Isolation of a Δ 7 -cholesterol desaturase from Tetrahymena thermophila

Abstract

Cell-free preparations of Tetrahymena thermophila catalyze the direct desaturation of cholesterol to delta7-dehydrocholesterol (provitamin D3). The activity was isolated in the microsomal fraction from Tetrahymena homogenates. Delta7-desaturase activity was stimulated fivefold by the addition of 6 mM ATP. Other cofactors assayed, including NAD, NADP, NADH or NADPH, had no significant effect. The activity was found in microsomes prepared from stationary-phase cultures of the ciliate, grown either with or without added cholesterol, thus indicating that it is constitutively expressed in T. thermophila cells.

Characterization and Properties of Cholesterol Desaturases from the Ciliate Tetrahymena thermophila

“…In conclusion, although we have previously demonstrated and preliminarily characterized a microsomal cholesterol desaturase activity (Valcarce, Florin-Christensen, and Nudel 2000), this is the first report to describe conditions for increased differential expression, solubilization, and reconstitution of C7(8) and C22(23) cholesterol desaturases in Tetrahymena cells. Moreover, the selective increase of D 5,7 cholesterol desaturase activity achieved by the addition of 22-DHC in culture medium, and the inhibition of competing D 5,22 desaturase activity by addition of b-ME, are encouraging results towards a practical application of the system for the biotransformation of cholesterol into pro-vitamin D 3 , either in foodstuffs or as an alternative vitamin D 3 production process.…”

“…Expression and isolation of cholesterol desaturases in Tetrahymena . In a previous study, T. thermophila cell‐free extracts were incubated aerobically with CHOL in the presence of ATP and reduced cofactors, and, upon incubation, a Δ 5,7 sterol metabolite was detected by HPLC analysis monitored at 205 and 285 nm, and unequivocally identified as 7‐DHC (pro‐vitamin D 3 ) by GC, HPLC, and MS (Valcarce, Florin‐Christensen, and Nudel 2000). We have now completed the identification of another sterol metabolite, also originated upon incubation with CHOL, corresponding to 22‐DHC.…”

“…This sterol separates cleanly from CHOL and its derivatives under the conditions used. The identity of each compound was established by GC-MS (Casabuono and Pomilio 1997;Valcarce, Florin-Christensen, and Nudel 2000).…”

“…While C22(23) desaturase activity in T. pyriformis has been analyzed in relation to substrate specificity and stereochemistry of the reaction (Conner and Landrey 1978; Zander and Caspi 1970), so far C7(8) cholesterol‐desaturase activity has not been studied in any living organism. Recently, we isolated and preliminarily characterized this activity in cell‐free extracts and microsomal preparations of Tetrahymena (Valcarce, Florin‐Christensen, and Nudel 2000). However, we still do not know how the expression of these enzymes is regulated.…”

Conservation and Innovation in Tetrahymena Membrane Traffic: Proteins, Lipids, and Compartments

“…In T. thermophila , the C-7 and C-22 sterol-desaturating activities, found mainly in a microsomal fraction, require cytochrome b 5 as shown by their inhibition with azide and cyanide(Nusblat et al, 2005; Valcarce et al, 2000). This cytochrome b 5 dependence is not characteristic of the C22 desaturases of plants and fungi, which require cytochrome P450.…”