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- PDB-6hyc: The structure of full-length human phenylalanine hydroxylase in c... -

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Basic information

Entry
Database: PDB / ID: 6hyc
TitleThe structure of full-length human phenylalanine hydroxylase in complex with the cofactor and negative regulator tetrahydrobiopterin
ComponentsPhenylalanine-4-hydroxylase
KeywordsOXIDOREDUCTASE / Tetrahydrobiopterin / phenylalanine hydroxylase / phenylketonuria / allostery
Function / homology
Function and homology information


Phenylketonuria / phenylalanine 4-monooxygenase / Phenylalanine metabolism / phenylalanine 4-monooxygenase activity / L-tyrosine biosynthetic process / catecholamine biosynthetic process / L-phenylalanine catabolic process / amino acid biosynthetic process / iron ion binding / cytosol
Similarity search - Function
Phenylalanine-4-hydroxylase, tetrameric form / Eukaryotic phenylalanine-4-hydroxylase, catalytic domain / Phenylalanine Hydroxylase / Aromatic amino acid hydroxylase / Tyrosine 3-monooxygenase-like / ACT domain / Aromatic amino acid hydroxylase, iron/copper binding site / Biopterin-dependent aromatic amino acid hydroxylases signature. / Aromatic amino acid hydroxylase / Aromatic amino acid hydroxylase, C-terminal ...Phenylalanine-4-hydroxylase, tetrameric form / Eukaryotic phenylalanine-4-hydroxylase, catalytic domain / Phenylalanine Hydroxylase / Aromatic amino acid hydroxylase / Tyrosine 3-monooxygenase-like / ACT domain / Aromatic amino acid hydroxylase, iron/copper binding site / Biopterin-dependent aromatic amino acid hydroxylases signature. / Aromatic amino acid hydroxylase / Aromatic amino acid hydroxylase, C-terminal / Aromatic amino acid monoxygenase, C-terminal domain superfamily / Aromatic amino acid hydroxylase superfamily / Biopterin-dependent aromatic amino acid hydroxylase / Biopterin-dependent aromatic amino acid hydroxylase family profile. / ACT domain profile. / ACT domain / ACT-like domain / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
5,6,7,8-TETRAHYDROBIOPTERIN / Phenylalanine-4-hydroxylase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.18 Å
AuthorsAlcorlo Pages, M. / Flydal, I.M.
Funding support Spain, Norway, 2items
OrganizationGrant numberCountry
Spanish Ministry of Economy and CompetitivenessBFU2014-59389-P and BFU2017-90030-P to JAH Spain
Research Council of NorwayPrograms Forny (248889/O30) and FRIMEDBIO (261826) to AM Norway
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Structure of full-length human phenylalanine hydroxylase in complex with tetrahydrobiopterin.
Authors: Marte Innselset Flydal / Martín Alcorlo-Pagés / Fredrik Gullaksen Johannessen / Siseth Martínez-Caballero / Lars Skjærven / Rafael Fernandez-Leiro / Aurora Martinez / Juan A Hermoso /
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 ...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 (BH) that partly acts as a pharmacological chaperone. Here we present structures of full-length human PAH (hPAH) both unbound and complexed with BH in the precatalytic state. Crystal structures, solved at 3.18-Å resolution, show the interactions between the cofactor and PAH, explaining the negative regulation exerted by BH BH forms several H-bonds with the N-terminal autoregulatory tail but is far from the catalytic Fe Upon BH binding a polar and salt-bridge interaction network links the three PAH domains, explaining the stability conferred by BH Importantly, BH 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 BH reveals a highly dynamic conformation for the tetramers. Structural analyses of the hPAH:BH subunits revealed that the substrate-induced movement of Tyr138 into the active site could be coupled to the displacement of BH 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.
History
DepositionOct 19, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 5, 2019Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2019Group: Data collection / Database references / Category: citation / citation_author / pdbx_database_proc
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
D: Phenylalanine-4-hydroxylase
A: Phenylalanine-4-hydroxylase
C: Phenylalanine-4-hydroxylase
B: Phenylalanine-4-hydroxylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)208,4397
Polymers207,7164
Non-polymers7243
Water905
1
D: Phenylalanine-4-hydroxylase
C: Phenylalanine-4-hydroxylase
hetero molecules

