PKU mutation (D143G) associated with an apparent high residual enzyme activity: Expression of a kinetic variant form of phenylalanine hydroxylase in three different systems

Knappskog, Per M.; Eiken, Hans Geir; Martı́nez, Aurora; Bruland, Ove; Apold, Jaran; Flatmark, Torgeir

doi:10.1002/(sici)1098-1004(1996)8:3<236::aid-humu7>3.0.co;2-7

Cited by 51 publications

(30 citation statements)

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“…The seven mutations were introduced into the wt-hPAH cDNA by PCR-based site-specific mutagenesis [10], using the pMAL-hPAH vector [4] as a template and the primers given in Table 1. The target sequence for mutagenesis was the XhoIϪBamHI fragment of the PAH cDNA, and for the G272X mutation the XhoIϪAflII fragment was used.…”

Section: Methodsmentioning

confidence: 95%

“…The target sequence for mutagenesis was the XhoIϪBamHI fragment of the PAH cDNA, and for the G272X mutation the XhoIϪAflII fragment was used. The authenticity of the mutagenesis was verified by DNA sequencing, and the PAH cDNA XhoIϪAflII mutant hPAH cDNA fragments were then subcloned into the pcDNA3-hPAH vector [4] which was used for expression in A293 cells and the TnT system.…”

Section: Methodsmentioning

confidence: 99%

“…Coupled in vitro transcription-translation of the wild-type and mutant forms hPAH was carried out by using the TnT-T7 reticulocyte lysate system and the pcDNA3-hPAH expression vector as recently described [4]. The amount of hPAH synthesized was measured as radioactivity by a β-scanner (Packard Instant Imager) following SDS/PAGE.…”

Section: Methodsmentioning

confidence: 99%

“…G46S [3], D143G [4] and Y204C [5]. In the present study we have extended these studies to include a panel of mutations that except for L255V are associated with severe forms of PKU (see PAH Mutation Analysis Consortium database : http ://www.…”

mentioning

confidence: 99%

“…The residues in the motif (252Ϫ272) are located near, or are part of, the active site [9], and the motif is important for folding and thereby the conformational stability of the enzyme. The selected seven missense point mutations (R252G/Q, L255V/ S, A259V/T and R270S) and a predicted termination mutation [4]. b Taken from [30].…”

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confidence: 99%

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Partial characterization and three‐dimensional‐structural localization of eight mutations in exon 7 of the human phenylalanine hydroxylase gene associated with phenylketonuria

