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- PDB-6u2x: Structure of ALDH7A1 mutant E399G complexed with NAD -

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Basic information

Entry
Database: PDB / ID: 6u2x
TitleStructure of ALDH7A1 mutant E399G 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 / betaine-aldehyde dehydrogenase activity / betaine-aldehyde dehydrogenase / glycine betaine biosynthetic process from choline / cellular aldehyde metabolic process / Lysine catabolism / aldehyde dehydrogenase (NAD+) ...L-aminoadipate-semialdehyde dehydrogenase activity / L-aminoadipate-semialdehyde dehydrogenase / Choline catabolism / choline catabolic process / betaine-aldehyde dehydrogenase activity / betaine-aldehyde dehydrogenase / glycine betaine biosynthetic process from choline / cellular aldehyde metabolic process / Lysine catabolism / aldehyde dehydrogenase (NAD+) / glyceraldehyde-3-phosphate dehydrogenase (NAD+) (non-phosphorylating) activity / 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 dehydrogenases glutamic acid active site. / Aldehyde dehydrogenase, 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 dehydrogenases glutamic acid active site. / Aldehyde dehydrogenase, 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 / MOLECULAR REPLACEMENT / Resolution: 2.15 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
DepositionAug 20, 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

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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
E: Alpha-aminoadipic semialdehyde dehydrogenase
F: Alpha-aminoadipic semialdehyde dehydrogenase
G: Alpha-aminoadipic semialdehyde dehydrogenase
H: Alpha-aminoadipic semialdehyde dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)449,69416
Polymers444,3868
Non-polymers5,3078
Water26,3921465
1
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)224,8478
Polymers222,1934
Non-polymers2,6544
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area24520 Å2
ΔGint-115 kcal/mol
Surface area60240 Å2
MethodPISA
2
E: Alpha-aminoadipic semialdehyde dehydrogenase
F: Alpha-aminoadipic semialdehyde dehydrogenase
G: Alpha-aminoadipic semialdehyde dehydrogenase
H: Alpha-aminoadipic semialdehyde dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)224,8478
Polymers222,1934
Non-polymers2,6544
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area23670 Å2
ΔGint-106 kcal/mol
Surface area60150 Å2
MethodPISA
Unit cell
Length a, b, c (Å)155.270, 161.650, 158.800
Angle α, β, γ (deg.)90.000, 94.410, 90.000
Int Tables number5
Space group name H-MC121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resid 3 through 19 or (resid 20...
21(chain B and (resid 3 through 9 or (resid 10...
31(chain C and ((resid 3 and (name N or name...
41(chain D and (resid 3 through 9 or (resid 10...
51(chain E and (resid 3 through 9 or (resid 10...
61(chain F and ((resid 3 and (name N or name...
71(chain G and (resid 3 through 9 or (resid 10...
81(chain H and ((resid 3 and (name N or name...

