Biosynthesis of 9-cis-retinoic acid in vivo - The roles of different retinol dehydrogenases and a structure-activity analysis of microsomal retinol dehydrogenases

Retinoic acid is generated by a two-step mechanism. First, retinol is conve rted into retinal by a retinol dehydrogenase, and, subsequently, retinoic a cid is formed by a retinal dehydrogenase. In vitro, several enzymes are sug gested to act in this metabolic pathway. However, little is known regarding their capacity to contribute to retinoic acid biosynthesis in vivo. We hav e developed a versatile cell reporter system to analyze the role of several of these enzymes in 9-cis-retinoic acid biosynthesis in vivo. Using a Gal4 -retinoid X receptor fusion protein-based luciferase reporter assay, the fo rmation of 9-cis-retinoic acid from g-cis-retinol was measured in cells tra nsfected with expression plasmids encoding different combinations of retino l and retinal dehydrogenases. The results suggested that efficient formatio n of 9-cis-retinoic acid required co-expression of retinol and retinal dehy drogenases. Interestingly, the cytosolic alcohol dehydrogenase 4 failed to efficiently catalyze 9-cis-retinol oxidation. A structure-activity analysis showed that mutants of two retinol dehydrogenases, devoid of the carboxyl- terminal cytoplasmic tails, displayed greatly reduced enzymatic activities in vivo, but were active in vitro. The cytoplasmic tails mediate efficient endoplasmic reticulum localization of the enzymes, suggesting that the uniq ue milieu in the endoplasmic reticulum compartment is necessary for in vivo activity of microsomal retinol dehydrogenases.