The structures of the unique sulfotransferase retinol dehydratase with product and inhibitors provide insight into enzyme mechanism and inhibition.

Cytosolic sulfotransferases (STs) catalyze the transfer of a sulfate group from the universal sulfate donor 3′-phosphoadenosine 5′-phosphosulfate (PAPS) onto a hydroxyl or amino group of a large number of structurally diverse acceptor molecules, such as steroids, hormones, neurotransmitters, and other endogenous or xenobiotic compounds (Weinshilboum et al. 1997; Strott 2002; Chapman et al. 2004). Sulfation of metabolites commonly leads to their inactivation and elimination by increasing their water solubility and decreasing biological activity (Falany 1997). Recent data have also implicated STs in a number of disease states, such as chronic inflammation (Kansas 1996), entry of HIV (Farzan et al. 1999; Cormier et al. 2000), and various forms of cancer (Falany 1997; Weinshilboum et al. 1997; Armstrong and Bertozzi 2000). For example, STs have been shown to produce highly reactive intermediates (e.g., N-hydroxy heterocyclic and aromatic arylamines) that bind DNA and promote mutations (Yamazoe et al. 1999). For this reason, there is growing interest in the study of substrate binding and inhibition mechanisms of STs. Crystal structures of several STs have been reported (Gamage et al. 2003, and references therein; Lee et al. 2003). Structural studies have shown that despite low to moderate sequence identity between ST subfamilies, the overall molecular fold of all known STs is surprisingly similar. As one might infer by the diverse substrates recognized by individual members of the superfamily, the acceptor substrate binding pockets are the least conserved parts of the ST molecules. Retinol dehydratase (DHR) from Spodeptera frugiperda is currently the only insect sulfotransferase for which a structure has been determined. The enzyme catalyzes the conversion of all-trans-retinol (RTL) to anhydroretinol (AR) (Fig. 1 ▶), a retro-retinoid that appears to play a role in morphogenesis. AR is also present in a number of mammalian cell types including liver, kidney, and lung (Buck et al. 1993). However, mammalian enzymes responsible for the production of AR have yet to be identified. Figure 1. Retinol dehydratase-catalyzed reaction. DHR has several unique features that distinguish it from other members of the ST family. The enzyme has low-sequence identity to the most homologous sulfotransferase rat aryl sulfotransferase (30%) and is significantly larger (41 kDa) than mammalian STs (30–36 kDa). However, sequence-alignment analyses and kinetic studies invariantly classify this enzyme as a member of the sulfotransferase superfamily (Vakiani et al. 1998). As a typical cytosolic sulfotransferase, DHR sulfonates a wide variety of different hydroxycompounds, such as p-nitrophenol, phenol, vanillin, and serotonin. Some steroids bind tightly to DHR, in particular hydrocortisone and androsterone. However, these compounds do not appear to be substrates, as no sulfonated products are detected under standard assay conditions (Vakiani et al. 1998). On the other hand, other sulfotransferases do not seem to utilize retinol or other retinoids as substrates (Vakiani et al. 1998). The feature that most distinguishes DHR from other STs is that the end product of the enzymatic reaction, anhydroretinol, is not sulfonated. Retinyl sulfate appears to be a transient intermediate in the transformation of retinol to anhydroretinol. We have previously shown (Pakhomova et al. 2001) that the insertion of the 32-residue helical “lid” domain into the canonical sulfotransferase fold is responsible for the functional transformation of a sulfotransferase to a dehydratase. We also showed that a DHR deletion mutant that lacks the lid domain retains sulfotransferase activity, but no longer functions as a dehydratase. On the basis of biochemical and structural data, a mechanism for the enzymatic reaction was proposed. However, structural information was limited to the enzyme:PAP:substrate complex. In this article, we present the results of three crystal structure determinations of ternary complexes of the enzyme with (1) the potent steroid inhibitor androsterone, (2) the product of the reaction anhydroretinol, and (3) the retinoid inhibitor (all-trans-4-oxoretinol) (Fig. 2 ▶), all in the presence of PAP. These structures provide a more detailed picture of the enzymatic reaction and help to explain differences between DHR and other members of the ST super-family. Figure 2. Chemical structures of androsterone, all-trans-retinol, anhydroretinol, and all-trans-4-oxoretinol.