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

• Published in: The Journal of biological chemistry 2008-07, Vol.283 (29), p.20299-20308
• Main Authors: Belyaeva, Olga V., Johnson, Mary P., Kedishvili, Natalia Y.
• Format: Article
• Language: English
• Subjects: Alcohol Oxidoreductases - chemistry; Alcohol Oxidoreductases - genetics; Alcohol Oxidoreductases - metabolism; Amino Acid Sequence; Animals; Cell Line; Cell Survival; Conserved Sequence; Down-Regulation; Gene Expression; Humans; Kinetics; Metabolism and Bioenergetics; Models, Molecular; Molecular Sequence Data; Protein Structure, Tertiary; Retinaldehyde - metabolism; RNA, Small Interfering - genetics; Sequence Alignment; Spodoptera; Substrate Specificity; Vitamin A - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M800019200

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+ (∼100 μm) but the lowest apparent Km value for all-trans-retinol (∼0.035 μm) 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 RNA-mediated 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.