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 K(m) value for NAD(+) (~100 microm) but the lowest apparent K(m) value for all-trans-retinol (~0.035 microm) 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.
Belyaeva
Olga V
OV
Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama, Birmingham, AL 35294, USA.
Johnson
Mary P
MP
Kedishvili
Natalia Y
NY
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D017124:Conserved Sequence; D015536:Down-Regulation; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000235:genetics; D012172:Retinaldehyde / Q000378:metabolism; D016415:Sequence Alignment; D018411:Spodoptera; D013379:Substrate Specificity; D014801:Vitamin A / Q000378:metabolism
D034741:RNA, Small Interfering; D014801:Vitamin A; D000429:Alcohol Oxidoreductases; C113992:trans-retinol dehydrogenase; C022520:retinol dehydrogenase; D012172:Retinaldehyde