Loss of Function in Phenylketonuria Is Caused by Impaired Molecular Motions and Conformational Instability

Gersting, Søren W.; Kemter, Kristina; Staudigl, Michael; Messing, Dunja D.; Danecka, Marta K.; Lagler, Florian B.; Sommerhoff, Christian P.; Roscher, Adelbert A.; Muntau, Ania C.

doi:10.1016/j.ajhg.2008.05.013

Cited by 105 publications

(103 citation statements)

References 38 publications

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“…Of those disease-associated variants that have been studied in vitro (e.g., ref. 10), some confound the allosteric response, and some are interpreted as structurally unstable. We also suggest that the activities of some disease-associated variants may be dysregulated by an altered equilibrium among conformers having different intrinsic levels of activity, arguing by analogy to the enzyme porphobilinogen synthase (PBGS) and its porphyriaassociated variants (11).…”

mentioning

confidence: 99%

First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

Arturo

Gupta

Héroux

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

119

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Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73-82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype-phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phestabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype-phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate.phenylalanine hydroxylase | phenylketonuria | X-ray crystallography | small-angle X-ray scattering | allosteric regulation M ammalian phenylalanine hydroxylase (PAH) (EC 1.14.16.1) is a multidomain homo-multimeric protein whose dysfunction causes the most common inborn error in amino acid metabolism, phenylketonuria (PKU), and milder forms of hyperphenylalaninemia (OMIM 261600) (1). PAH catalyzes the hydroxylation of phenylalanine (Phe) to tyrosine, using nonheme iron and the cosubstrates tetrahydrobiopterin and molecular oxygen (2, 3). A detailed kinetic mechanism has recently been derived from elegant single-turnover studies (4). PAH activity must be carefully regulated, because although Phe is an essential amino acid, high Phe levels are neurotoxic. Thus, Phe allosterically activates PAH by binding to a regulatory domain. Phosphorylation at Ser16 potentiates the effects of Phe, with phosphorylated PAH achieving full activation at lower Phe concentrations than the unphosphorylated protein (5, 6). Allosteric activation by Phe is accompanied by a major conformational change, as evidenced by changes in protein fluorescence and proteolytic susceptibility, and by stabilization of a tetrameric confo...

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mentioning

confidence: 99%

First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

Arturo

Gupta

Héroux

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

119

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“…Mutations in the human PAH-cDNA sequence were introduced by site-directed mutagenesis, using QuikChange XL kit from Agilent Technologies (Santa Clara, CA, USA) and confirmed by sequencing analysis using BigDye Terminator Cycle sequencing v1.1 (Applied Biosystems) on an ABI Prism 3100 Sequencer. 6 …”

Section: Expression Plasmid Preparationmentioning

confidence: 99%

“…The fully functional homotetrameric PAH catalyzes hydroxylation of Phe to tyrosine (Tyr) in the presence of cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH 4 ) and molecular oxygen [3,4]. According to the Locus Knowledgebase (PAHdb, www.pahdb.mcgill.ca), about 60% of mutations in the PAH gene are missense mutations, which may lead to a misfolding of the protein [5,6], disturbing the complex enzyme regulation and changes in kinetics, due to altered affinities for the Phe substrate and the BH 4 cofactor.…”

Section: Introductionmentioning

confidence: 99%

Quantification of phenylalanine hydroxylase activity by isotope-dilution liquid chromatography–electrospray ionization tandem mass spectrometry

