The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Paul F. Fitzpatrick - One of the best experts on this subject based on the ideXlab platform.
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the phenylketonuria associated substitution r68s converts Phenylalanine Hydroxylase to a constitutively active enzyme but reduces its stability
Journal of Biological Chemistry, 2019Co-Authors: Crystal A. Khan, Steve P Meisburger, Nozomi Ando, Paul F. FitzpatrickAbstract:The naturally occurring R68S substitution of Phenylalanine Hydroxylase (PheH) causes phenylketonuria (PKU). However, the molecular basis for how the R68S variant leads to PKU remains unclear. Kinetic characterization of R68S PheH establishes that the enzyme is fully active in the absence of allosteric binding of Phenylalanine, in contrast to the WT enzyme. Analytical ultracentrifugation establishes that the isolated regulatory domain of R68S PheH is predominantly monomeric in the absence of Phenylalanine and dimerizes in its presence, similar to the regulatory domain of the WT enzyme. Fluorescence and small-angle X-ray scattering analyses establish that the overall conformation of the resting form of R68S PheH is different from that of the WT enzyme. The data are consistent with the substitution disrupting the interface between the catalytic and regulatory domains of the enzyme, shifting the equilibrium between the resting and activated forms ∼200-fold, so that the resting form of R68S PheH is ∼70% in the activated conformation. However, R68S PheH loses activity 2 orders of magnitude more rapidly than the WT enzyme at 37 °C and is significantly more sensitive to proteolysis. We propose that, even though this substitution converts the enzyme to a constitutively active enzyme, it results in PKU because of the decrease in protein stability.
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phosphorylation of Phenylalanine Hydroxylase increases the rate constant for formation of the activated conformation of the enzyme
Biochemistry, 2018Co-Authors: Crystal A. Khan, Paul F. FitzpatrickAbstract:Liver Phenylalanine Hydroxylase (PheH) is an allosteric enzyme that is activated by Phenylalanine. The enzyme is also phosphorylated by protein kinase A, but the effects of phosphorylation are unclear. Recent structural studies ( Meisburger et al. ( 2016 ) J. Amer. Chem. Soc. 138 , 6506 - 6516 ) support a model in which activation of the enzyme involves dimerization of the regulatory domains, creating the allosteric site for Phenylalanine at the dimer interface. This conformational change also results in a change in the fluorescence of the protein that can be used to monitor activation. The kinetics of activation of PheH are biphasic over a range of Phenylalanine concentrations. These data are well-described by a model involving an initial equilibrium between the resting form and the activated conformation, with a value of the equilibrium constant for formation of the activated conformation, L, equal to 0.007, followed by binding of two molecules of Phenylalanine. Phosphorylation increases L 10-fold by increasing the rate constant for conversion of the resting form to the activated form. The results provide functional support for the previous structural model, identify the specific effect of phosphorylation on the enzyme, and rationalize the lack of change in the protein structure upon phosphorylation.
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domain movements upon activation of Phenylalanine Hydroxylase characterized by crystallography and chromatography coupled small angle x ray scattering
Journal of the American Chemical Society, 2016Co-Authors: Steve P Meisburger, Paul F. Fitzpatrick, Crystal A. Khan, Shengnan Zhang, Alexander B Taylor, Nozomi AndoAbstract:Mammalian Phenylalanine Hydroxylase (PheH) is an allosteric enzyme that catalyzes the first step in the catabolism of the amino acid Phenylalanine. Following allosteric activation by high Phenylalanine levels, the enzyme catalyzes the pterin-dependent conversion of Phenylalanine to tyrosine. Inability to control elevated Phenylalanine levels in the blood leads to increased risk of mental disabilities commonly associated with the inherited metabolic disorder, phenylketonuria. Although extensively studied, structural changes associated with allosteric activation in mammalian PheH have been elusive. Here, we examine the complex allosteric mechanisms of rat PheH using X-ray crystallography, isothermal titration calorimetry (ITC), and small-angle X-ray scattering (SAXS). We describe crystal structures of the preactivated state of the PheH tetramer depicting the regulatory domains docked against the catalytic domains and preventing substrate binding. Using SAXS, we further describe the domain movements involved...