Bjørgo

Knappskog

Martı́nez

et al. 1998

European Journal of Biochemistry

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The molecular basis for the metabolic defect in patients with phenylketonuria has been characterized for seven missense point mutations (R252G/Q, L255V/S, A259V/T and R270S) and a termination mutation (G272X) in an evolutionarily conserved motif of exon 7 in the catalytic domain of the human phenylalanine hydroxylase (hPAH) gene. The mutations were expressed in three heterologous in vitro systems. When expressed as fusion proteins with maltose-binding protein in Escherichia coli five of the mutant proteins demonstrated a defect in the normal ability of hPAH to fold and assemble as homotetramer/dimer, and they were mostly recovered as inactive aggregated forms. Only for the R252Q and L255V mutants were catalytically active tetramer and dimer recovered and for R252G some dimer, i.e. 20% (R252Q, tetramer), 44% (L255V, tetramer) and 4.4 % (R252G, dimer) of the activity for the respective wild-type (wt) forms. When expressed by a coupled in vitro transcription-translation system, all the mutant enzymes were recovered as a mixture of non-phosphorylated and phosphorylated forms with a low homospecific activity (i.e. maximum 11% of wt-hPAH for the L255V mutant). When transiently expressed in human embryonic kidney (A293) cells a very low level of immunoreactive PAH protein was recovered in spite of normal PAH mRNA levels. All these mutations resulted in variant hPAH proteins which revealed a defect in oligomerization, an increased sensitivity to limited proteolysis in vitro, reduced cellular stability and a variable reduction in their catalytic activity. All these effects seem to result from structural perturbations of the monomer, and based on the crystal structure of the catalytic domain of hPAH, an explanation is provided for the impact of the mutations on the folding and oligomerization of the monomers.Keywords : phenylalanine hydroxylase; mutation; phenylketonuria expression ; protein stability ; threedimensional structure.Hepatic phenylalanine hydroxylase (PAH, phenylalanine 4-monooxygenase catalyses the hydroxylation of the essential amino acid L-phenylalanine (L-Phe) to form L-tyrosine (L-Tyr) in the presence of tetrahydrobiopterin (H 4 biopterin) and dioxygen. PAH is a soluble cytosolic enzyme which is deficient in phenylketonuria (PKU), an autosomal recessive human disorder, and more than 280 different mutant alleles have been identified in the hPAH gene (for review, see [1] Enzyme. Phenylalanine 3-monooxygenase (EC 1.14.16.1). Note. The coordinates for the crystal structure of the catalytic domain of the dimeric truncated form (residues 103Ϫ452) of wild-type hPAH have the PDB accession code 1PAH. zygous for two different mutations. Our current understanding of the disorder is that the metabolic and clinical phenotypes are mainly determined by the PAH genotype (for review, see [2]).At this point the molecular mechanism of the reduced catalytic activity of mutant hPAH enzymes has been throughly characterized for only a few selected missense mutations, i.e. G46S [3], D143G [4] and Y204C [5]. In the present study...

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Section: Methodsmentioning

confidence: 95%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Partial characterization and three‐dimensional‐structural localization of eight mutations in exon 7 of the human phenylalanine hydroxylase gene associated with phenylketonuria

Bjørgo

Knappskog

Martı́nez

et al. 1998

European Journal of Biochemistry

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Phenylketonuria splice mutation (EXON6nt-96Ag) masquerading as missense mutation (Y204C)

1997

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In vitro expression analysis of mutations in phenylalanine hydroxylase: Linking genotype to phenotype and structure to function

et al. 1998

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Mutations in the human phenylalanine hydroxylase gene (PAH) altering the expressed cDNA nucleotide sequence (GenBank U49897) can impair activity of the corresponding enzyme product (hepatic phenylalanine hydroxylase, PAH) and cause hyperphenylalaninemia (HPA), a metabolic phenotype for which the major disease form is phenylketonuria (PKU; OMIM 261600). In vitro expression analysis of inherited human mutations in eukaryotic, prokaryotic, and cell‐free systems is informative about the mechanisms of mutation effects on enzymatic activity and their predicted effect on the metabolic phenotype. Corresponding analysis of site‐directed mutations in rat Pah cDNA has assigned critical functional roles to individual amino acid residues within the best understood species of phenylalanine hydroxylase. Data on in vitro expression of 35 inherited human mutations and 22 created rat mutations are reviewed here. The core data are accessible at the PAH Mutation Analysis Consortium Web site (http://www.mcgill.ca/pahdb). Hum Mutat 11:4–17, 1998. © 1998 Wiley‐Liss, Inc.

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PKU mutation (D143G) associated with an apparent high residual enzyme activity: Expression of a kinetic variant form of phenylalanine hydroxylase in three different systems

Cited by 51 publications

References 29 publications

Partial characterization and three‐dimensional‐structural localization of eight mutations in exon 7 of the human phenylalanine hydroxylase gene associated with phenylketonuria

Partial characterization and three‐dimensional‐structural localization of eight mutations in exon 7 of the human phenylalanine hydroxylase gene associated with phenylketonuria

Phenylketonuria splice mutation (EXON6nt-96Ag) masquerading as missense mutation (Y204C)

In vitro expression analysis of mutations in phenylalanine hydroxylase: Linking genotype to phenotype and structure to function

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