Catalytic contribution of threonine 244 in human ALDH2

Abstract

Amongst the numerous conserved residues in the aldehyde dehydrogenase superfamily, the precise role of Thr-244 remains enigmatic. Crystal structures show that this residue lies at the interface between the coenzyme-binding and substrate-binding sites with the side chain methyl substituent oriented toward the B-face of the nicotinamide ring of the NAD(P)(+) coenzyme, when in position for hydride transfer. Site-directed mutagenesis in ALDH1A1 and GAPN has suggested a role for Thr-244 in stabilizing the nicotinamide ring for efficient hydride transfer. Additionally, these studies also revealed a negative effect on cofactor binding which is not fully explained by the interaction with the nicotinamide ring. However, it is suggestive that Thr-244 immediately precedes helix αG, which forms one-half of the primary binding interface for the coenzyme. Hence, in order to more fully investigate the role of this highly conserved residue, we generated valine, alanine, glycine and serine substitutions for Thr-244 in human ALDH2. All four substituted enzymes exhibited reduced catalytic efficiency toward substrate and coenzyme. We also determined the crystal structure of the T244A enzyme in the absence and presence of coenzyme. In the apo-enzyme, the alpha G helix, which is key to NAD binding, exhibits increased temperature factors accompanied by a small displacement toward the active site cysteine. This structural perturbation was reversed in the coenzyme-bound complex. Our studies confirm a role for the Thr-244 beta methyl in the accurate positioning of the nicotinamide ring for efficient catalysis. We also identify a new role for Thr-244 in the stabilization of the N-terminal end of helix αG. This suggests that Thr-244, although less critical than Glu-487, is also an important contributor toward coenzyme binding.

Fig. 1

NAD binding in the ALDH superfamily. (A) ALDH2 tetramer with helices αF and αG shown as ribbons and NAD as vdw spheres (PDB code 1o02). (B) Stereo view of the cofactor conformations observed in the wild-type ALDH2-NAD complex (PDB code 1o00), in the E268A GAPN-NADP complex (PDB code 2esd, ref. 16), and the T244S GAPN-NADP complex (PDB code 2id2, ref. 19). Legend refers to color of nicotinamide ring, surface representation shown for 1o00 only. All molecular figures were generated using PyMol [38].

Fig. 2

Stereo views of the active site of ALDH2 with butyraldehyde modeled in (A) PDB code 1o04 with a transparent vdw surface shown for Thr-244 Cγ2. (B) PDB code 1o01 (absent non-covalent crotonaldehyde). Alternate structures with Glu in “down” position: Apo wt 1o05 (subunits BCEFH) and SM-NAD wt 1cw3 (subunits ADEH).

Fig. 3

Stereo views of the interactions N- and C-terminal to αG. (A) Apo T244A and wild-type ALDH2 (PDB codes 3n81 and 3n80) (B) NADH complexes of T244A and wild-type (PDB codes 3n82 and 1o02). (C) NAD complexes of T244A and C302S ALDH2 (PDB codes 3n83 and 1o04). 2F_o-F_c electron density maps contoured at 1.0 σ. Legend refers to color of the electron density (hash-mark color) and corresponding model (text color).

Fig. 4

Relative variation in average B-factor as a function of residue number. Delta-B values were calculated as the difference between the refined values in each of the T244A structures and the refined values in the wild-type apo ALDH2 structure, after baseline correction using the overall B for each structure.

Scheme 1

General mechanism for the dehydrogenase reaction catalyzed by ALDH.