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Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase
Journal of Biological Chemistry, 2016Co-Authors: Shengnan Zhang, Paul F. FitzpatrickAbstract:Abstract Liver Phenylalanine Hydroxylase (PheH) is an allosteric enzyme that requires activation by Phenylalanine for full activity. The location of the allosteric site for Phenylalanine has not been established. NMR spectroscopy of the isolated regulatory domain (RDPheH(25–117) is the regulatory domain of PheH lacking residues 1–24) of the rat enzyme in the presence of Phenylalanine is consistent with formation of a side-by-side ACT dimer. Six residues in RDPheH(25–117) were identified as being in the Phenylalanine-binding site on the basis of intermolecular NOEs between unlabeled Phenylalanine and isotopically labeled protein. The location of these residues is consistent with two allosteric sites per dimer, with each site containing residues from both monomers. Site-specific variants of five of the residues (E44Q, A47G, L48V, L62V, and H64N) decreased the affinity of RDPheH(25–117) for Phenylalanine based on the ability to stabilize the dimer. Incorporation of the A47G, L48V, and H64N mutations into the intact protein increased the concentration of Phenylalanine required for activation. The results identify the location of the allosteric site as the interface of the regulatory domain dimer formed in activated PheH.
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the amino acid specificity for activation of Phenylalanine Hydroxylase matches the specificity for stabilization of regulatory domain dimers
Biochemistry, 2015Co-Authors: Shengnan Zhang, Andrew P Hinck, Paul F. FitzpatrickAbstract:Liver Phenylalanine Hydroxylase is allosterically activated by Phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of Phenylalanine on the isolated regulatory domain of the enzyme support a model in which Phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and Phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10-50 mM, d-Phenylalanine, l-methionine, l-norleucine, and (S)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-Phenylalanine. Lower levels of activation were seen with l-4-aminoPhenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of Phenylalanine Hydroxylase is linked to dimerization of regulatory domains.
Aurora Martinez - One of the best experts on this subject based on the ideXlab platform.
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structure of full length human Phenylalanine Hydroxylase in complex with tetrahydrobiopterin
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Marte Innselset Flydal, Aurora Martinez, Martin Alcorlopages, Fredrik Gullaksen Johannessen, Siseth Martinezcaballero, Lars Skjaerven, Rafael Fernandezleiro, Juan A HermosoAbstract:Phenylalanine Hydroxylase (PAH) is a key enzyme in the catabolism of Phenylalanine, and mutations in this enzyme cause phenylketonuria (PKU), a genetic disorder that leads to brain damage and mental retardation if untreated. Some patients benefit from supplementation with a synthetic formulation of the cofactor tetrahydrobiopterin (BH4) that partly acts as a pharmacological chaperone. Here we present structures of full-length human PAH (hPAH) both unbound and complexed with BH4 in the precatalytic state. Crystal structures, solved at 3.18-A resolution, show the interactions between the cofactor and PAH, explaining the negative regulation exerted by BH4. BH4 forms several H-bonds with the N-terminal autoregulatory tail but is far from the catalytic FeII. Upon BH4 binding a polar and salt-bridge interaction network links the three PAH domains, explaining the stability conferred by BH4. Importantly, BH4 binding modulates the interaction between subunits, providing information about PAH allostery. Moreover, we also show that the cryo-EM structure of hPAH in absence of BH4 reveals a highly dynamic conformation for the tetramers. Structural analyses of the hPAH:BH4 subunits revealed that the substrate-induced movement of Tyr138 into the active site could be coupled to the displacement of BH4 from the precatalytic toward the active conformation, a molecular mechanism that was supported by site-directed mutagenesis and targeted molecular dynamics simulations. Finally, comparison of the rat and human PAH structures show that hPAH is more dynamic, which is related to amino acid substitutions that enhance the flexibility of hPAH and may increase the susceptibility to PKU-associated mutations.
