petr Kulhánek & Lenka Fajkusová

  2. National Center for Biomolecular Research, Faculty of Science, Masaryk University, Kotlářská 2, 611 37, Brno, Czech Republic

    Petr Kulhánek

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  1. Kamila Réblová
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  2. Petr Kulhánek
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Correspondence to Kamila Réblová.

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Réblová, K., Kulhánek, P. & Fajkusová, L. Computational study of missense mutations in phenylalanine hydroxylase. J Mol Model 21, 70 (2015). https://doi.org/10.1007/s00894-015-2620-6

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