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111(chain A and (resid 3 through 19 or (resid 20...A3 - 19
121(chain A and (resid 3 through 19 or (resid 20...A20 - 21
131(chain A and (resid 3 through 19 or (resid 20...A3 - 601
141(chain A and (resid 3 through 19 or (resid 20...A3 - 601
151(chain A and (resid 3 through 19 or (resid 20...A3 - 601
161(chain A and (resid 3 through 19 or (resid 20...A3 - 601
211(chain B and (resid 3 through 9 or (resid 10...B3 - 9
221(chain B and (resid 3 through 9 or (resid 10...B10
231(chain B and (resid 3 through 9 or (resid 10...B3 - 511
311(chain C and ((resid 3 and (name N or name...C3
321(chain C and ((resid 3 and (name N or name...C3 - 511
331(chain C and ((resid 3 and (name N or name...C3 - 511
341(chain C and ((resid 3 and (name N or name...C3 - 511
351(chain C and ((resid 3 and (name N or name...C3 - 511
411(chain D and (resid 3 through 9 or (resid 10...D3 - 9
421(chain D and (resid 3 through 9 or (resid 10...D10
431(chain D and (resid 3 through 9 or (resid 10...D3 - 601
441(chain D and (resid 3 through 9 or (resid 10...D3 - 601
451(chain D and (resid 3 through 9 or (resid 10...D3 - 601
461(chain D and (resid 3 through 9 or (resid 10...D3 - 601
511(chain E and (resid 3 through 9 or (resid 10...E3 - 9
521(chain E and (resid 3 through 9 or (resid 10...E10
531(chain E and (resid 3 through 9 or (resid 10...E3 - 601
541(chain E and (resid 3 through 9 or (resid 10...E3 - 601
551(chain E and (resid 3 through 9 or (resid 10...E3 - 601
561(chain E and (resid 3 through 9 or (resid 10...E3 - 601
611(chain F and ((resid 3 and (name N or name...F3
621(chain F and ((resid 3 and (name N or name...F3 - 601
631(chain F and ((resid 3 and (name N or name...F3 - 601
641(chain F and ((resid 3 and (name N or name...F3 - 601
651(chain F and ((resid 3 and (name N or name...F3 - 601
711(chain G and (resid 3 through 9 or (resid 10...G3 - 9
721(chain G and (resid 3 through 9 or (resid 10...G10
731(chain G and (resid 3 through 9 or (resid 10...G3 - 501
741(chain G and (resid 3 through 9 or (resid 10...G3 - 501
751(chain G and (resid 3 through 9 or (resid 10...G3 - 501
811(chain H and ((resid 3 and (name N or name...H3
821(chain H and ((resid 3 and (name N or name...H3 - 601
831(chain H and ((resid 3 and (name N or name...H3 - 601
841(chain H and ((resid 3 and (name N or name...H3 - 601
851(chain H and ((resid 3 and (name N or name...H3 - 601

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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


Mass: 55548.305 Da / Num. of mol.: 8 / Mutation: E399G
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-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE / Nicotinamide adenine dinucleotide


Mass: 663.425 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: NAD*YM
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1465 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 2.24 Å3/Da / Density % sol: 44.98 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: 25% PEG 3350, 0.2 M sodium chloride, 0.1 M Bis-Tris pH 6.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 4.2.2 / Wavelength: 1 Å
DetectorType: RDI CMOS_8M / Detector: CMOS / Date: Mar 18, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.15→49.16 Å / Num. obs: 207132 / % possible obs: 97.9 % / Redundancy: 9.9 % / CC1/2: 0.99 / Rmerge(I) obs: 0.214 / Rpim(I) all: 0.072 / Rrim(I) all: 0.226 / Net I/σ(I): 9.1 / Num. measured all: 2054217 / Scaling rejects: 1053
Reflection shellResolution: 2.15→2.19 Å / Redundancy: 7.8 % / Rmerge(I) obs: 1.323 / Num. measured all: 79131 / Num. unique obs: 10198 / CC1/2: 0.688 / Rpim(I) all: 0.508 / Rrim(I) all: 1.419 / Net I/σ(I) obs: 1.5 / % possible all: 97.7

