Structural Basis of Substrate Recognition by Aldehyde Dehydrogenase 7A1

Abstract

Aldehyde dehydrogenase 7A1 (ALDH7A1) is part of lysine catabolism and catalyzes the NAD(+)-dependent oxidation of α-aminoadipate semialdehyde to α-aminoadipate. Herein, we describe a structural study of human ALDH7A1 focused on substrate recognition. Five crystal structures and small-angle X-ray scattering data are reported, including the first crystal structure of any ALDH7 family member complexed with α-aminoadipate. The product binds with the ε-carboxylate in the oxyanion hole, the aliphatic chain packed into an aromatic box, and the distal end of the product anchored by electrostatic interactions with five conserved residues. This binding mode resembles that of glutamate bound to the proline catabolic enzyme ALDH4A1. Analysis of ALDH7A1 and ALDH4A1 structures suggests key interactions that underlie substrate discrimination. Structures of apo ALDH7A1 reveal dramatic conformational differences from the product complex. Product binding is associated with a 16 Å movement of the C-terminus into the active site, which stabilizes the active conformation of the aldehyde substrate anchor loop. The fact that the C-terminus is part of the active site was hitherto unknown. Interestingly, the C-terminus and aldehyde anchor loop are disordered in a new tetragonal crystal form of the apoenzyme, implying that these parts of the enzyme are highly flexible. Our results suggest that the active site of ALDH7A1 is disassembled when the aldehyde site is vacant, and the C-terminus is a mobile element that forms quaternary structural interactions that aid aldehyde binding. These results are relevant to the c.1512delG genetic deletion associated with pyridoxine-dependent epilepsy, which alters the C-terminus of ALDH7A1.

Figure 1

Reactions related to ALDH7A1 and PDE. (A) Reaction catalyzed by ALDH7A1. (B) Inactivation of pyridoxal 5′-phosphate by Δ¹-piperideine-6-carboxylic acid.

Figure 2

Structure of ALDH7A1. (A) Protomer structure of ALDH7A1 highlighting domain architecture. The three domains are colored red (NAD⁺-binding), blue (catalytic), and green (oligomerization). One of the polypeptide sections that links the catalytic and NAD⁺-binding domains is colored gold. AA is colored pink. (B) Three orthogonal views of the ALDH7A1 dimer-of-dimers tetramer. One protomer is colored according to domains as in panel A, while the other three protomers each have a different color (gray, pink, or wheat). The three orientations correspond to viewing along the three mutually perpendicular 2-fold axes of the tetramer, which are labeled P, Q, and R.

Figure 3

SAXS analysis of ALDH7A1. (A) Comparison of the experimental SAXS curve with those calculated from ALDH7A1 oligomers using FoXS. The fits have χ values of 1.5 for the tetramer, 12.4 for the dimer, and 1.2 for the 96%/4% tetramer/dimer ensemble. The inset shows a Guinier plot, which spans the qR_g range of 0.356–1.273. (B) Superposition of the ALDH7A1 tetramer with the envelope from shape reconstruction calculations.

Figure 4

Electron density and interactions of AA bound to ALDH7A1. The mesh represents a simulated annealing F₀ – F_c omit map contoured at 2.5σ.

Figure 5

Conformational variation of the C-terminus. (A) Dimer of ALDH7A1 complexed with AA (pink). The domains are colored as in Figure 2A, with the NAD⁺-binding domain colored red, the catalytic domain blue, and the oligomerization domain green. Gln506 is shown as sticks. (B) Close-up of the quaternary structural interactions that stabilize the aldehyde-binding site in the AA complex. One protomer of the dimer is colored white with bound AA colored pink. The C-terminus of the other protomer is colored green. (C) Superposition of the dimers of the ALDH7A1–AA complex (gray with a cyan C-terminus), apo ALDH7A1 in space group C2 (gray with a red C-terminus), and the ALDH7A1–NAD⁺ complex in space group C2 (also gray with a red C-terminus). The arrow indicates the 16 Å movement of the C-terminus from the open conformation (AA-free) to the closed conformation (AA-bound). (D) Close-up of a superposition of the AA complex (cyan) and the apoenzyme in space group C2 (gold). The arrows indicate movement of the active site from the open conformation (AA-free) to the closed conformation (AA-bound). AA is colored pink.

Figure 6

Crystal packing of ALDH7A1 in P42₁2. The lattice is viewed down the a-axis, with the b-axis horizontal and c-axis vertical. The two chains of the asymmetric unit are colored gold and blue. Neighboring chains are colored pale cyan. The C-terminus from the C2 structure (open conformation) has been grafted onto the P42₁2 structure and is colored red. Note that the modeled C-terminus occupies a solvent channel.

Figure 7

Comparison of substrate recognition in ALDH7A1 and ALDH4A1. (A) Superposition of ALDH7A1 complexed with AA (white) and ALDH4A1 complexed with Glu (green). Black dashes indicate interactions that are unique to ALDH7A1. Magenta dashes denote those unique to ALDH4A1. (B) Substrate interaction diagrams for ALDH7A1 and ALDH4A1 inferred from the enzyme–product complex structures. Dashes denote hydrogen bonds and ion pairs. Squares denote van der Waals interactions between aromatic box residues and the aliphatic chain of the substrate. Interactions unique to either enzyme are colored red. W in a circle represents water-mediated interactions.

Figure 8

Sequence alignment of the C-termini of wild-type ALDH7A1 and the c.1512delG deletion mutant, which has been implicated in PDE.