Characterization of key residues and membrane association domains in retinol dehydrogenase 10.

It is well known that retinoid (vitamin A derivatives) metabolism is essential not only for normal vision but also for embryonic development and growth. A number of enzymes, retinoid-binding proteins and transporters are involved in modification, conversion and circulation of retinoids [1–3]. Vitamin A (all-trans retinol, atROL) is bound by retinol-binding protein (RBP) and transported from the liver to ocular tissues and non-ocular tissues. 11-cis retinal (11cRAL) is covalently bound to opsin molecule and served as chromophore for rod and cone visual pigments. A single photon isomerizes 11cRAL to all-trans retinal (atRAL), which triggers the activation of the phototransduction cascade and generates vision [4, 5]. Subsequently, 11cRAL is regenerated in the retinal pigment epithelium (RPE) through the retinoid visual cycle which involves a number of enzymes and retinoid binding proteins [6, 7]. An intact visual cycle is essential for normal vision [8]. Retinoid metabolism is also essential for the generation of retinoic acid which plays an important role in regulating development and cell differentiation [1, 2]. In certain tissues, atROL is first oxidized to atRAL which is then oxidized to all-trans retinoic acid (atRA) by a known enzyme, retinaldehyde dehydrogenase (Raldh). AtRA is known to regulate cell differentiation, development and morphogenesis through the retinoic acid receptors or retinoid X receptors [1–3]. Retinol dehydrogenases (RDH) are the enzymes to catalyze oxidation of retinol into retinal or reduction of retinal into retinol in a cofactor dependent manner in both vision and retinoid signaling. The RDH activity is observed in two distinct classes of enzymes, microsomal retinol dehydrogenases (RoDH) in the short-chain dehydrogenase/reductase (SDR) family and cytosolic alcohol dehydrogenases (ADH) in the medium-chain dehydrogenase/reductase (MDR) family [9–15]. More than 20,000 sequences (including species variants) of SDR have been deposited in the database [15]. A number of studies have been carried out to identify the functionally important residues among the highly conserved residues in the family by evaluating the impacts of mutations of the residues using site-directed mutagenesis [9, 16–23] and crystal structure analyses of the SDR family members such as drosophila alcohol dehydrogenase (DADH [24]), human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD-1 [25]) and bacterial 3β/17β-HSD [26]. Most of the mutations of the conserved residues abolish their enzymatic activities [9, 16–23]. These studies elucidated that the highly conserved residues in SDRs are the key residues and suggested that a triad of Ser-Tyr-Lys residues form the catalytic site, in which Tyr functions as the catalytic base, whereas Ser stabilizes the substrate, and Lys interacts with cofactor and lowers the pKa of the Tyr-OH. The conserved Asn residue interacts with the active site Lys via a water molecule to form a tetrad Asn-Ser-Tyr-Lys [10, 17, 27]. Although the SDR family members share low (approximately 15–30%) sequence identities, functionally important residues, such as catalytic sites (Asn-Ser-Tyr-Lys) and cofactor-binding motifs (Gly-X3-Gly-X-Gly), are highly conserved in the SDR family [9–11, 17]. The available 3-D structures of SDRs have revealed relatively similar α/β folding patterns of a Rossmann-fold [9, 10, 24, 25, 27]. Likewise, a number of studies had been carried out to investigate cofactor specificities of the SDR family. The residues between the first β-sheet and the third α-helix of SDRs are potentially important determinants of the specificity of cofactor toward either NADP(H) or NAD(H) [9, 19, 24, 28–34]. Further, RoDHs (membrane-bound SDR) have been shown to possess hydrophobic domains at the N-terminal and/or C-terminal regions, which may anchor the proteins into the membrane [11, 35–39]. RDH10 (retinol dehydrogenase class 10) was first cloned from bovine, mouse and human RPE [40]. The amino acid sequence homologies among RDH10 from different species are exceptionally high, with a 100% identity between bovine and human RDH10 and a 99% identity between mouse and human RDH10 at the amino acid level [40]. The highly conserved sequence of RDH10 across the evolution suggests its functional significance. We also showed that RDH10 is predominantly localized in the microsomal fraction, similar to other membrane-bound RDHs [40]. A recent study showed that RDH10 is highly expressed in many other non-ocular tissues during forelimb and hindlimb differentiation, whereas other RDHs (RDH1, 5, 6, 7 and 11) have significantly lower expression than RDH10 at this stage [41]. Moreover, mice carrying a miss-sense mutation of RDH10 have an embryonic lethal phenotype, indicating an important role of RDH10 in the early embryonic development [42]. Furthermore, the lethal phenotype of this RDH10 mutant mouse can be rescued by RA treatment of the pregnant mother, suggesting that RDH10 is essential for RA generation at early stages of embryonic development [42]. Although its exact function in RA generation is uncertain, RDH10 is likely to function as the enzyme to convert atROL to atRAL which is the substrate for Raldh2 to synthesize RA [2, 11]. This assumption is supported by a recent study showing that RDH10 and Raldh colocalize in tissues during embryogenesis and organ differentiation [43]. To further study the structure and function of this important enzyme, the present study has characterized the conserved key residues in the predicted catalytic center, cofactor-binding site, and membrane association domain in RDH10.