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

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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


rt:r-hsa-71182: / Phenylketonuria / ec:1.14.16.1: / phenylalanine 4-monooxygenase activity / tyrosine biosynthetic process / catecholamine biosynthetic process / neurotransmitter biosynthetic process / L-phenylalanine catabolic process / cellular amino acid biosynthetic process / iron ion binding / cytosol
Tyrosine 3-monooxygenase-like / Aromatic amino acid hydroxylase superfamily / ACT domain profile. / Biopterin-dependent aromatic amino acid hydroxylase family profile. / Biopterin-dependent aromatic amino acid hydroxylases signature. / ACT domain / Biopterin-dependent aromatic amino acid hydroxylase / Aromatic amino acid hydroxylase / ACT domain / Phenylalanine-4-hydroxylase, tetrameric form ...Tyrosine 3-monooxygenase-like / Aromatic amino acid hydroxylase superfamily / ACT domain profile. / Biopterin-dependent aromatic amino acid hydroxylase family profile. / Biopterin-dependent aromatic amino acid hydroxylases signature. / ACT domain / Biopterin-dependent aromatic amino acid hydroxylase / Aromatic amino acid hydroxylase / ACT domain / Phenylalanine-4-hydroxylase, tetrameric form / Aromatic amino acid hydroxylase, iron/copper binding site / Eukaryotic phenylalanine-4-hydroxylase, catalytic domain / Aromatic amino acid hydroxylase, C-terminal / Aromatic amino acid monoxygenase, C-terminal domain superfamily
Phenylalanine-4-hydroxylase
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.18 Å
AuthorsAlcorlo Pages, M. / Flydal, I.M.
Funding supportSpain , Norway , 2件
OrganizationGrant numberCountry
Spanish Ministry of Economy and CompetitivenessBFU2014-59389-P and BFU2017-90030-P to JAHSpain
Research Council of NorwayPrograms Forny (248889/O30) and FRIMEDBIO (261826) to AMNorway
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 19, 2018 / Release: Jun 5, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jun 5, 2019Structure modelrepositoryInitial release
1.1Jun 19, 2019Structure modelData collection / Database referencescitation / citation_author / pdbx_database_proc_citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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
γ
α
β
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/peptide
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, EC: 1.14.16.1
#2: Chemical ChemComp-H4B / 5,6,7,8-TETRAHYDROBIOPTERIN


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


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

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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
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

Refinement-ID: X-RAY DIFFRACTION

TypeDev idealNumber
f_bond_d0.00614227
f_angle_d1.0619257
f_dihedral_angle_d3.6039378
f_chiral_restr0.0582078
f_plane_restr0.0062488
LS refinement shell

Refinement-ID: X-RAY DIFFRACTION

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
3.1801-3.28260.38961250.3063264286
3.2826-3.39990.30521420.2784277393
3.3999-3.53580.31651880.2694287899
3.5358-3.69660.29941660.26553004100
3.6966-3.89120.30611780.25512995100
3.8912-4.13460.29571680.24863011100
4.1346-4.45320.33151590.24173053100
4.4532-4.90010.31741300.25443022100
4.9001-5.60650.422960.27093102100
5.6065-7.05310.3571550.3143054100
7.0531-33.92570.24151020.2385312199

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