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- PDB-6o4l: Structure of ALDH7A1 mutant E399D complexed with NAD -

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Basic information

Entry
Database: PDB / ID: 6o4l
TitleStructure of ALDH7A1 mutant E399D complexed with NAD
ComponentsAlpha-aminoadipic semialdehyde dehydrogenase
KeywordsOXIDOREDUCTASE / ALDEHYDE DEHYDROGENASE / NAD / LYSINE CATABOLISM
Function / homology
Function and homology information


L-aminoadipate-semialdehyde dehydrogenase activity / L-aminoadipate-semialdehyde dehydrogenase / Choline catabolism / choline catabolic process / Lysine catabolism / betaine-aldehyde dehydrogenase (NAD+) activity / betaine-aldehyde dehydrogenase / aldehyde metabolic process / glycine betaine biosynthetic process from choline / aldehyde dehydrogenase (NAD+) ...L-aminoadipate-semialdehyde dehydrogenase activity / L-aminoadipate-semialdehyde dehydrogenase / Choline catabolism / choline catabolic process / Lysine catabolism / betaine-aldehyde dehydrogenase (NAD+) activity / betaine-aldehyde dehydrogenase / aldehyde metabolic process / glycine betaine biosynthetic process from choline / aldehyde dehydrogenase (NAD+) / aldehyde dehydrogenase (NAD+) activity / sensory perception of sound / mitochondrial matrix / mitochondrion / extracellular exosome / identical protein binding / nucleus / cytosol
Similarity search - Function
Aldehyde dehydrogenase family 7 member A1-like / Aldehyde Dehydrogenase; Chain A, domain 2 / Aldehyde Dehydrogenase; Chain A, domain 2 / Aldehyde Dehydrogenase; Chain A, domain 1 / Aldehyde Dehydrogenase; Chain A, domain 1 / Aldehyde dehydrogenase, glutamic acid active site / Aldehyde dehydrogenases glutamic acid active site. / Aldehyde dehydrogenase domain / Aldehyde dehydrogenase family / Aldehyde dehydrogenase, C-terminal ...Aldehyde dehydrogenase family 7 member A1-like / Aldehyde Dehydrogenase; Chain A, domain 2 / Aldehyde Dehydrogenase; Chain A, domain 2 / Aldehyde Dehydrogenase; Chain A, domain 1 / Aldehyde Dehydrogenase; Chain A, domain 1 / Aldehyde dehydrogenase, glutamic acid active site / Aldehyde dehydrogenases glutamic acid active site. / Aldehyde dehydrogenase domain / Aldehyde dehydrogenase family / Aldehyde dehydrogenase, C-terminal / Aldehyde dehydrogenase, N-terminal / Aldehyde/histidinol dehydrogenase / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / Alpha-aminoadipic semialdehyde dehydrogenase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 1.85 Å
AuthorsTanner, J.J. / Korasick, D.A. / Laciak, A.R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM093123 United States
CitationJournal: J Inherit Metab Dis / Year: 2020
Title: Structural analysis of pathogenic mutations targeting Glu427 of ALDH7A1, the hot spot residue of pyridoxine-dependent epilepsy.
Authors: Adrian R Laciak / David A Korasick / Kent S Gates / John J Tanner /
Abstract: Certain loss-of-function mutations in the gene encoding the lysine catabolic enzyme aldehyde dehydrogenase 7A1 (ALDH7A1) cause pyridoxine-dependent epilepsy (PDE). Missense mutations of Glu427, ...Certain loss-of-function mutations in the gene encoding the lysine catabolic enzyme aldehyde dehydrogenase 7A1 (ALDH7A1) cause pyridoxine-dependent epilepsy (PDE). Missense mutations of Glu427, especially Glu427Gln, account for ~30% of the mutated alleles in PDE patients, and thus Glu427 has been referred to as a mutation hot spot of PDE. Glu427 is invariant in the ALDH superfamily and forms ionic hydrogen bonds with the nicotinamide ribose of the NAD cofactor. Here we report the first crystal structures of ALDH7A1 containing pathogenic mutations targeting Glu427. The mutant enzymes E427Q, Glu427Asp, and Glu427Gly were expressed in Escherichia coli and purified. The recombinant enzymes displayed negligible catalytic activity compared to the wild-type enzyme. The crystal structures of the mutant enzymes complexed with NAD were determined to understand how the mutations impact NAD binding. In the E427Q and E427G structures, the nicotinamide mononucleotide is highly flexible and lacks a defined binding pose. In E427D, the bound NAD adopts a "retracted" conformation in which the nicotinamide ring is too far from the catalytic Cys residue for hydride transfer. Thus, the structures revealed a shared mechanism for loss of function: none of the variants are able to stabilise the nicotinamide of NAD in the pose required for catalysis. We also show that these mutations reduce the amount of active tetrameric ALDH7A1 at the concentration of NAD tested. Altogether, our results provide the three-dimensional molecular structural basis of the most common pathogenic variants of PDE and implicate strong (ionic) hydrogen bonds in the aetiology of a human disease.
History
DepositionFeb 28, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 6, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2May 20, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.3Oct 11, 2023Group: 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