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the structure of full length human Phenylalanine Hydroxylase in complex with tetrahydrobiopterin
bioRxiv, 2019Co-Authors: Marte Innselset Flydal, Aurora Martinez, Martin Alcorlopages, Fredrik Gullaksen Johannessen, Siseth Martinezcaballero, Lars Skjaerven, Rafael Fernandezleiro, Juan A HermosoAbstract:Abstract Phenylalanine Hydroxylase (PAH) is a key enzyme in the catabolism of Phenylalanine, and mutations in this enzyme cause phenylketonuria (PKU), a genetic disorder that leads to brain damage and mental retardation if untreated. Some patients benefit from supplementation with a synthetic formulation of the cofactor tetrahydrobiopterin (BH4) that partly acts as a pharmacological chaperone. Here we present the first structures of full-length human PAH (hPAH) both unbound and complexed with BH4 in the pre-catalytic state. Crystal structures, solved at 3.18 A resolution, show the interactions between the cofactor and PAH, explaining the negative regulation exerted by BH4. BH4 forms several H-bonds with the N-terminal autoregulatory tail but is far from the catalytic FeII. Upon BH4 binding a polar and salt-bridge interaction network links the three PAH domains, explaining the stability conferred by BH4. Importantly, BH4 binding modulates the interaction between subunits, providing information about PAH allostery. Moreover, we also show that the cryo-EM structure of hPAH in absence of BH4 reveals a highly dynamic conformation for the tetramers. Structural analyses of the hPAH:BH4 subunits revealed that the substrate-induced movement of Tyr138 into the active site could be coupled to the displacement of BH4 from the pre-catalytic towards the active conformation, a molecular mechanism that was supported by site-directed mutagenesis and targeted MD simulations. Finally, comparison of the rat and human PAH structures show that hPAH is more dynamic, which is related to amino acid substitutions that enhance the flexibility of hPAH and may increase the susceptibility to PKU-associated mutations. Significance Statement The present crystal structure of Phenylalanine Hydroxylase (PAH) provides the first and long-awaited 3D-structure of the full-length human PAH, both unbound and complexed with the tetrahydrobiopterin (BH4) cofactor. The BH4-bound state is physiologically relevant, keeping PAH stable and in a pre-catalytic state at low L-Phe concentration. Furthermore, a synthetic form of BH4 (Kuvan®) is the only drug-based therapy for a subset of phenylketonuria patients. We found two tetramer conformations in the same crystal, depending on the active site occupation by BH4, which aid to understand the stabilization by BH4 and the allosteric mechanisms in PAH. The structure also reveals the increased mobility of human-compared with rat PAH, in line with an increased predisposition to disease-associated mutations in human.
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Phenylalanine Hydroxylase function structure and regulation
Iubmb Life, 2013Co-Authors: Marte Innselset Flydal, Aurora MartinezAbstract:Mammalian Phenylalanine Hydroxylase (PAH) catalyzes the rate-limiting step in the Phenylalanine catabolism, consuming about 75% of the Phenylalanine input from the diet and protein catabolism under physiological conditions. In humans, mutations in the PAH gene lead to phenylketonuria (PKU), and most mutations are mainly associated with PAH misfolding and instability. The established treatment for PKU is a Phenylalanine-restricted diet and, recently, supplementation with preparations of the natural tetrahydrobiopterin cofactor also shows effectiveness for some patients. Since 1997 there has been a significant increase in the understanding of the structure, catalytic mechanism, and regulation of PAH by its substrate and cofactor, in addition to improved correlations between genotype and phenotype in PKU. Importantly, there has also been an increased number of studies on the structure and function of PAH from bacteria and lower eukaryote organisms, revealing an additional anabolic role of the enzyme in the synthesis of melanin-like pigments. In this review, we discuss these recent studies, which contribute to define the evolutionary adaptation of the PAH structure and function leading to sophisticated regulation for effective catabolic processing of Phenylalanine in mammalian organisms.
