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

Abstract

Certain loss-of-function mutations in the gene encoding the lysine catabolic enzyme aldehyde dehydrogenase 7A1 (ALDH7A1) cause pyridoxinedependent 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. K E Y W O R D SALDH7A1, enzyme kinetics, missense mutation, PDE, pyridoxine-dependent epilepsy, X-ray crystallography Abbreviations: AA, α-aminoadipate; AASAL, α-aminoadipate semialdehyde; ALDH, aldehyde dehydrogenase; ALDH7A1, aldehyde dehydrogenase 7A1; NMN, nicotinamide mononucleotide; P6C, Δ 1 -piperideine-6-carboxylic acid; PA, L-pipecolic acid; PDE, pyridoxine-dependent epilepsy; PLP, pyridoxal 5 0 -phosphate.

Insights into Aldehyde Dehydrogenase Enzymes: A Structural Perspective

“…The most common missense mutation in ALDH7A1 has been reported as E427G Coughlin et al (2018), occurring in 30% of PDE patients Mills et al (2006), Plecko et al (2007), Mills et al (2010, a mutational hotspot for disease. A study investigating structural changes of ALDH7A1, exhibiting mutations E427G, E427Q and E427D, demonstrated a catalytic defect and a non-natural conformation of the NAD cofactor and as a result no catalytic activity (Laciak et al, 2020). The NAD adopts a flexible conformation and lacks a defined pose for E427G and E427Q variants whereas an inactive pose is demonstrated for E427D, compromising catalysis due to lack of stabilisation and increased distance of the cofactor from active site Cys.…”

Case report: Fatal outcome of pyridoxine-dependent epilepsy presenting as respiratory distress followed by a circulatory collapse

“…The p.Glu427Gln variant has been referred to as a mutational hotspot of the disease ( 18 ) and the region of ALDH7A1, where this variant is located, is considered to be essential to protein folding and stability ( 19 ). In addition, other variants characterized by an amino acid change in the same position (pGlu427Gly) have been associated with the disease suggesting that a change in this position may not be tolerated ( 18 ).…”

Structural Insight into the Catalytic Mechanisms of an L‐Sorbosone Dehydrogenase

“…[ 20 , 21 ] Interestingly, the substrate channels in the ALDH subfamily are very different, giving them high substrate diversity. [ 20 , 22 ] Some studies have also found a physically linked state of ALDH and ADH domains that can form biologically active higher‐order spirosome structures. [ 23 ] At present, there is no report on the structure of the SNDH subfamily, and its catalytic mechanism and the substrate‐specific structural basis are still unknown.…”