Characterisation of a Novel Desaturase Complex from the Ciliate Tetrahymena thermophila for the Biotransformation of Cholesterol into pro-Vitamin D.

A. NUSBLAT; D. BOCHOEYER; C. B. NUDEL

New Orleans, EEUU

Congreso; American Society for Microbiology (ASM) 104th General Meeting; 2004

American Society for Microbiology

In response to the addition of cholesterol to the growth medium Tetrahymena suppresses the formation of tetrahymanol and replaces this compound by the unsaturated derivatives D 7,22 bis-dehydrocholesterol (pro-vitamin D2 analog), D 22 dehydrocholesterol and D 7 dehydrocholesterol (pro-vitamin D3). In these desaturations of the cholesterol moiety, essentially two enzymatic activities are involved: C7(8) and C22(23) cholesterol desaturases. 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. Differential expression of C7(8) or C22(23) desaturase in live Tetrahymena was 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 increased C22(23) desaturase expression. Both C7(8) and C22(23) cholesterol desaturases were isolated and characterised in microsomal fractions. The microsomes were solubilized with the aid of a detergent and the enzymatic activity could be reconstituted in liposomes made of phosphatydilcholine. Both C7(8) and C22(23) cholesterol desaturases require O2 and reduced cofactors in the form of NADH or NADPH for their activity. Also cytochome (cyt) b5 is required, as shown by the inhibition of the desaturase activity upon azide or cianide treatment. For C22(23) cholesterol desaturase activity NAD could be used with similar efficiency. None of the desaturases displayed inhibition towards azole compounds. These experiments led to the conclusion that C7(8) and C22(23) cholesterol desaturases from Tetrahymena behave similarly to D 7 sterol C5(6) desaturase and C4 sterol oxidases from plants, yeast and mamalian cells, as well as to D 9 fatty acid desaturase from Tetrahymena. On the other hand they show significant differences with C22(23) sterol desaturases from yeasts, that belong to the cyt P450 superfamily.