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Phenylalanine Hydroxylase Misfolding and Pharmacological Chaperones
Current topics in medicinal chemistry, 2013Co-Authors: Jarl Underhaug, Oscar Aubi, Aurora MartinezAbstract:Phenylketonuria (PKU) is a loss-of-function inborn error of metabolism. As many other inherited diseases the main pathologic mechanism in PKU is an enhanced tendency of the mutant Phenylalanine Hydroxylase (PAH) to misfold and undergo ubiquitin-dependent degradation. Recent alternative approaches with therapeutic potential for PKU aim at correcting the PAH misfolding, and in this respect pharmacological chaperones are the focus of increasing interest. These compounds, which often resemble the natural ligands and show mild competitive inhibition, can rescue the misfolded proteins by stimulating their renaturation in vivo. For PKU, a few studies have proven the stabilization of PKU-mutants in vitro, in cells, and in mice by pharmacological chaperones, which have been found either by using the tetrahydrobiopterin (BH4) cofactor as query structure for shape-focused virtual screening or by high-throughput screening of small compound libraries. Both approaches have revealed a number of compounds, most of which bind at the iron-binding site, competitively with respect to BH4. Furthermore, PAH shares a number of ligands, such as BH4, amino acid substrates and inhibitors, with the other aromatic amino acid Hydroxylases: the neuronal/neuroendocrine enzymes tyrosine Hydroxylase (TH) and the tryptophan Hydroxylases (TPHs). Recent results indicate that the PAH-targeted pharmacological chaperones should also be tested on TH and the TPHs, and eventually be derivatized to avoid unwanted interactions with these other enzymes. After derivatization and validation in animal models, the PAH-chaperoning compounds represent novel possibilities in the treatment of PKU.
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quantification of Phenylalanine Hydroxylase activity by isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry
Molecular Genetics and Metabolism, 2012Co-Authors: Caroline Heintz, Aurora Martinez, Beat Thony, Heinz Troxler, Nenad BlauAbstract:Abstract 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 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.
Raymond C Stevens - One of the best experts on this subject based on the ideXlab platform.
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molecular genetics of tetrahydrobiopterin responsive Phenylalanine Hydroxylase deficiency
Human Mutation, 2008Co-Authors: Marcel R Zurfluh, Johannes Zschocke, François Feillet, Raymond C Stevens, Alberto Burlina, Martin Lindner, Celine Chery, Beat Thony, Nenad BlauAbstract:Mutations in the Phenylalanine Hydroxylase (PAH) gene result in phenylketonuria (PKU). Tetrahydrobiopterin (BH(4))-responsive hyperPhenylalaninemia has been recently described as a variant of PAH deficiency caused by specific mutations in the PAH gene. It has been suggested that BH(4)-responsiveness may be predicted from the corresponding genotypes. Data from BH(4) loading tests indicated an incidence of BH(4)-responsiveness of >40% in the general PKU population and >80% in mild PKU patients. The current project entailed genotype analysis of 315 BH(4)-responsive patients tabulated in the BIOPKUdb database and comparison with the data from the PAHdb locus-specific knowledgebase, as well as with previously published PAH mutations for several European countries, Northern China, and South Korea. We identified 57 mutations, presenting with a substantial residual PAH activity (average approximately 47%), presumed to be associated with BH(4)-responsiveness. More than 89% of patients are found to be compound heterozygotes. The three most common mutations found in >5% of BH(4)-responsive patients are p.A403 V, p.R261Q, and p.Y414C. Using the Hardy-Weinberg formula the predicted average frequency of BH(4)-responsiveness in European populations was calculated to be 55% (range 17-79%, lowest in Baltic countries and Poland and highest in Spain), 57% in Northern China, and 55% for South Korea. The genotype-predicted prevalence of BH(4)-responsiveness was higher than prevalence data obtained from BH(4) loading tests. Inconsistent results were observed for mutations p.L48S, p.I65 T, p.R158Q, p.R261Q, and p.Y414C. Our data suggest that BH(4)-responsiveness may be more common than assumed and to some extent may be predicted or excluded from the patient's genotype.
