Importance of the C-Terminus of Aldehyde Dehydrogenase 7A1 for Oligomerization and Catalytic Activity

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.

Figure 1

Chemical reactions relevant to ALDH7A1 and PDE. (A) Reaction catalyzed by ALDH7A1. (B) Reaction between P6C and PLP, which results in covalent inactivation of PLP. P6C forms a nonenzymatic equilibrium with AASAL.

Figure 2

Structural and genetic contexts of the mutations studied. (A) Protomer of ALDH7A1 with the mutated residues Ala505 and Gln506 marked with spheres (PDB entry 4ZUL). The three domains are colored red (NAD⁺-binding), blue (catalytic), and green (oligomerization). The residues deleted in truncation mutant Δ504–511 are colored gold. The product AA is colored pink. (B) Superposition of the open and closed ALDH7A1 dimers [PDB entries 4ZUK (open) and 4ZUL (closed)]. The domains are colored as in panel A. The arrows indicate the 16 Å motion of the C-terminus from the open (out) to the closed (in) state. (C) Dimer-of-dimers tetramer of ALDH7A1. One protomer is colored according to domains as in panel A. The other three protomers have individual colors (aquamarine, gray, and violet). (D) Close-up of the quaternary structural interactions that stabilize the aldehyde-binding site in the closed state (AA complex, PDB entry 4ZUL). One protomer of the dimer is colored white, with bound AA colored pink. The C-terminus of the other protomer is colored gold. Note the interactions of Ala505 and Gln506 with the active site. This panel also includes a sequence alignment of the C-termini of wild-type ALDH7A1 and the c.1512delG deletion mutant, which has been implicated in PDE.

Figure 3

Steady-state kinetics of wild-type and mutant ALDH7A1 using AASAL as the variable substrate at a fixed NAD⁺ concentration of 2.5 mM (26 °C, pH 8.0). (A) Initial velocity data for wild-type ALDH7A1 (black) and A505P (red). The curves are fits to the Michaelis–Menten equation. (B) Initial velocity data for Q506K (blue). The line was calculated from linear regression. The data for wild-type ALDH7A1 are shown for comparison. (C) Initial velocity data for A505P/Q506K (purple). The line was calculated from linear regression. The data for wild-type ALDH7A1 are shown for comparison. (D) Initial velocity data for Δ504–511 (green). The line was calculated from linear regression. The data for wild-type ALDH7A1 are shown for comparison.

Figure 4

Sedimentation equilibrium analysis of wild-type and mutant ALDH7A1. For each protein, the three panels show three different protein concentrations: 0.2 mg mL⁻¹ (left), 0.4 mg mL⁻¹ (middle), and 0.8 mg mL⁻¹ (right). Within each graph, the three data sets correspond to centrifugation speeds of 6000 (squares), 9000 (circles), and 12000 (triangles) rpm. The red curves for wild-type ALDH7A1 represent a global fit of the data to a previously described dimer–tetramer oligomerization equilibrium model. The red curves for the mutant enzymes are from global fits to a single-species model.

Figure 5

Sedimentation velocity analysis of wild-type ALDH7A1 and its C-terminal point mutants. (A) Apparent sedimentation coefficient distributions for wild-type ALDH7A1 (dashed black), A505P (red), Q506K (blue), or A505P/Q506K (purple). (B) Apparent M_r distributions for wild-type ALDH7A1 (dashed black), A505P (red), Q506K (blue), or A505P/Q506K (purple). All enzymes were used at 4.5 mg mL⁻¹ (40 μM using dimer M_r).

Figure 6

Concentration dependence of ALDH7A1 Δ504–511 self-association. Sedimentation velocity analysis was conducted on Δ504–511 at the indicated concentrations (8–93 μM). Concentrations were determined using the dimer M_r (109 kDa). Each panel shows the distribution of apparent molecular masses in solution at the indicated concentration. For reference and ease in identifying the peak corresponding to the ALDH7A1 tetramer, the black dashed curve shows the distribution of molecular masses of wild-type ALDH7A1 at 40 μM.

Figure 7

High-concentration sedimentation equilibrium analysis of ALDH7A1 Δ504–511. Sedimentation equilibrium analysis was conducted on ALDH7A1 Δ504–511 using Raleigh Interference optics at 2 mg mL⁻¹ (left panel), 4 mg mL⁻¹ (center panel), and 8 mg mL⁻¹ (right panel) at three rotor speeds: 6000 (squares), 9000 (circles), and 12000 (triangles) rpm. The red curves represent global fits to a dimer–tetramer equilibrium model. We note that the center and right panels are missing data from faster rotor speeds due to the limits of detection at the apparent concentrations within the sedimentation cell. Data were graphed and analyzed in Origin 2017.

Figure 8

SAXS analysis of ALDH7A1 Δ504–511. (A) Experimental SAXS curves for ALDH7A1 Δ504–511 (○) at four concentrations: 1, 2, 4, and 6 mg mL⁻¹ (from bottom to top, respectively) (9–55 μM using dimer M_r). The theoretical SAXS curves were calculated using MultiFoXS from a two-body ensemble consisting of the domain-swapped dimer and the tetramer of ALDH7A1 (PDB entry 4ZUK) lacking the eight terminal residues. The χ values from MultiFoXS are listed. An arbitrary scale factor has been applied for curve separation. The inset shows Guinier plots. (B) Analysis of the SAXS data from panel A using a model of the trimer of Δ504–511. The blue and green curves show the two-body fit obtained with a dimer–trimer ensemble. The orange and red curves show the single-body fit obtained with a trimer. (C) Experimental distance distribution functions calculated with GNOM via PRIMUS.