the molecular basis of phenylketonuria...•eisensmith and woo. gene therapy for phenylketonuria....
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The Molecular Basis ofThe Molecular Basis ofPhenylketonuriaPhenylketonuria
Rebecca Siemer
2/26/01
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Background
• What is it?– Phenylalanine
– Untreated …
– Restricted Diet
• Autosomal Recessive– Carriers: 1/50
– Frequency: 1/10,000
– Screening: 1960’s
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Phenylalanine HydroxylaseActivity
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Phenylalanine Toxicity
• Unknown• Serotonin and Catecholamines
– Inhibits tyrosine and tryptophan transport intoneurons
• Phenylpyruvic acid– Pyruvate decarboxylase inhibitor
• Myelin Synthesis• Alterations in brain architecture
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Phenylalanine hydroxylase
• 75% phenylalanine’sdisposal
• Converts phe to tyr
• 12q24.1
• 1% map to genes forBH4
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Phenylalanine hydroxylase
• Domains– N-terminus (1-142)
– Catalytic (143-410)
– Tetramerization(411-452)
• Active Site– Non heme iron
• Iron III resting state
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Active site
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Phenylalanine hydroxylase
• pH dependentequilibrium– Homodimers
– Homotetramers• Antiparallel coiled-coil
core
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Mutations
• Catalytic– 209
• Regulatory– 49
• Tetramerization– 10
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Active-site Mutations
• T278I, T278A, andT278N– Thr278 location
– H-bond to Glu280
• E280K– Electrostatic potential
altered
• F254I– pi-stacks with pterin ring
– Interfere with pterin binding
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Active-site Mutations (cont…)
• P281L– Defines shape near iron
• F331C and F331L– Abolish pi-stacking
interactions that stabilizethe active site wall
• S349P– Located near active site
– Total alteration of activesite shape
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Fe3+
BH4
Sub.
Fe3+
Sub.
BH4
e-Fe2+
Sub.
BH4ox
O2
Fe
Sub.
BH4
-O-O Fe
Sub.
BH2
-O Fe3+
Product
BH2
Normal PAH Mechanism
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Fe3+
BH4
Sub.
Fe3+
Sub.
BH4
e-Fe2+
Sub.
BH4ox
O2
Fe
Sub.
BH4
-O-O Fe
Sub.
BH2
-O Fe3+
Sub.
BH2
S349P Mutation Uncoupling
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Regulatory Domain
• Regulation of PAH
• G46S Mutation– Distorts secondary structure
– Inactive aggregates formed
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Tetramerization DomainMutations
• IVS12 + 1g � a in intron12– Most prevalent mutation
among Caucasians
– Truncated form of PAH
• Lacks last 52 aminoacids
– Unstable protein
• pro407-arg408-pro409– Hinge region
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The Heterozygote Advantage
• Ochratoxin A– Aspergillus and Penicillium– N-acyl derivative of phenylalanine– Stops Protein Synthesis
• Competes for phenylalanyl-tRNA synthetase
• Celtic Origin– Mild, wet climate– Famine, economic hardships
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Treatment
• Diet– Non compliance
• Gene Therapy– Far Future
• Enzyme Replacement Therapy– PAL
• Phenylalanine � trans-cinnamic acid
– Oral
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An Interesting Tangent
• Phenylacetate– Damage to immature brain– Inhibits protein prenylation– Other mechanisms?
• Primary brain tumors are very similar toimmature CNS
• Significant tumor suppression with noapparent toxicity to the host
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Conclusions
• PAH deficiency
• Autosomal recessive– Heterozygote advantage
• Almost 400 known mutations
• Future– Most promising: Enzyme replacement therapy
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References
• Eisensmith and Woo. Gene Therapy for Phenylketonuria. EuropeanJournal of Pediatrics 155: [suppl 1] S16-S19. 1996.
• Ellis, Daubner, and Fitzpatrick. Mutation of Serine 395 of TyrosineHydroxylase Decouples Oxygen-Oxygen Bond Cleavage and TyrosineHydroxylation. Biochemistry 39: (14) 4174-4181. 2000
• Erlandsen, Fusetti, Martinez, Hough, Flatmark, and Stevens. CrystalStructure of the Catalytic Domain of Human PhenylalanineHydroxylase Reveals the Structural Basis for Phenylketonuria. NatureStructural Biology 4: (12) 995-1000. Dec, 1997.
• Erlandsen and Stevens. The Structural Basis of Phenylketonuria.Molecular Genetics and Metabolism 68: (2) 103-125. Oct, 1999.
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References (cont…)
• Fusetti, Erlandsen, Flatmark and Stevens. Structure of TetramericHuman Phenylalanine Hydroxylase and its Implications forPhenylketonuria. Journal of Biological Chemistry 273: (27) 16962-16967. July 3, 1998.
• Gamez, Perez, Ugarte, and Desviat. Expression Analysis ofPhenylketonuria Mutations. Journal of Biological Chemistry 275: (38)29737-29742. Sept 22, 2000.
• Samid, Ram, Hudgins, Shack, Liu, Walbridge, Oldfield, Myers.Selective Activity of Phenylacetate Against Malignant Gliomas:Resemblance to Fetal Brain Damage in Phenylketonuria. CancerResearch 54: (4) 891-895. Feb 15, 1994.
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References (cont…)
• Sarkissian, Shao, Blain, Peevers, Su, Heft, Chang, Scriver. A DifferentApproach to Treatment of Phenylketonuria: PhenylalanineDegradation with Recombinant Phenylalanine Ammonia Lyase.Medical Sciences 96: (5) 2339-2344 Mar 2, 1999.
• Smith, Howells, Hyland. Pteridines and Mono-amines: Relevance toNeurological Damage. Postgraduate Medical Journal 62: (724) 113-123. Feb, 1986.
• Woolf. The Heterozygoete Advantage in Phenylketonuria. AmericanJournal of Human Genetics 38: 773-775, 1986.