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Biopterin responsive Phenylalanine Hydroxylase deficiency
Genetics in Medicine, 2004Co-Authors: Reuben Matalon, Kathryn Moseley, Heidi Erlandsen, Raymond C Stevens, Richard Koch, Kimberlee Michals-matalon, Sankar Surendran, Stephen Tyring, Alejandra Gamez, Anne RomstadAbstract:Purpose: Phenylketonuria (PKU) is an autosomal recessive disorder caused by mutations in the Phenylalanine Hydroxylase (PAH) gene. There have been more than 400 mutations identified in the PAH gene leading to variable degrees of deficiency in PAH activity, and consequently a wide spectrum of clinical severity. A pilot study was undertaken to examine the response to 6-R-l-erythro-5,6,7,8-tetrahydrobiopterin (BH_4) in patients with atypical and classical PKU. Methods: PAH gene mutation analysis was performed using denaturing gradient gel electrophoresis and gene sequencing. Patients with classical, atypical, or mild PKU were orally given BH4 10 mg/kg. Blood Phenylalanine and tyrosine levels were determined using tandem MS/MS at 0 hours, 4 hours, 8 hours, and 24 hours intervals. Results: Thirty-six patients were given a single oral dose of 10 mg/kg of BH_4. Twenty one patients (58.33%) responded with a decrease in blood Phenylalanine level. Of the patients that responded, 12 were classical, 7 atypical, and 2 mild. The mean decline in blood Phenylalanine at 24 hours was > 30% of baseline. There were 15 patients who did not respond to the BH_4 challenge, 14 of those had classical and one had atypical PKU. Mapping the mutations that responded to BH_4 on the PAH enzyme showed that mutations were in the catalytic, regulatory, oligomerization, and BH_4 binding domains. Five patients responding to BH_4 had mutations not previously identified. Conclusion: The data presented suggest higher than anticipated number of PKU mutations respond to BH_4, and such mutations are on all the domains of PAH.
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toward pku enzyme replacement therapy pegylation with activity retention for three forms of recombinant Phenylalanine Hydroxylase
Molecular Therapy, 2004Co-Authors: Alejandra Gamez, Marianne G Patch, Mary Straub, Lin Wang, Raymond C StevensAbstract:Abstract Phenylketonuria (PKU) is a disease in which Phenylalanine and Phenylalanine-derived metabolites build up to neurotoxic levels due to mutations in the Phenylalanine Hydroxylase gene ( PAH ). Enzyme replacement therapy is a viable option to supply active PAH. However, the inherent protease sensitivity and potential immunogenicity of PAH have precluded adoption of this approach. In this report, we have used polyethylene glycol derivatization (PEGylation) to produce protected forms of PAH for potential therapeutic use. Three recombinantly produced PAH enzymes were reacted with activated PEG species, with the aim of developing a stable and active PKU enzyme replacement. Tetrameric full-length human PAH, dimeric double-truncated (ΔN102–ΔC428) human PAH, and monomeric Chromobacterium violaceum PAH were PEGylated with succinimidyl succinate polyethylene glycol of molecular weight 5000 or 20,000 Da. Characterization of the PEGylated species was accomplished with MALDI-TOF mass spectrometry, SDS–PAGE, and specific activity measurements using ESI mass spectrometry. All PEG-derivatized PAH species retained catalytic activity, and, at low numbers of PEG molecules attached, these PEGylated PAH proteins were found to be more active and more stable than their nonderivatized PAH counterparts.
