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Specificity of Zebrafish Retinol Saturase: Formation of All-trans-13,14-dihydroretinol and All-trans-7,8- dihydroretinol^†

Alexander R. Moise,*^‡ Andrea Isken,^§ Marta Domínguez, Angel R. de Lera, Johannes von Lintig,^§ and Krzysztof Palczewski*^‡

Department of Pharmacology, Case School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965, Institut fr Biologie I, Universitt Freiburg, Hauptstrasse 1, D-79104 Freiburg, Germany, and Departamento de Qumica Orgnica, Facultade de Qumica, Universidade de Vigo, 36310 Vigo, Spain

Biochemistry, 2007, 46 (7), pp 1811–1820

DOI: 10.1021/bi062147u

Publication Date (Web): January 25, 2007

†

This research was supported in part by grants EY015399 and EY009339 from the National Eye Institute and National Institutes of Health, and SAF04- 07131 from MEC-FEDER (Spain).

To whom correspondence should be addressed. Phone: 216-368-4631. Fax: 216-368-1300. E-mail: ram50@case.edu (A.R.M.); kxp65@case.edu (K.P.).

‡

Case Western Reserve University.

Universität Freiburg.

Universidade de Vigo.

Abstract

Metabolism of vitamin A, all-trans-retinol, leads to the formation of 11-cis-retinaldehyde, the visual chromophore, and all-trans-retinoic acid, which is involved in the regulation of gene expression through the retinoic acid receptor. Enzymes and binding proteins involved in retinoid metabolism are highly conserved across species. We previously described a novel mammalian enzyme that saturates the 13−14 double bond of all-trans-retinol to produce all-trans-13,14-dihydroretinol, which then follows the same metabolic fate as that of all-trans-retinol. Specifically, all-trans-13,14-dihydroretinol is transiently oxidized to all-trans-13,14-dihydroretinoic acid before being oxidized further by Cyp26 enzymes. Here, we report the identification of two putative RetSat homologues in zebrafish, one of which, zebrafish RetSat A (zRetSat A), also had retinol saturase activity, whereas zebrafish RetSat B (zRetSat B) was inactive under similar conditions. Unlike mouse RetSat (mRetSat), zRetSat A had an altered bond specificity saturating either the 13−14 or 7−8 double bonds of all-trans-retinol to produce either all-trans-13,14-dihydroretinol or all-trans-7,8-dihydroretinol, respectively. zRetSat A also saturated the 13−14 or 7−8 double bonds of all-trans-3,4-didehydroretinol (vitamin A2), a second endogenous form of vitamin A in zebrafish. The dual enzymatic activity of zRetSat A displays a newly acquired specificity for the 13−14 double bond retained in higher vertebrates and also the evolutionarily preserved activity of bacterial phytoene desaturases and plant carotenoid isomerases. Expression of zRetSat A was restricted to the liver and intestine of hatchlings and adult zebrafish, whereas zRetSat B was expressed in the same tissues but at earlier developmental stages. Exogenous all-trans-retinol, all-trans-13,14-dihydroretinol, or all-trans-7,8-dihydroretinol led to the strong induction of the expression of the retinoic acid-metabolizing enzyme, Cyp26A1, arguing for an active signaling function of dihydroretinoid metabolites in zebrafish. These findings point to a conserved function but altered specificity of RetSat in vertebrates, leading to the generation of various dihydroretinoid compounds, some of which could have signaling functions.