D: Phenylalanine-4-hydroxylase
C: Phenylalanine-4-hydroxylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)208,6818
Polymers207,7164
Non-polymers9654
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_556-x,y,-z+11
Buried area14710 Å2
ΔGint-57 kcal/mol
Surface area69200 Å2
2
A: Phenylalanine-4-hydroxylase
B: Phenylalanine-4-hydroxylase
hetero molecules

A: Phenylalanine-4-hydroxylase
B: Phenylalanine-4-hydroxylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)208,1986
Polymers207,7164
Non-polymers4822
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area14290 Å2
ΔGint-50 kcal/mol
Surface area68990 Å2
Unit cell
Length a, b, c (Å)101.940, 101.368, 203.543
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number5
Space group name H-MC121

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Components

#1: Protein
Phenylalanine-4-hydroxylase / PAH / Phe-4-monooxygenase


Mass: 51928.906 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PAH / Production host: Escherichia coli #1/H766 (bacteria) / References: UniProt: P00439, phenylalanine 4-monooxygenase
#2: Chemical ChemComp-H4B / 5,6,7,8-TETRAHYDROBIOPTERIN


Mass: 241.247 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C9H15N5O3
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.48 Å3/Da / Density % sol: 50.35 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 7
Details: 1.5 M DL Malic Acid pH=7.0, 100 mM Bis-Tris Propane pH=6.2, 0.9 mM Thesit, 0.98 mM BH4, 25 mM DTT and 1 mM reduced glutathione

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALBA / Beamline: XALOC / Wavelength: 0.97926 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Oct 19, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97926 Å / Relative weight: 1
ReflectionResolution: 3.18→33.92 Å / Num. obs: 34280 / % possible obs: 94.3 % / Redundancy: 3 % / Rmerge(I) obs: 0.181 / Rpim(I) all: 0.1314 / Net I/σ(I): 2.4
Reflection shellResolution: 3.18→3.34 Å / Num. unique obs: 3926

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Processing

Software
NameVersionClassification
PHENIX(1.13_2998: ???)refinement
XDSdata reduction
Aimlessdata scaling
MoRDaphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5den

5den


Resolution: 3.18→33.92 Å / SU ML: 0.43 / Cross valid method: FREE R-VALUE / σ(F): 0 / Phase error: 31.58
RfactorNum. reflection% reflection
Rfree0.3107 1609 4.7 %
Rwork0.2619 --
obs0.2641 34264 97.77 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 3.18→33.92 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms13792 0 51 5 13848
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00614227
X-RAY DIFFRACTIONf_angle_d1.0619257
X-RAY DIFFRACTIONf_dihedral_angle_d3.6039378
X-RAY DIFFRACTIONf_chiral_restr0.0582078
X-RAY DIFFRACTIONf_plane_restr0.0062488
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.1801-3.28260.38961250.30632642X-RAY DIFFRACTION86
3.2826-3.39990.30521420.27842773X-RAY DIFFRACTION93
3.3999-3.53580.31651880.26942878X-RAY DIFFRACTION99
3.5358-3.69660.29941660.26553004X-RAY DIFFRACTION100
3.6966-3.89120.30611780.25512995X-RAY DIFFRACTION100
3.8912-4.13460.29571680.24863011X-RAY DIFFRACTION100
4.1346-4.45320.33151590.24173053X-RAY DIFFRACTION100
4.4532-4.90010.31741300.25443022X-RAY DIFFRACTION100
4.9001-5.60650.422960.27093102X-RAY DIFFRACTION100
5.6065-7.05310.3571550.3143054X-RAY DIFFRACTION100
7.0531-33.92570.24151020.23853121X-RAY DIFFRACTION99

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