Kinetic Analysis of Human Enzyme RDH10 Defines the Characteristics of a Physiologically Relevant Retinol Dehydrogenase

Abstract

Human retinol dehydrogenase 10 (RDH10) was implicated in the oxidation of all-trans-retinol for biosynthesis of all-transretinoic acid, however, initial assays suggested that RDH10 prefers NADP ؉ as a cofactor, undermining its role as an oxidative enzyme. Here, we present evidence that RDH10 is, in fact, a strictly NAD ؉ -dependent enzyme with multisubstrate specificity that recognizes cis-retinols as well as all-trans-retinol as substrates. RDH10 has a relatively high apparent K m value for NAD ؉ (ϳ100 M) but the lowest apparent K m value for alltrans-retinol (ϳ0.035 M) among all NAD ؉ -dependent retinoid oxidoreductases. Due to its high affinity for all-trans-retinol, RDH10 exhibits a greater rate of retinol oxidation in the presence of cellular retinol-binding protein type I (CRBPI) than human microsomal RoDH4, but like RoDH4, RDH10 does not recognize retinol bound to CRBPI as a substrate. Consistent with its preference for NAD ؉ , RDH10 functions exclusively in the oxidative direction in the cells, increasing the levels of retinaldehyde and retinoic acid. Targeted small interfering RNAmediated silencing of endogenous RDH10 or RoDH4 expression in human cells results in a significant decrease in retinoic acid production from retinol, identifying both human enzymes as physiologically relevant retinol dehydrogenases. The dual cis/ trans substrate specificity suggests a dual physiological role for RDH10: in the biosynthesis of 11-cis-retinaldehyde for vision as well as the biosynthesis of all-trans-retinoic acid for differentiation and development.Retinoic acid is a small lipophilic molecule derived from vitamin A that regulates gene expression through binding to nuclear transcription factors, retinoic acid receptors (1). The oxidation of retinol to retinaldehyde is the rate-limiting step in retinoic acid biosynthesis (2); however, the identity of the physiologically relevant retinol dehydrogenase(s) has long been controversial. Two types of enzymes have been implicated in the oxidation of retinol: cytosolic alcohol dehydrogenases (ADH) 2 of the medium-chain alcohol dehydrogenase superfamily and microsomal dehydrogenases (RoDH) of the short-chain dehydrogenase/reductase (SDR) superfamily (reviewed in Ref.3). Several criteria have been suggested for evaluation of the candidate retinoid oxidoreductases. First of all, it was proposed that to function in the oxidative direction in vivo, the enzyme has to prefer NAD ϩ as a cofactor (reviewed in Ref. 4), because unlike NADP ϩ , NAD ϩ exists mostly in the oxidized form in the majority of cells (5). Second, it was argued that the physiologically relevant retinol dehydrogenase must recognize retinol bound to cellular retinol-binding protein type I (holoCRBPI) as a substrate, because holoCRBPI is the predominant form of retinol in the cells (2). Finally, the enzyme essential for the oxidation of retinol to retinaldehyde was expected to be expressed in the "hot spots" of retinoic acid biosynthesis during various stages of development and be relatively well conserved to supp...

Enzymology of retinoic acid biosynthesis and degradation

“…Biochemical analysis carried out in our laboratory revealed that human ortholog of RDH10 (SDR16C4) recognizes not only all-trans-retinol but also 11-cis and 9-cis retinols as substrates and strongly prefers NAD + as a cofactor, in agreement with the structural determinants of SDR cofactor specifi city ( 47 ). This fi nding contradicted the previous report that human RDH10 has a signifi cant activity with NADP + ( 43 ).…”

“…In comparison with SDR9C all-trans -retinol/sterol dehydrogenases, human RDH10 is not active toward 3 ␣ -hydroxysteroids, and it exhibits a much higher affi nity for all-trans -retinol ( ‫ف‬ 28-fold) than does either RoDH4 or RL-HSD ( 47 ). When overexpressed in a model of human organotypic skin raft culture, only RDH10 [not RoDH4, RL-HSD, or DHRS9 (RDHL)] induces a phenotype consistent with overproduction of atRA, which is characterized by an increased proliferation and reduced differentiation of keratinocytes ( 48 ).…”

Conditional Ablation of Retinol Dehydrogenase 10 in the Retinal Pigmented Epithelium Causes Delayed Dark Adaption in Mice

“…RDH10 was found to be a strictly NAD ϩ -specific enzyme with dual specificity for trans-and cis-retinol, consistent with earlier findings (18). In cells, the concentration of NAD ϩ is higher than that of NADP ϩ , which could cause Rdh10 to evolve its strict specificity toward NAD ϩ (29,30).…”

“…Wu et al (13) reported that RDH10 is an efficient all-trans-RDH with NADP ϩ as its preferred cofactor. However, Belyaeva et al (18) demonstrated that RDH10 is an efficient 11-cis-RDH with NAD ϩ as its preferred cofactor. The in vivo role of RDH10 in the visual cycle has not been reported.…”

“…In cells, the concentration of NAD ϩ is higher than that of NADP ϩ , which could cause Rdh10 to evolve its strict specificity toward NAD ϩ (29,30). Through homology modeling, Belyaeva et al (18) proposed a steric hindrance for NADP ϩ but not NAD ϩ in the cofactor-binding pocket of Rdh10. Although RDH10 catalyzed the conversion of 11-cis-retinol to 11-cis-retinal, its specific activity with both all-trans-retinol and 9-cisretinol was higher.…”