br Conclusions br Introduction During numerous

Conclusions
Introduction During numerous physiological processes, aldehydes are generated from a variety of endogenous and exogenous precursors including amino acids, carbohydrates, and lipids [1]. Most aldehydes are highly reactive and cytotoxic due to the strong electrophilic nature of the terminal carbonyl groups [2]. In contrast to free radicals, aldehydes are comparatively long-lasting and can affect targets over a long distance via diffusion and transportation [3]. Although most aldehydes are transformed to less toxic aldehyde intermediates by various reductive and oxidative enzymes, including alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) [4], some of the resulting aldehyde intermediates also play important roles in vision, embryonic development, and neurotransmission [4]. Retinoic beta lactamase inhibitor (REA), a metabolite of vitamin A (retinol), is one of the aldehyde intermediates that is essential for a wide range of biological processes in vertebrates, including cell proliferation, differentiation, and morphogenesis [5], [6], [7]. The biosynthesis of REA from retinol is carried out by two enzymes. Retinal is first synthesized by the oxidation of retinol by retinol dehydrogenase, and is then converted to REA through oxidation by retinal dehydrogenase, which belongs to the ALDH family. REA is also one of the most important ingredients in cosmetics based on its anti-photoaging role in protecting the skin from the effects of UV radiation [8]. Our group has sought to develop a novel biocatalyst to produce REA from bacterial resources, which can be more amenable to industrial engineering procedures and conditions. Recently, we have cloned and characterized a novel bacterial ALDH from Bacillus cereus (BcALDH) that can oxidize all-trans-retinal to all-trans-REA using either NAD+ or NADP+ with equal efficiency and in addition atypically can reduce all-trans-retinal to all-trans-retinol using NADPH (Fig. 1b) [9]. ALDHs oxidize aldehyde substrates by reducing the cofactor NAD(P)+. The catalytic mechanism involves two major steps of acylation and deacylation [10]. ALDHs are classified into two different families based on the acyl acceptor used in the second catalytic step of deacylation: the phosphorylating ALDH family uses inorganic phosphate and the non-phosphorylating ALDH family uses water or the coenzyme A (CoA) molecule as an acyl acceptor. BcALDH belongs to the non-phosphorylating CoA-independent ALDH family [9]. In the first acylation step, the activated catalytic Cys, Cys300 in BcALDH, attacks the substrate aldehyde group to form a thio-hemiacetal intermediate [11] (Fig. 1a). The oxidized NAD(P)+ cofactor takes up a hydride ion from the thio-hemiacetal intermediate, which transforms it to a thioester intermediate [12]. This intermediate is then deacylated by a water molecule [13], in which the additional catalytic residue Glu, Glu266 in BcALDH, activates the hydrolytic water molecule by abstracting a proton [14]. Finally, the reduced NAD(P)H is released [15], [16]. In ALDH crystal structures, the nicotinamide moiety in NAD(P)+ cofactor can adopt diverse conformations such as extended, contracted, flipped, and enzyme surface-faced conformations and the conformational change has been proposed to be important in the catalytic mechanism [10], [17], [18], [19], [20], [21], [22], [23]. In contrast, the AMP moiety of the cofactor has a relatively conserved static conformation. In this study, we determined the crystal structure of BcALDH alone, and in complex with NAD+ and NADP+ cofactors. This is the first ALDH structure complexed with both NAD+ and NADP+ cofactors. The structures explain the flexible cofactor-binding pocket of BcALDH, which will provide useful structural information to understand the cofactor specificity of ALDHs and to develop a new biocatalyst of bacterial origin that can produce REA and related high-end products.
Materials and methods
Results
Discussion The enzymatic activities of human ALDHs have been well studied because of their important roles in development, neurotransmission, oxidative stress, and cancer [4]. Human ALDH2 (PDB ID: 4FR8) has small aldehydes as substrates and is involved in the oxidation of acetaldehyde during ethanol metabolism [27]. Human ALDH2 has a bulky Met124 residue at the mouth of its substrate-binding pocket, as one of the three signature residues for substrate specificity. Met124 has a steric clash with the β-ionone ring of REA (Fig. S4). In contrast, BcALDH has the smaller Ala123 residue at the same mouth position, and hence, there is no steric clash with REA. BcALDH also has the non-hydrophobic Glu457 residue in the neck (the middle) of the substrate pocket (Fig. 3c and Fig. S4). The E457V variant of BcALDH has a 6- and 7-fold higher activity (kcat/Km) in both the oxidizing and reduction reactions with NAD+ and NADPH, respectively [9]. A more hydrophobic substrate-binding pocket in BcALDH could therefore increase the binding affinity for retinal.