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structural studies on Phenylalanine Hydroxylase and implications toward understanding and treating phenylketonuria
Pediatrics, 2003Co-Authors: Heidi Erlandsen, Alejandra Gamez, Marianne G Patch, Mary Straub, Raymond C StevensAbstract:Mutations in the gene encoding for Phenylalanine Hydroxylase (PAH) result in phenylketonuria (PKU) or hyperPhenylalaninemia (HPA). Several 3-dimensional structures of truncated forms of PAH have been determined in our laboratory and by others, using x-ray crystallographic techniques. These structures have allowed for a detailed mapping of the >250 missense mutations known to cause PKU or HPA found throughout the 3 domains of PAH. This structural information has helped formulate rules that might aid in predicting the likely effects of unclassified or newly discovered PAH mutations. Also, with the aid of recent crystal structure determinations of co-factor and substrate analogs bound at the PAH active site, the recently discovered tetrahydrobiopterin-responsive PKU/HPA genotypes can be mapped onto the PAH structure, providing a molecular basis for this tetrahydrobiopterin response.
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Structural basis of autoregulation of Phenylalanine Hydroxylase
Nature structural biology, 1999Co-Authors: Bostjan Kobe, Richard G. H. Cotton, Ian G Jennings, Raymond C Stevens, Colin M. House, Belinda J. Michell, Kenneth E. Goodwill, Bernard D. Santarsiero, Bruce E. KempAbstract:Phenylalanine Hydroxylase converts Phenylalanine to tyrosine, a rate-limiting step in Phenylalanine catabolism and protein and neurotransmitter biosynthesis. It is tightly regulated by the substrates Phenylalanine and tetrahydrobiopterin and by phosphorylation. We present the crystal structures of dephosphorylated and phosphorylated forms of a dimeric enzyme with catalytic and regulatory properties of the wild-type protein. The structures reveal a catalytic domain flexibly linked to a regulatory domain. The latter consists of an N-terminal autoregulatory sequence (containing Ser 16, which is the site of phosphorylation) that extends over the active site pocket, and an alpha-beta sandwich core that is, unexpectedly, structurally related to both pterin dehydratase and the regulatory domains of metabolic enzymes. Phosphorylation has no major structural effects in the absence of Phenylalanine, suggesting that Phenylalanine and phosphorylation act in concert to activate the enzyme through a combination of intrasteric and possibly allosteric mechanisms.
Charles R Scriver - One of the best experts on this subject based on the ideXlab platform.
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Phenylalanine Hydroxylase deficiency
Genetics in Medicine, 2011Co-Authors: John J Mitchell, Yannis J Trakadis, Charles R ScriverAbstract:Phenylalanine Hydroxylase deficiency is an autosomal recessive disorder that results in intolerance to the dietary intake of the essential amino acid Phenylalanine. It occurs in approximately 1:15,000 individuals. Deficiency of this enzyme produces a spectrum of disorders including classic phenylketonuria, mild phenylketonuria, and mild hyperPhenylalaninemia. Classic phenylketonuria is caused by a complete or near-complete deficiency of Phenylalanine Hydroxylase activity and without dietary restriction of Phenylalanine most children will develop profound and irreversible intellectual disability. Mild phenylketonuria and mild hyperPhenylalaninemia are associated with lower risk of impaired cognitive development in the absence of treatment. Phenylalanine Hydroxylase deficiency can be diagnosed by newborn screening based on detection of the presence of hyperPhenylalaninemia using the Guthrie microbial inhibition assay or other assays on a blood spot obtained from a heel prick. Since the introduction of newborn screening, the major neurologic consequences of hyperPhenylalaninemia have been largely eradicated. Affected individuals can lead normal lives. However, recent data suggest that homeostasis is not fully restored with current therapy. Treated individuals have a higher incidence of neuropsychological problems. The mainstay of treatment for hyperPhenylalaninemia involves a low-protein diet and use of a Phenylalanine-free medical formula. This treatment must commence as soon as possible after birth and should continue for life. Regular monitoring of plasma Phenylalanine and tyrosine concentrations is necessary. Targets of plasma Phenylalanine of 120–360 μmol/L (2–6 mg/dL) in the first decade of life are essential for optimal outcome. Phenylalanine targets in adolescence and adulthood are less clear. A significant proportion of patients with phenylketonuria may benefit from adjuvant therapy with 6R-tetrahydrobiopterin stereoisomer. Special consideration must be given to adult women with hyperPhenylalaninemia because of the teratogenic effects of Phenylalanine. Women with Phenylalanine Hydroxylase deficiency considering pregnancy should follow special guidelines and assure adequate energy intake with the proper proportion of protein, fat, and carbohydrates to minimize risks to the developing fetus. Molecular genetic testing of the Phenylalanine Hydroxylase gene is available for genetic counseling purposes to determine carrier status of at-risk relatives and for prenatal testing.
