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J. Biol. Chem., Vol. 283, Issue 29, 20299-20308, July 18, 2008

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

From the Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama, Birmingham, Alabama 35294

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 µM) but the lowest apparent K_m 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.

Received for publication, January 2, 2008 , and in revised form, May 22, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant AA12153 from the NIAAA (to N. Y. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: 720 20th St. South, 440B Kaul Genetics Bldg., Birmingham, AL 35294. Tel.: 205-996-4023; Fax: 205-934-0758; E-mail: nkedishvili{at}uab.edu.

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				I. Strate, T. H. Min, D. Iliev, and E. M. Pera Retinol dehydrogenase 10 is a feedback regulator of retinoic acid signalling during axis formation and patterning of the central nervous system Development, February 1, 2009; 136(3): 461 - 472. [Abstract] [Full Text] [PDF]