Abstract
Aldehyde dehydrogenase 7A1 (ALDH7A1) catalyzes the terminal step of lysine catabolism, the NAD+-dependent oxidation of α-aminoadipate semialdehyde to α-aminoadipate. Structures of ALDH7A1 reveal the C-terminus is a gate that opens and closes in response to the binding of α-aminoadipate. In the closed state, the C-terminus of one protomer stabilizes the active site of the neighboring protomer in the dimer-of-dimers tetramer. Specifically, Ala505 and Gln506 interact with the conserved aldehyde anchor loop structure in the closed state. The apparent involvement of these residues in catalysis is significant because they are replaced by Pro505 and Lys506 in a genetic deletion (c.1512delG) that causes pyridoxine-dependent epilepsy. Inspired by the c.1512delG defect, we generated variant proteins harboring either A505P, Q506K, or both mutations (A505P/Q506K). Additionally, a C-terminal truncation mutant lacking the last eight residues was prepared. The catalytic behaviors of the variants were examined in steady-state kinetic assays, and their quaternary structures were examined by analytical ultracentrifugation. The mutant enzymes exhibit a profound kinetic defect characterized by markedly elevated Michaelis constants for α-aminoadipate semialdehyde, suggesting that the mutated residues are important for substrate binding. Furthermore, analyses of the in-solution oligomeric states revealed that the mutant enzymes are defective in tetramer formation. Overall, these results suggest that the C-terminus of ALDH7A1 is crucial for the maintenance of both the oligomeric state and the catalytic activity.