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in vitro expression analysis of mutations in Phenylalanine Hydroxylase linking genotype to phenotype and structure to function
Human Mutation, 1998Co-Authors: Paula J Waters, Michael A Parniak, Piotr Nowacki, Charles R ScriverAbstract: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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human Phenylalanine Hydroxylase mutations and hyperPhenylalaninemia phenotypes a metanalysis of genotype phenotype correlations
American Journal of Human Genetics, 1997Co-Authors: Emre Kayaalp, Paula J Waters, Piotr Nowacki, Eileen P Treacy, Susan Byck, Charles R ScriverAbstract:Summary We analyzed correlations between mutant genotypes at the human Phenylalanine Hydroxylase locus (gene symbol PAH ) and the corresponding hyperPhenylalaninemia (HPA) phenotypes (notably, phenylketonuria [OMIM 261600]). We used reports, both published and in the PAH Mutation Analysis Consortium Database, on 365 patients harboring 73 different PAH mutations in 161 different genotypes. HPA phenotypes were classified as phenylketonuria (PKU), variant PKU, and non-PKU HPA. By analysis both of homoallelic mutant genotypes and of "functionally hemizygous" heteroallelic genotypes, we characterized the phenotypic effect of 48 of the 73 different, largely missense mutations. Among those with consistent in vivo expression, 24 caused PKU, 3 caused variant PKU, and 10 caused non-PKU HPA. However, 11 mutations were inconsistent in their effect: 9 appeared in two different phenotype classes, and 2 (I65T and Y414C) appeared in all three classes. Seven mutations were inconsistent in phenotypic effect when in vitro (unit-protein) expression was compared with the corresponding in vivo phenotype (an emergent property). We conclude that the majority of PAH mutations confer a consistent phenotype and that this is concordant with their effects, when known, predicted from in vitro expression analysis. However, significant inconsistencies, both between in vitro and in vivo phenotypes and between different individuals with similar PAH genotypes, reveal that the HPA-phenotype is more complex than that predicted by Mendelian inheritance of alleles at the PAH locus.
Savio L. C. Woo - One of the best experts on this subject based on the ideXlab platform.
