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The Molecular Basis ofThe Molecular Basis ofPhenylketonuriaPhenylketonuria

Rebecca Siemer

2/26/01

Background

• What is it?– Phenylalanine

– Untreated …

– Restricted Diet

• Autosomal Recessive– Carriers: 1/50

– Frequency: 1/10,000

– Screening: 1960’s

Phenylalanine HydroxylaseActivity

Phenylalanine Toxicity

• Unknown• Serotonin and Catecholamines

– Inhibits tyrosine and tryptophan transport intoneurons

• Phenylpyruvic acid– Pyruvate decarboxylase inhibitor

• Myelin Synthesis• Alterations in brain architecture

Phenylalanine hydroxylase

• 75% phenylalanine’sdisposal

• Converts phe to tyr

• 12q24.1

• 1% map to genes forBH4

Phenylalanine hydroxylase

• Domains– N-terminus (1-142)

– Catalytic (143-410)

– Tetramerization(411-452)

• Active Site– Non heme iron

• Iron III resting state

Active site

Phenylalanine hydroxylase

• pH dependentequilibrium– Homodimers

– Homotetramers• Antiparallel coiled-coil

core

Mutations

• Catalytic– 209

• Regulatory– 49

• Tetramerization– 10

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

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

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

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

Regulatory Domain

• Regulation of PAH

• G46S Mutation– Distorts secondary structure

– Inactive aggregates formed

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

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

Treatment

• Diet– Non compliance

• Gene Therapy– Far Future

• Enzyme Replacement Therapy– PAL

• Phenylalanine � trans-cinnamic acid

– Oral

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

Conclusions

• PAH deficiency

• Autosomal recessive– Heterozygote advantage

• Almost 400 known mutations

• Future– Most promising: Enzyme replacement therapy

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.

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.

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.