Kinetic analysis of human enzyme RDH10 defines the characteristics of a physiologically relevant retinol dehydrogenase

Human retinol dehydrogenase 10 (RDH10) was implicated in the oxidation of all-trans-retinol for biosynthesis of all-trans-retinoic 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 Km value for NAD(+) (similar to 100 mu M) but the lowest apparent Km value for all-trans-retinol (similar to 0.035 mu 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.