NAD+ promotes assembly of the active tetramer of aldehyde dehydrogenase 7A1

Abstract

Nicotinamide adenine dinucleotide (NAD) is the redox cofactor of many enzymes, including the vast aldehyde dehydrogenase (ALDH) superfamily. Although the function of NAD(H) in hydride transfer is established, its influence on protein structure is less understood. Herein, we show that NAD⁺ -binding promotes assembly of the ALDH7A1 tetramer. Multiangle light scattering, small-angle X-ray scattering, and sedimentation velocity all show a pronounced shift of the dimer-tetramer equilibrium toward the tetramer when NAD⁺ is present. Furthermore, electron microscopy shows that cofactor binding enhances tetramer formation even at the low enzyme concentration used in activity assays, suggesting the tetramer is the active species. Altogether, our results suggest that the catalytically active oligomer of ALDH7A1 is assembled on demand in response to cofactor availability.

Keywords: aldehyde dehydrogenase; analytical ultracentrifugation; electron microscopy; enzyme oligomerization; nicotinamide adenine dinucleotide; small-angle X-ray scattering.

Fig. 1.

Reactions related to ALDH7A1 biology. ALDH7A1 catalyzes the oxidation of α-aminoadipate semialdehyde to α-aminoadipate, which is in non-enzymatic equilibrium with L-Δ¹-piperideine-6-carboxylate.

Fig. 2.

SEC-SAXS analysis of ALDH7A1 in the presence and absence of NAD⁺. Each panel shows SEC-SAXS experimental data (open circles) and the corresponding FoXS single-body fit to the ALDH7A1 crystallographic tetramer (PDB ID: 4ZUK; cyan curve) or the MultiFoXS two-body ensemble fit to a theoretical mixture of ALDH7A1 crystallographic dimer and tetramer (PDB ID: 4ZUK; red curve). The top three panels display three increasing nominal concentrations (from left to right) of apo ALDH7A1. The bottom three panels display three increasing nominal concentrations (from left to right) of ALDH7A1 in the presence of 1 mM NAD⁺. The complete experimental SAXS data and Guinier plots are included in Fig. S2 for reference.

Fig. 3.

Sedimentation velocity analysis of ALDH7A1 in the absence (black) and presence (red) of NAD⁺. (A) The distribution of sedimentation coefficients, c(s). (B) The distribution of molecular masses, c(M). In both panels, the black curve depicts apo ALDH7A1 at 9 μM (dimer M_r), and the red curve depicts ALDH7A1 in the presence of 1 mM NAD⁺ at 9 μM (dimer M_r).

Fig. 4.

Negative-Stain EM of ALDH7A1 in the presence and absence of NAD⁺. (A) The crystal structure of ALDH7A1 (PDB ID: 4ZUK) shown as a ribbon in red with an overlaid envelope representing a 20 Å resolution view of both the dimer and tetramer. The 20 Å resolution envelope was generated using Chimera [49]. (B) Two-dimensional class averages for apo ALDH7A1 at 90 nM. (C) Two-dimensional class averages for 90 nM ALDH7A1 in the presence of 1 mM NAD⁺. For panels B and C, the letters ‘D’ or ‘T’ in the inset of each class average indicate classes that visually resemble the dimer or tetramer, respectively. Classes that lack a letter in the inset were not clearly dimeric or tetrameric.