A Novel Desaturase Complex from the Ciliate Tetrahymena thermophila

ALEJANDRO NUSBLAT; DIEGO BOCHOEYER; CLARA NUDEL

Santiago de Chile, Chile

Congreso; 12 th International biotechnology symposium and exhibition; 2004

Conicyt - IUPAC

A novel desaturase complex displaying C7(8) and C22(23) cholesterol desaturase activities was isolated in Tetrahymena thermophila microsomes. Differential expression of both desaturases in live Tetrahymena can be achieved by culturing the organism with different sterols; i.e, addition of 22- dehydrocholesterol to the culture medium increased C7(8) desaturase expression, while addition of cholesterol or 7-dehydrocholesterol increased C22(23) desaturase expression. Cultures with no sterols showed almost undetectable any desaturase activity. The microsomal complex desaturates cholesterol to yield 7-dehydrocholesterol and/or 22- dehydrocholesterol with addition of exogenous NAD(P)H. Selectivity towards one product can be achieved with the addition of specific compounds, i.e., b-mercaptoethanol  inhibited completely C22(23) desaturase, while ethanol increased selectively the same. Detergent -solubilized microsomal fractions showed no desaturase activities; however, the addition of dilauroyl phosphatidil choline liposomes restored partially the enzyme activity (25%) Both cholesterol desaturases require reduced cofactors, molecular oxygen and Cytochrome b5. NADH and NADPH can serve as reduced cofactors, presumably delivered either by CYT b5-reductase or CYT P-450-reductase, present in the microsomal fraction (measured activities were 120 and 55.2 nmol.min-1. mg-1, respectively). Azide and KCN but not azole compounds inhibited these desaturases, suggesting a key role for Cytochrome b5 in these reactions, and ruling out the possibility that they might belong to the CYT P-450 superfamily. The said biotransformation of cholesterol has potential for biotechnology purposes, as it can be used for decreasing cholesterol content in foodstuffs of animal origin with simultaneous enrichment in pro vitamin D.