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- PDB-6hpo: Crystallographic structure of the catalytic domain of Human Pheny... -

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Entry
Database: PDB / ID: 6hpo
TitleCrystallographic structure of the catalytic domain of Human Phenylalanine Hydroxylase (hPAH CD) in complex with iron at 1.6 Angstrom
ComponentsPhenylalanine-4-hydroxylase
KeywordsOXIDOREDUCTASE / Tetrahydrobiopterin / amino acid hydroxylases / phenylketonuria / allosteric regulation / METAL BINDING PROTEIN / oxidoreductase
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: 1.67 Å
AuthorsAlcorlo Pages, M. / Innselset Flydal, 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: Sep 21, 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

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Assembly

Deposited unit
A: Phenylalanine-4-hydroxylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,9852
Polymers51,9291
Non-polymers561
Water5,296294
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area140 Å2
ΔGint-13 kcal/mol
Surface area14520 Å2
MethodPISA
Unit cell
γ
α
β
Length a, b, c (Å)65.856, 107.549, 124.016
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221

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Components

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


Mass: 51928.906 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PAH / Production host: Escherichia coli (E. coli) / References: UniProt: P00439, EC: 1.14.16.1
#2: Chemical ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe / Iron
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 294 / 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 / Details: 40 mM PIPES, 20% PEG 2000, pH 6.8

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALBA / Beamline: XALOC / Wavelength: 0.9792 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Mar 1, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 1.67→32.77 Å / Num. obs: 51389 / % possible obs: 99.96 % / Redundancy: 13.4 % / Rmerge(I) obs: 0.084 / Rpim(I) all: 0.026 / Net I/σ(I): 16.2
Reflection shellResolution: 1.67→1.73 Å / Rmerge(I) obs: 1.921 / Num. unique obs: 2603 / Rpim(I) all: 0.574

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Processing

Software
NameVersionClassification
PHENIX(1.13_2998: ???)refinement
XDSdata reduction
Aimlessdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4ANP
Resolution: 1.67→32.77 Å / SU ML: 0.16 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 19.41
RfactorNum. reflection% reflection
Rfree0.177 2541 4.95 %
Rwork0.1607 --
Obs0.1615 51335 99.92 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 1.67→32.77 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2540 0 1 294 2835
Refine LS restraints

Refinement-ID: X-RAY DIFFRACTION

TypeDev idealNumber
f_bond_d0.0062680
f_angle_d0.7733651
f_dihedral_angle_d5.8822208
f_chiral_restr0.05382
f_plane_restr0.005476
LS refinement shell

Refinement-ID: X-RAY DIFFRACTION / % reflection obs: 100 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork
1.67-1.70210.30071450.27322706
1.7021-1.73690.28131310.25322671
1.7369-1.77460.23471360.2252656
1.7746-1.81590.22341320.21072696
1.8159-1.86130.21071130.19992718
1.8613-1.91160.20941460.18822681
1.9116-1.96790.20441270.1672706
1.9679-2.03140.1791400.15962681
2.0314-2.1040.17261610.15852687
2.104-2.18820.18221520.16262660
2.1882-2.28780.19891410.15832709
2.2878-2.40840.18331490.15962680
2.4084-2.55920.17731440.15642721
2.5592-2.75670.20381450.16512726
2.7567-3.0340.18211530.1632708
3.034-3.47260.17671330.15682754
3.4726-4.37340.16011590.13662739
4.3734-32.93440.14021340.15452895
Refinement TLS params.Method: refined / Origin x: 0.1353 Å / Origin y: 24.8754 Å / Origin z: 9.606 Å
111213212223313233
T0.2107 Å20.0059 Å20.0035 Å2-0.2229 Å2-0.0025 Å2--0.2037 Å2
L1.071 °2-0.209 °2-0.1755 °2-0.7995 °20.1815 °2--0.6804 °2
S-0.0217 Å °-0.0939 Å °-0.0357 Å °-0.0221 Å °0.0171 Å °-0.0176 Å °-0.0102 Å °-0.0493 Å °0.0063 Å °
Refinement TLS groupSelection details: all

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