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Processing

Software
NameVersionClassification
Aimless0.7.3data scaling
PHENIX1.14_3260: ???refinement
PDB_EXTRACT3.25data extraction
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4zuk
Resolution: 2.15→49.159 Å / SU ML: 0.3 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 23.75
RfactorNum. reflection% reflection
Rfree0.2325 10028 4.99 %
Rwork0.1741 --
obs0.177 200818 94.97 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 92.46 Å2 / Biso mean: 31.516 Å2 / Biso min: 8.1 Å2
Refinement stepCycle: final / Resolution: 2.15→49.159 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms30492 0 297 1465 32254
Biso mean--34.49 30.45 -
Num. residues----4062
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A18187X-RAY DIFFRACTION7.035TORSIONAL
12B18187X-RAY DIFFRACTION7.035TORSIONAL
13C18187X-RAY DIFFRACTION7.035TORSIONAL
14D18187X-RAY DIFFRACTION7.035TORSIONAL
15E18187X-RAY DIFFRACTION7.035TORSIONAL
16F18187X-RAY DIFFRACTION7.035TORSIONAL
17G18187X-RAY DIFFRACTION7.035TORSIONAL
18H18187X-RAY DIFFRACTION7.035TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.15-2.17440.32753200.2646652398
2.1744-2.20.34623250.2662659698
2.2-2.22680.5488970.4802181127
2.2268-2.2550.50122920.4247548982
2.255-2.28470.50872750.3733488272
2.2847-2.3160.3173340.2191649598
2.316-2.34910.27293650.2013653298
2.3491-2.38420.26723590.1978655298
2.3842-2.42140.27033780.1962651098
2.4214-2.46110.26923530.1961655898
2.4611-2.50350.26383940.1962650898
2.5035-2.54910.26463540.1976660498
2.5491-2.59810.27413860.1935653399
2.5981-2.65110.24973230.1838659998
2.6511-2.70880.25123260.1865659499
2.7088-2.77180.24753410.1841660399
2.7718-2.84110.25523220.1788660499
2.8411-2.91790.25693420.1739664999
2.9179-3.00370.23383660.1744661999
3.0037-3.10070.25693770.1827660499
3.1007-3.21150.25433450.1766663099
3.2115-3.340.21823690.1632658499
3.34-3.4920.18893440.1551670799
3.492-3.6760.21913160.1598663499
3.676-3.90630.21012770.154673999
3.9063-4.20770.18433340.1341667899
4.2077-4.63090.15663570.11786704100
4.6309-5.30030.15593680.12086677100
5.3003-6.67520.20613480.15266747100
6.6752-49.1590.16633410.14026825100
Refinement TLS params.

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

IDL112)L122)L132)L222)L232)L332)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T112)T122)T132)T222)T232)T332)Origin x (Å)Origin y (Å)Origin z (Å)
10.3550.12950.08050.51820.20510.78320.0181-0.05160.0340.00650.0589-0.1529-0.03520.151-0.0680.1550.00590.0030.1822-0.05970.289656.842858.624734.1439
20.8688-0.25120.15530.68430.11120.47050.0176-0.0820.0640.0565-0.03780.0912-0.009-0.06990.02430.1696-0.03480.0320.193-0.06180.226522.330358.279643.2758
30.8119-0.11540.08270.5020.11510.52320.02150.0791-0.1594-0.0402-0.02480.01480.038-0.03070.00190.1770-00.1539-0.01480.253545.483818.780423.5828
40.67340.18-0.11310.567-0.21850.60370.0515-0.1873-0.11110.1474-0.0308-0.09260.03780.0766-0.02280.2203-0.027-0.02740.23430.01480.214736.091220.467158.1742
50.84970.4906-0.060.4681-0.05920.4169-0.05210.0601-0.0595-0.02990.02350.03620.0386-0.09080.0250.1912-0.01430.02470.2263-0.10170.267723.821398.50333.1499
60.2838-0.1159-0.06950.3778-0.0580.3239-0.0120.011-0.07080.0423-0.0174-0.090.00860.02090.03430.2078-0.04380.0060.2415-0.08360.306658.264999.145243.2722
70.4455-0.10640.04310.3864-0.09570.5118-0.0155-0.06950.1413-0.0367-0.02710.0781-0.0746-0.11020.03560.1960.0178-0.01280.2151-0.09290.26935.5968138.146120.5347
80.6931-0.0234-0.15840.37450.24260.48890.0115-0.2150.10660.06690.0167-0.0005-0.0778-0.0433-0.0280.233-0.00440.01810.2778-0.08030.230944.0697137.387156.4431
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1(chain 'A')A0
2X-RAY DIFFRACTION2(chain 'B)B0
3X-RAY DIFFRACTION3(chain 'C')C0
4X-RAY DIFFRACTION4(chain 'D')D0
5X-RAY DIFFRACTION5(chain 'E')E0
6X-RAY DIFFRACTION6(chain 'F')F0
7X-RAY DIFFRACTION7(chain 'G')G0
8X-RAY DIFFRACTION8(chain 'H')H0

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