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complete and persistent phenotypic correction of phenylketonuria in mice by site specific genome integration of murine Phenylalanine Hydroxylase cdna
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Li Chen, Savio L. C. WooAbstract:We explored the potential of using a bacteriophage integrase system to achieve site-specific genome integration of murine Phenylalanine Hydroxylase cDNA in the livers of phenylketonuric (PKU) mice. The phiBT1 phage integrase is an enzyme that catalyses the efficient recombination between unique sequences in the phage and bacterial genomes, leading to the site-specific integration of the former into the latter in a unidirectional manner. Here we showed that this phage integrase functions efficiently in mouse cells, and several naturally occurring pseudo-attP sites located in the intergenic regions of the mouse genome have been identified and molecularly characterized. We further demonstrated that the addition of nuclear localization signal sequences to the C terminus of the phage integrase enhanced the efficiency for transgene integration into the mouse genome. Using this phage integration system, we delivered mouse Phenylalanine Hydroxylase cDNA to the livers of PKU mice by hydrodynamic injection of plasmid DNA and showed that the severity of the hyperPhenylalaninemic phenotype in the treated mice decreased significantly. After three applications, serum Phenylalanine levels in all treated PKU mice were reduced to the normal range and remained stable thereafter. Their fur color also changed from gray to black, indicating the reconstitution of melanin biosynthesis as a result of available tyrosine derived from reconstituted Phenylalanine hydroxylation in the liver. Thus, the phiBT1 bacteriophage integrase represents an effective site-specific genome integration system in mammalian cells and can be of great value in DNA-mediated gene therapy for a multitude of genetic disorders.
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associations between mutations and a vntr in the human Phenylalanine Hydroxylase gene
American Journal of Human Genetics, 1992Co-Authors: Alexei A Goltsov, Randy C Eisensmith, D S Konecki, Uta Lichterkonecki, Savio L. C. WooAbstract:The HindIII RFLP in the human Phenylalanine Hydroxylase (PAH) gene is caused by the presence of an AT-rich (70%) minisatellite region. This region contains various multiple of 30-bp tandem repeats and is located 3 kb downstream of the final exon of the gene. PCR-mediated amplification of this region from haplotyped PAH chromosomes indicates that the previously reported 4.0-kb HindIII allele contains three of these repeats, while the 4.4-kb HindIII allele contains 12 of these repeats. The 4.2-kb HindIII fragment can contain six, seven, eight, or nine copies of this repeat. These variations permit more detailed analysis of mutant haplotypes 1, 5, 6, and, possibly, others. Kindred analysis in phenylketonuria families demonstrates Mendelian segregation of these VNTR alleles, as well as associations between these alleles and certain PAH mutations. The R261Q mutation, associated with haplotype 1, is associated almost exclusively with an allele containing eight repeats; the R408W mutation, when occurring on a haplotype 1 background, may also be associated with the eight-repeat VNTR allele. Other PAH mutations associated with haplotype 1, R252W and P281L, do not appear to segregate with specific VNTR alleles. The IVS-10 mutation, when associated with haplotype 6, is associated exclusively with an allele containing seven repeats. The combined use of this VNTR system and the existing RFLP haplotype system will increase the performance of prenatal diagnostic tests based on haplotype analysis. In addition, this VNTR may prove useful in studies concerning the origins and distributions of PAH mutations in different human populations.
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molecular basis of phenylketonuria and related hyperPhenylalaninemias mutations and polymorphisms in the human Phenylalanine Hydroxylase gene
Human Mutation, 1992Co-Authors: Randy C Eisensmith, Savio L. C. WooAbstract:Mutations in the human Phenylalanine Hydroxylase gene producing phenylketonuria or hyperPhenylalaninemia have now been identified in many patients from various ethnic groups. These mutations all exhibit a high degree of association with specific restriction fragment-length polymorphism haplotypes at the PAH locus. About 50 of these mutations are single-base substitutions, including six nonsense mutations and eight splicing mutations, with the remainder being missense mutations. One splicing mutation results in a 3 amino acid in-frame insertion. Two or 3 large deletions, 2 single codon deletions, and 2 single base deletions have been found. Twelve of the missense mutations apparently result from the methylation and subsequent deamination of highly mutagenic CpG dinucleotides. Recurrent mutation has been observed at several of these sites, producing associations with different haplotypes in different populations. About half of all missense mutations have been examined by in vitro expression analysis, and a significant correlation has been observed between residual PAH activity and disease phenotype. Since continuing advances in molecular methodologies have dramatically accelerated the rate in which new mutations are being identified and characterized, this register of mutations will be updated periodically. © 1992 Wiley-Liss, Inc.