Downloads & links

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Assembly

Deposited unit
A: Alpha-aminoadipic semialdehyde dehydrogenase
B: Alpha-aminoadipic semialdehyde dehydrogenase
C: Alpha-aminoadipic semialdehyde dehydrogenase
D: Alpha-aminoadipic semialdehyde dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)225,39814
Polymers222,4254
Non-polymers2,97310
Water13,637757
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area27200 Å2
ΔGint-146 kcal/mol
Surface area59180 Å2
MethodPISA
Unit cell
Length a, b, c (Å)81.790, 127.500, 88.390
Angle α, β, γ (deg.)90.000, 101.260, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein
Alpha-aminoadipic semialdehyde dehydrogenase / Alpha-AASA dehydrogenase / Aldehyde dehydrogenase family 7 member A1 / Antiquitin-1 / Betaine ...Alpha-AASA dehydrogenase / Aldehyde dehydrogenase family 7 member A1 / Antiquitin-1 / Betaine aldehyde dehydrogenase / Delta1-piperideine-6-carboxylate dehydrogenase / P6c dehydrogenase / ALDH7A1


Mass: 55606.340 Da / Num. of mol.: 4 / Mutation: E399D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ALDH7A1, ATQ1 / Production host: Escherichia coli (E. coli)
References: UniProt: P49419, L-aminoadipate-semialdehyde dehydrogenase, aldehyde dehydrogenase (NAD+), betaine-aldehyde dehydrogenase
#2: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H6O2
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#4: Chemical
ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE


Mass: 663.425 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: NAD*YM
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 757 / 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: 2.03 Å3/Da / Density % sol: 39.47 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 5.7
Details: 0.2 M MgCl2, 21% (w/v) PEG 3350, and 0.1 M Bis-Tris pH 5.7

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Data collection

DiffractionSerial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 4.2.2 / Wavelength: 1 Å
DetectorType: RDI CMOS_8M / Detector: CMOS / Date: May 29, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.85→45.72 Å / Num. obs: 149120 / % possible obs: 98.7 % / Redundancy: 10 % / CC1/2: 0.999 / Rmerge(I) obs: 0.104 / Rpim(I) all: 0.034 / Rrim(I) all: 0.11 / Net I/σ(I): 16.7 / Num. measured all: 1488879 / Scaling rejects: 354
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) all% possible all
1.85-1.886.72.38264780.4840.9742.58487.2
10.13-45.7210.70.0389500.9990.0120.0498.6

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Processing

Software
NameVersionClassification
Aimless0.7.1data scaling
PHENIXrefinement
PDB_EXTRACT3.24data extraction
XDSdata reduction
PHENIXphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: 4zuk

4zuk


Resolution: 1.85→45.72 Å / SU ML: 0.24 / Cross valid method: THROUGHOUT / σ(F): 0.31 / Phase error: 24.81
RfactorNum. reflection% reflection
Rfree0.2202 13805 4.84 %
Rwork0.1739 --
obs0.1761 149120 95.39 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 98.3 Å2 / Biso mean: 36.0412 Å2 / Biso min: 13.32 Å2
Refinement stepCycle: final / Resolution: 1.85→45.72 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms15318 0 194 757 16269
Biso mean--43.72 34.53 -
Num. residues----2032
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.85-1.8710.33463560.31186990734674
1.871-1.8930.32893880.28867300768878
1.893-1.91610.30123690.26937917828683
1.9161-1.94040.32484720.26938117858987
1.9404-1.96590.29654600.25278816927692
1.9659-1.99280.30744720.24819219969198
1.9928-2.02130.29414950.24029158965398
2.0213-2.05150.30444680.23519296976498
2.0515-2.08350.30554520.23649265971798
2.0835-2.11770.30094340.23179264969898
2.1177-2.15420.27214950.22049277977297
2.1542-2.19340.26184440.20949150959497
2.1934-2.23560.24784390.20279069950896
2.2356-2.28120.25464420.20158990943294
2.2812-2.33080.24124540.19688960941495
2.3308-2.3850.2514170.19679149956696
2.385-2.44470.26054570.20069171962897
2.4447-2.51080.25874550.1929316977198
2.5108-2.58460.25214730.18159444991799
2.5846-2.66810.24225360.181594169952100
2.6681-2.76340.24295560.183494279983100
2.7634-2.8740.23835110.18194349945100
2.874-3.00480.23095010.178893979898100
3.0048-3.16320.23284760.169295019977100
3.1632-3.36130.18234140.16299403981799
3.3613-3.62070.1974700.15469369983999
3.6207-3.98490.184630.14319378984198
3.9849-4.56110.16564670.12659333980099
4.5611-5.74470.16494200.12899490991099
5.7447-45.73430.16765490.139593629911100
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.92390.1133-0.3390.4735-0.27331.0706-0.03920.1412-0.14210.0032-0.0557-0.03690.16080.15060.07690.1450.0148-0.01570.2284-0.02270.200236.87131.30196.1831
20.6490.01830.04570.8325-0.32441.1103-0.00340.06670.14730.13940.04980.0897-0.3787-0.157-0.03020.240.0528-0.01010.19590.01760.21663.726828.17157.549
30.9074-0.1065-0.2120.67010.17140.63030.0324-0.34650.18980.05390.0426-0.1281-0.24130.2617-0.03050.2903-0.0927-0.01280.3287-0.06680.19941.429917.124438.178
41.0240.2782-0.2420.61130.07820.89170.0617-0.22020.03480.1338-0.06920.0991-0.0802-0.1259-0.00550.26130.02280.00760.2241-0.0060.191-0.62249.642338.0431
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1peptide and chain AA0
2X-RAY DIFFRACTION2peptide and chain BB0
3X-RAY DIFFRACTION3peptide and chain CC0
4X-RAY DIFFRACTION4peptide and chain DD0

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