Substrate Specificity and Kinetic Mechanism of the Insect Sulfotransferase, Retinol Dehydratase
Journal of Biological Chemistry
Spodoptera frugiperda retinol dehydratase catalyzes the conversion of retinol to the retro-retinoid anhydroretinol. It shares sequence homology with the family of mammalian cytosolic sulfotransferases and provides the first link between sulfotransferases and retinol metabolism. In this study the enzymatic properties of retinol dehydratase were examined using bacterially expressed protein. We show that retinol dehydratase can catalyze the transfer of the sulfonate moiety to small phenolic
... ds and exhibits many functional similarities to the mammalian cytosolic sulfotransferases. The bisubstrate reaction that it catalyzes between retinol and the universal sulfonate donor 3-phosphoadenosine 5-phosphosulfate seems to involve ternary complex formation and to proceed via a Random Bi Bi mechanism. In addition to the low nanomolar K m value for free retinol, retinol dehydratase is strongly inhibited by retinol metabolites, suggesting a preference for retinoids. Conversely, a number of tested mammalian cytosolic sulfotransferases do not utilize retinol, indicating that retinol is not a general substrate for sulfotransferases. Vitamin A (retinol) regulates cellular function in multiple ways. Since it is not known to have biological activity itself, it is thought to serve as the parent compound for the biosynthesis of a number of retinoid metabolites. 11-cis-Retinaldehyde, for example, has been established as the essential chromophore for vision (1), and all-trans-retinoic acid (RA) 1 has been widely studied for its roles in cellular differentiation (2) and morphogenesis (3). The two retro-retinoids, 14-hydroxy-4,14-retro-retinol (14-HRR) (4) and anhydroretinol (AR) (5), are among the most recently characterized bioactive retinoids. They are physiologically present in a number of insect and mammalian cell types, and evidence to date suggests they play a role in cell survival (reviewed in Ref. 6). 14-HRR appears to be essential in lymphocyte and fibroblast activation and can prevent cell death in retinol-dependent cell lines grown in serum-free medium. AR can competitively inhibit the growth-supportive effects of retinol and 14-HRR. These retro-retinoids seem to define a novel class of messenger molecules. Progress in the characterization of retinol metabolizing enzymes and isolation of their genes has lagged behind the identification of the various bioactive metabolites. Although most studies have focused on alcohol and aldehyde dehydrogenases and their potential role in the oxidation of retinol and retinaldehyde to RA (reviewed in Ref . 7) , other types of enzymes are required for the complete array of retinol metabolism. The cloning of retinol dehydratase (8) raises the possibility that sulfotransferases, a class of enzymes not previously linked to vitamin A metabolism, may play a role in the biosynthesis of certain retinol metabolites. Retinol dehydratase is the enzyme responsible for the conversion of retinol to the retro-retinoid AR (see Fig. 1 ) and was cloned from the insect cell line Spodoptera frugiperda (Sf)-21. It shares sequence homology to the family of mammalian (reviewed in Refs. 9 -11) and plant (reviewed in Ref. 12) cytosolic sulfotransferases, and it represents the first insect homologue of this growing family of enzymes. Like all sulfotransferases, retinol dehydratase uses 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS) as co-substrate (Fig. 1) . Cytosolic sulfotransferases catalyze bisubstrate reactions where the sulfonate moiety from the universal sulfonate donor PAPS is transferred to small acceptor molecules, such as steroids and catecholamines. Many of the putative substrates bind with high K m or K i values, and only a few physiological substrates (i.e. endogenous substances that serve as substrates at their physiological concentrations, e.g. 17␤-estradiol) have been described. A single enzymatic mechanism for the sulfonation of these compounds has not been established. A number of kinetic studies point toward a sequential mechanism (13-19), whereas the first sulfotransferase crystal structure suggested that at least some sulfotransferase reactions may follow a ping-pong mechanism (20). S. frugiperda retinol dehydratase differs from the described cytosolic sulfotransferases in several important ways: its molecular mass (41 kDa) is higher than that of the known cytosolic sulfotransferases (30 -36 kDa), and its end product, AR, is not sulfonated. In addition, retinol dehydratase catalyzes the formation of a putative signaling molecule, and its low K m value for its apparent substrate, free retinol, indicates a specific interaction between enzyme and substrate. We were interested in examining whether retinol dehydratase functions as a sulfotransferase and in establishing the specificity of interaction between retinol dehydratase and retinoids. Our studies of the enzymatic properties of retinol dehydratase are also important in understanding the mechanism of AR formation and the role of sulfotransferases in retinol metabolism.