Disruption of the coenzyme binding site and dimer interface revealed in the crystal structure of mitochondrial aldehyde dehydrogenase "Asian" variant

Abstract

Mitochondrial aldehyde dehydrogenase (ALDH2) is the major enzyme that oxidizes ethanol-derived acetaldehyde. A nearly inactive form of the enzyme, ALDH2*2, is found in about 40% of the East Asian population. This variant enzyme is defined by a glutamate to lysine substitution at residue 487 located within the oligomerization domain. ALDH2*2 has an increased Km for its coenzyme, NAD+, and a decreased kcat, which lead to low activity in vivo. Here we report the 2.1 A crystal structure of ALDH2*2. The structure shows a large disordered region located at the dimer interface that includes much of the coenzyme binding cleft and a loop of residues that form the base of the active site. As a consequence of these structural changes, the variant enzyme exhibits rigid body rotations of its catalytic and coenzyme-binding domains relative to the oligomerization domain. These structural perturbations are the direct result of the inability of lysine 487 to form important stabilizing hydrogen bonds with arginines 264 and 475. Thus, the elevated Km for coenzyme exhibited by this variant probably reflects the energetic penalty for reestablishing this site for productive coenzyme binding, whereas the structural alterations near the active site are consistent with the lowered Vmax.

Figure 1

The ALDH2*2 homotetramer. Blue and violet subunits make one dimer pair (subunits A and B, respectively) and gold and green the other (subunits C and D, respectively). The remaining two tetramers in the asymmetric unit are comprised of subunits E–H and I–L.

Figure 2

A single subunit of ALDH2: a) Wild-type subunit with its three major domains labeled. The secondary structure features are color coded: navy blue, αF; gold, β10; red, αG; green, β11; orange, loop at residues 269–273; violet, loop at residues 463–478. Residues 264, 487, and 475, shown in space-filled representation, are labeled and colored green, pink, and violet, respectively. The catalytic nucleophile, C302, is labeled and denoted by black ribbon. b) An alignment of ALDH2*2 subunits A and B to wild type. In this dimer representation, the ALDH2*2 αG helix is shown in red, wild type in blue. The helix of ALDH2*2 shifts 3 Å into the NAD⁺-binding cleft. Residues 264, 487, and 475 from the wild-type structure are colored as in Figure 2a.

Figure 3

2F_O−F_C electron density maps contoured at one standard deviation of the map: a) For residue K487, nearly all atoms are well ordered with N_ζ and C_ɛ being the exceptions in some subunits. b) The β10 strand and αG helix. The electron density maps taper at the C-terminal end of the β10 strand. For eleven of the twelve subunits observed, there is no interpretable density for most of the αG helix.

Figure 4

A stereo diagram of side-chain and main-chain shifts from the dimer interface to the NAD⁺-binding cleft. NAD⁺-bound wild type (violet, subunit A; blue, subunit B) and apoenzyme ALDH2*2 (red) are aligned.

Figure 5

The ALDH2*2 enzyme (red) aligned to wild-type (blue). The view shown is rotated about 90° with respect to Figure 2: a) Monomer view—alignment of the coenzyme-binding and catalytic domains. The 2.5º rotation between the coenzyme-binding and oligomerization domains is illustrated by the shift in the oligomerization domain. b) Dimer view—alignment of the oligomerization domains. The 2.5º rotation causes the coenzyme-binding and catalytic domains to fall away from the interface at the αG helices.