Crystal structure of aldehyde dehydrogenase from Bacillus cereus and its atypical bidirectional oxidizing and reducing activities for all-trans-retinal

Aldehyde dehydrogenases (ALDHs) are NAD(P)⁺-dependent oxidoreductases that catalyze the oxidation of a variety of aldehydes to their acid forms. ALDH from Bacillus cereus (BcALDH) oxidizes all-trans-retinal to all-trans-retinoic acid with NAD⁺ and NADP⁺. The enzyme produced 208 μM all-trans-retinoic acid from 400 μM all-trans-retinal in the presence of 5 mM NAD⁺ for 240 min, with a molar conversion yield of 52% and a volumetric productivity of 35 μM/h. In addition, BcALDH atypically reduces all-trans-retinal to all-trans-retinol with NADPH. The crystal structures of BcALDH were determined alone and in complex with NAD⁺ or NADPH. Both NAD⁺ and NADPH cofactors were in the contracted conformation. In the NADPH-bound structure, the ADP-binding pocket became wider mainly due to the bulkier size of the additional 2′-phosphate attached to the ADP-ribose, and the steric interaction pushed up the ADP part by 1.1 Å compared with that of NAD⁺. E366A and C300A mutants of BcALDH lost the canonical oxidation activity for the all-trans-retinal. However, they still showed the reduction activity. E194S mutant, having a wider ADP-binding pocket than wild-type, showed the 15-fold higher reducing catalytic efficiency (k_cat/K_m) with NADPH. E457V mutant, having a complete hydrophobic substrate channel for the better binding of all-trans-retinal, showed 6-fold higher activities for both oxidation and reduction reactions.