Atypically BcALDH has a minor

Atypically, BcALDH has a minor reducing activity for all-trans-retinal, which requires the binding of the reduced form of the NADP+ (NADPH) cofactor that is a side-product of the canonical oxidation activity of BcALDH. The reduction activity that converts all-trans-retinal to all-trans-retinol was rarely studied, even in mammalian ALDHs. The bidirectional oxidation and reduction reactions in BcALDH are not reversible reactions that convert a substrate to a product, and vice-versa, at equilibrium. Instead, BcALDH converts the same substrate retinal into either REA through oxidation using NAD(P)+ or retinol through reduction using NADPH (Fig. 1b). The oxidation of all-trans-retinal is the main reaction catalyzed by ALDH, whereas the reduction of all-trans-retinal to all-trans-retinol is the backward activity of the canonical oxidation reaction of alcohol dehydrogenase (ADH), not ALDH. The simplest plausible mechanism for the atypical reduction activity of BcALDH is the backwards mechanism of ADH’s oxidation reaction, which is the direct hydride transfer from the reduced nicotinamide of NADPH to the aldehyde of all-trans-retinal followed by proton uptake by the aldehyde group from a nearby sta 90 australia (Fig. 1a). For the simplest plausible mechanism, two essential requirements should be satisfied. First, the reduced NADPH cofactor should be able to access the substrate aldehyde group in the active site to deliver a hydride ion before the substrate aldehyde group is converted to the thiohemiacetal intermediate through Cys300 in the canonical oxidative reaction. Second, the proton donor should be close to the substrate aldehyde group to change it to the alcohol group. In the active site, the closest acid is the conserved catalytic residue Glu266 (Fig. 5). The E266A variant of BcALDH has a reduction in catalytic activity of more than 12-fold [9], which supports the notion that an acidic residue near the substrate aldehyde binding site is important for the atypical reduction activity and further that Glu266 may be a candidate catalytic acid. In order to confirm this hypothesis of the atypical reducing activity, the structure of BcALDH in complex with both NADPH and retinal is critical. In this study, we determined the crystal structures of the bacterial retinal-converting enzyme, BcALDH, that has a bidirectional oxidation and reduction activity for all-trans-retinal using NAD(P)+ and NADPH, respectively. The structures showed the tight but flexible cofactor-binding site that allows both NAD+ and NADP+ binding and there is a close relationship between the cofactor-binding and substrate-binding sites. This structure should be useful in developing a new biocatalyst to produce REA or other related high-end products.
PDB references The atomic coordinates and structural factors for the reported crystal structures have been deposited with the Protein Data Bank under the accession codes 4PS9 (apo), 4PT0 (NAD+-bound), and 4PT3 (NADP+-bound).
Conflict of interest
Acknowledgements We are grateful to the staff members of Beamline 5C at the Pohang Accelerator Laboratory (PAL), Republic of Korea. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2015R1A2A2A01004375), by “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01103101)” Rural Development Administration, Republic of Korea, and by WTU Joint Research Grants of Konkuk University.
Introduction Aldehyde dehydrogenases (ALDHs), broadly defined as a superfamily of NADP(+)-dependent enzymes, participate in aldehyde metabolism, catalysing the oxidation of exogenous aldehydes (drugs and ethanol) and endogenous aldehydes (lipid, amino acids, or vitamins) into their corresponding carboxylic acids [1]. The primary toxicity of aldehydes can induce enzyme inactivation, DNA damage, impaired cellular homeostasis and even cell death by forming adducts with various cellular targets including glutathione, nucleic acids and amino acids [2], [3]. Deficiency and polymorphisms of ALDHs in organisms are related to diseases such as Parkinson's disease, Type II hyperprolinaemia, hypertension and Sjögren–Larsson syndrome, and may even contribute to the occurrence of carcinoma [4], [5], [6], [7], [8]. Numerous studies have indicated that tumours with high malignancy have high levels of ALDHs [9], [10].