Structural and biochemical consequences of pyridoxine‐dependent epilepsy mutations that target the aldehyde binding site of aldehyde dehydrogenase ALDH7A1

Abstract

In humans, certain mutations in the gene encoding aldehyde dehydrogenase 7A1 are associated with pyridoxine‐dependent epilepsy (PDE). Understanding the impact of PDE‐causing mutations on the structure and activity of ALDH7A1 could allow for the prediction of symptom‐severity and aid the development of patient‐specific medical treatments. Herein, we investigate the biochemical and structural consequences of PDE missense mutations targeting residues in the aldehyde substrate binding site: N167S, P169S, A171V, G174V, and W175G. All but G174V could be purified for biochemical and X‐ray crystallographic analysis. W175G has a relatively mild kinetic defect, exhibiting a fivefold decrease in kcat with no change in Km. P169S and N167S have moderate defects, characterized by catalytic efficiencies of 20‐ and 100‐times lower than wild‐type, respectively. A171V has a profound functional defect, with catalytic efficiency 2000‐times lower than wild‐type. The crystal structures of the variants are the first for any PDE‐associated mutant of ALDH7A1. The structures show that missense mutations that decrease the steric bulk of the side chain tend to create a cavity in the active site. The protein responds by relaxing into the vacant space, and this structural perturbation appears to cause misalignment of the aldehyde substrate in W175G and N167S. The P169S structure is nearly identical to that of the wild‐type enzyme; however, analysis of B‐factors suggests the catalytic defect may result from altered protein dynamics. The A171V structure suggests that the potential for steric clash with Val171 prevents Glu121 from ion pairing with the amino group of the aldehyde substrate. Enzymes Aldehyde dehydrogenase 7A1 (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/2/1/31.html). Databases Coordinates have been deposited in the Protein Data Bank under the following accession codes: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6O4B, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6O4C, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6O4D, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6O4E, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6O4F, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6O4G, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6O4H.

Highlighting Human Enzymes Active in Different Metabolic Pathways and Diseases: The Case Study of EC 1.2.3.1 and EC 2.3.1.9

“…Here we focus on a complete form of the biological unit (PDB code: 4ZUL), a homotetramer solved with a resolution of 0.170 nm and with the maximal coverage with the sequence P49419, without the mitochondrial target peptide [32]. Recently, important variants of the protein, associated with PDE and hampering its activity, have been also solved with atomic resolution [33]. Finally, the protein, as a major feature, according to the MobiDB database [34], does not have intrinsically disordered regions (IDPs).…”

“…This suggests that disease may be also due to an unpaired translocation of the protein to the mitochondrial compartment. For the sake of comparison, in Table S2 (Supplementary Materials), we label, in red, some PDE disease-related variations, known to occur in the aldehyde substrate binding site (N195S, P197S, A199V, G202V, W203G) and recently detailed with atomic resolution on their effect on the protein structure and function [33]. INPS predicts P197S, G202V, W203G as perturbing the protein stability (Table S2).…”

Structural analysis of pathogenic mutations targeting Glu427 of ALDH7A1, the hot spot residue of pyridoxine‐dependent epilepsy

“…The tetramer observed in the crystal has been shown to be the active form of ALDH7A1 . We note this is the same crystal form that has been used to solve structures of wild‐type ALDH7A1 and several PDE mutant variants …”

“…We note that the NMN is also disordered in wild‐type ALDH7A1 structures when NAD + is included in the crystallisation at 5 mM (eg, see PDB code 4ZUK) . However, the cofactor is fully resolved by strong electron density when included in the crystallisation in the higher concentration range of 11 to 15 mM (PDB codes 2J6L, 6O4B‐C, 6O4E, 6O4H) . Because 15 mM NAD + was used for crystallisation of E427Q, the lack of electron density for the NMN is meaningful.…”

“…The mutations were verified by Sanger sequencing. Wild‐type ALDH7A1 and the mutant variants were expressed in E. coli and purified as described previously …”