Heintz

Troxler

Martı́nez

et al. 2012

Molecular Genetics and Metabolism

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BACKGROUND: Residual phenylalanine hydroxylase (PAH) activity is the key determinant for the phenotype severity in phenylketonuria (PKU) patients and correlates with the patient's genotype. Activity of in vitro expressed mutant PAH may predict the patient's phenotype and response to tetrahydrobiopterin (BH(4)), the cofactor of PAH. METHODS: A robust LC-ESI-MSMS PAH assay for the quantification of phenylalanine and tyrosine was developed. We measured PAH activity a) of the PAH mutations p.Y417C, p.I65T, p.R261Q, p.E280A, p.R158Q, p.R408W, and p.E390G expressed in eukaryotic COS-1 cells; b) in different cell lines (e.g. Huh-7, Hep3B); and c) in liver, brain, and kidney tissue from wild-type and PKU mice. RESULTS: The PAH assay was linear for phenylalanine and tyrosine (r(2) 0.99), with a detection limit of 105 nmol/L for Phe and 398 nmol/L for Tyr. Intra-assay and inter-assay coefficients of variation were <5.3% and <6.2%, respectively, for the p.R158Q variant in lower tyrosine range. Recovery of tyrosine was 100%. Compared to the wild-type enzyme, the highest PAH activity at standard conditions (1 mmol/L L-Phe; 200 mol/L BH(4)) was found for the mutant p.Y417C (76%), followed by p.E390G (54%), p.R261Q (43%), p.I65T (33%), p.E280A (15%), p.R158Q (5%), and p.R408W (2%). A relative high PAH activity was found in kidney (33% of the liver activity), but none in brain. CONCLUSIONS: This novel method is highly sensitive, specific, reproducible, and efficient, allowing the quantification of PAH activity in different cells or tissue extracts using minimum amounts of samples under standardized conditions

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“…The key to comprehend genotype-phenotype relationships and its inconsistencies lies within understanding PKU disease mechanisms: gene variants within the catalytic domain may directly abolish PAH enzyme function, 7 while splice-site variants can result in a non-functional truncated protein. 8 Missense variants have been reported to primarily result in misfolding of the protein [9][10][11] which impairs protein stability 9,11 and oligomer assembly. 9 Misfolded proteins were observed to aggregate in prokaryote hosts 4 and to be degraded in cellular models 12 leading to diminished PAH activity.…”

Section: Introductionmentioning

confidence: 99%

Linking genotypes database with locus-specific database and genotype–phenotype correlation in phenylketonuria

et al. 2014

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The wide range of metabolic phenotypes in phenylketonuria is due to a large number of variants causing variable impairment in phenylalanine hydroxylase function. A total of 834 phenylalanine hydroxylase gene variants from the locus-specific database PAHvdb and genotypes of 4181 phenylketonuria patients from the BIOPKU database were characterized using FoldX, SIFT Blink, Polyphen-2 and SNPs3D algorithms. Obtained data was correlated with residual enzyme activity, patients' phenotype and tetrahydrobiopterin responsiveness. A descriptive analysis of both databases was compiled and an interactive viewer in PAHvdb database was implemented for structure visualization of missense variants. We found a quantitative relationship between phenylalanine hydroxylase protein stability and enzyme activity (r s ¼ 0.479), between protein stability and allelic phenotype (r s ¼ À0.458), as well as between enzyme activity and allelic phenotype (r s ¼ 0.799). Enzyme stability algorithms (FoldX and SNPs3D), allelic phenotype and enzyme activity were most powerful to predict patients' phenotype and tetrahydrobiopterin response. Phenotype prediction was most accurate in deleterious genotypes (E100%), followed by homozygous (92.9%), hemizygous (94.8%), and compound heterozygous genotypes (77.9%), while tetrahydrobiopterin response was correctly predicted in 71.0% of all cases. To our knowledge this is the largest study using algorithms for the prediction of patients' phenotype and tetrahydrobiopterin responsiveness in phenylketonuria patients, using data from the locus-specific and genotypes database.

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Loss of Function in Phenylketonuria Is Caused by Impaired Molecular Motions and Conformational Instability

Cited by 105 publications

References 38 publications

First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

Quantification of phenylalanine hydroxylase activity by isotope-dilution liquid chromatography–electrospray ionization tandem mass spectrometry

Linking genotypes database with locus-specific database and genotype–phenotype correlation in phenylketonuria

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