You are here

Home > Structure Details

Human aldehyde dehydrogenase 7A1

Please contact us for any questions or request for reagents for this structure.

Click on the iSee icon to launch an enhanced annotation with interactive 3D representations and animated transitions. Please note that a web plugin (activeICM) is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available.

PDB Code 2J6L Target Class Oxidoreductases

Target ALDH7A1A
Alias ATQ1, EPD, PDE
Disease Area/Function neurobiology, metabolism
Date Deposited 2006-09-29
Authors G.BUNKOCZI, K.GUO, J.E.DEBRECZENI, C.SMEE, C.ARROWSMITH, A.EDWARDS, M.SUNDSTROM, J.WEIGELT, F.VON DELFT, U.OPPERMANN

Struc Details Tabs

Structure Details
Human Aldehyde dehydrogenase 7A1 (ALDH7A1), also known as antiquitin, has recently been shown to be an important component of the lysine catabolic routes involving the L-pipecolic acid pathway. In this reaction sequence, ALDH7A1 catalyzes the final oxidation of α-aminoadipic semialdehyde to α-aminoadipate. The α-aminoadipic semialdehyde forms an intramolecular Schiff base resulting in L-Δ1-piperidine-6 carboxylate (P6C), and these two components are in an equilibrium in solution. P6C can act in a Knoevenagel condensation reaction with pyridoxal 5'-phosphate, thus depleting the intracellular levels of this essential prosthetic group, which is involved among others in neurotransmitter metabolism.

Through this mechanism, the neurological symptoms observed in a rare neurological disorder are explained. Mutations in the ALDH7A1 gene (located on human chromosome 5q31) were recently shown to be involved in the pathophysiology of pyridoxine-dependent seizures (PDS). Classical PDS appears within four weeks of birth and can be treated by pyridoxine supplementation; however, ALDH7A1 deficiency with milder manifestations might be involved in thus far intractable forms of epilepsy.

ALDH7A1 is a member of the ALDH superfamily and shows the typical conserved sequence and active site features, and the mutations found in PDS map to several essential sites within the enzyme structure.

Materials & Methods
StructureALDH7A1
PDB Code2J6L
Entry clone accession BC002515
Entry clone source MGC
Tag N-terminal TEV-cleavable (at *) his-tag with the following sequence mhhhhhhssgvdlgtenlyfq*s
Construct sequencemhhhhhhssgvdlgtenlyfqSMSTLLIN QPQYAWLKELGLREENEGVYNGSWGGRGE VITTYCPANNEPIARVRQASVA
DYEETVK KAREAWKIWADIPAPKRGEIVRQIGDALR EKIQVLGSLVSLEMGKILVEGVGEVQEYV DICDYAVGLSRMIGG
PILPSERSGHALIE QWNPVGLVGIITAFNFPVAVYGWNNAIAM ICGNVCLWKGAPTTSLISVAVTKIIAKVL EDNKLPGA
ICSLTCGGADIGTAMAKDERV NLLSFTGSTQVGKQVGLMVQERFGRSLLE LGGNNAIIAFEDADLSLVVPSALFAAVGT A
GQRCTTARRLFIHESIHDEVVNRLKKAY AQIRVGNPWDPNVLYGPLHTKQAVSMFLG AVEEAKKEGGTVVYGGKVMDRPG
NYVEPT IVTGLGHDASIAHTETFAPILYVFKFQNE EEVFAWNNEVKQGLSSSIFTKDLGRIFRW LGPKGSDCGIVNVNIP
TSGAEIGGAFGGE KHTGGGRESGSDAWKQYMRRSTCTINYS
Vector pNIC28-Bsa4
Expression host BL21(DE3)-R3
Growth method An overnight culture (10 mL) was used to innoculate 1L TB with 50 µg/mL of Kanamycin (total 6L). The cells were cultured at 37°C until the OD reached 0.65 and then the temperature was decreased to 18°C. IPTG was added to 0.5 mM final concentration and the culture kept at 18°C overnight.
Extraction buffers500 mM NaCl, 5% glycerol, 50 mM HEPES pH 7.5, 5 mM imidazole.
Extraction procedure The cells were harvested by centrifugation at 4,000 g for 10 min. The pellet from 1 L of culture was resuspended in 25 mL of extraction buffer. The sample was homogenized by using the EmulsiFlex-05 homogenizer (Glen Creston) and then centrifuged at 37000 xg. The supernatant was kept for further purification.
Purification buffersBinding buffer: 500 mM NaCl, 5% glycerol, 50 mM HEPES pH 7.5, 5 mM imidazole.
Washing Buffer: 500 mM NaCl, 5% glycerol, 50 mM HEPES pH 7.5, 30 mM imidazole.
Elution Buffer: 500 mM NaCl, 5% glycerol, 50 mM HEPES pH 7.5, 250 mM imidazole.
Gel filtration and column 3 buffer: 500 mM NaCl, 5% glycerol, 50 mM HEPES pH 7.5, 0.5 mM TCEP
Purification procedure Column 1: Ni-Sepharose
The column was packed with 2 mL of Ni-Sepharose slurry and equilibrated with 15 mL of binding buffer. The supernatant was loaded onto the column and the flow through was collected. The column was washed with 50 mL of binding buffer and then 50 mL of washing buffer. The protein was eluted with 12 mL of elution buffer and collected in 1.5 mL fractions.

Column 2: Superdex 200 Hiload 16/60
Procedure: An AKTA Purifier system was used. The fractions were analyzed by SDS - PAGE and combined together for TEV cleavage.

Enzymatic treatment : 300 µl of TEV protease were added into the the sample after gel filtration. The sample was incubated at 4°C overnight

Column 3: Ni - Sepharose
Procedure: After treatment with TEV protease, the sample was loaded onto the column (packed with 0.5 mL of Ni-Sepharose slurry). The flow through was collected and the column was then washed with 3 mL of the buffer (also collected).

Concentration: The protein was concentrated in Amicon to 38.8 mg/mL.

Mass spec The experimentally determined mass of 54115 agrees well with the expected mass of 54113.8.
Crystallization Crystals were grown by vapour diffusion at 20°C in 300 nl sitting drops. NADH to a final concentration of 11 mM was added to the protein just prior to crystallisation. The drops were prepared by mixing 150 nl of protein solution (30 mg/mL) and 150 nl of buffer consisting of 0.2 M NaBr , 0.1 M Bis-Tris Propane pH 6.5 and 20% PEG 3350 and 10% EtGly. Crystals were transferred to a cryo-protectant consisting of 20% glycerol, 80 % well solution before flash-cooling in liquid nitrogen.
Landing Page