Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line

POKLEPOVICH T.J; URTASUN, N; MIRANDA, V; A. D. NUSBLAT; B.C. NUDEL

STEROIDS

ELSEVIER SCIENCE INC

Lugar: Amsterdam; Año: 2015 p. 132 - 139

0039-128X

Tetrahymena thermophila transforms exogenous cholesterol into pro-vitamin D3 (7-dehydrocholesterol)with remarkable efficiency in a one-step reaction carried out by a C-7 cholesterol desaturase. The enzymeDES7 is encoded by the gene TTHERM_00310640, identified with RNAi and gene knock-out experiments,but has not yet been heterologously expressed actively in any organism. A model derived from its aminoacid sequence classified DES7p as a Rieske-type oxygenase with transmembrane localization. The proteinhas catalytic activity, sequence and topological similarity to DAF-36/Neverland proteins involved in thesynthesis of steroid hormones in insects and nematodes. Due to their structural and functional similarity,we analyzed the expression of a codon optimized DES7 gene from Tetrahymena in the insect Sf9 cell line,identified and measured the steroid metabolites formed, and extended the actual knowledge on itslocalization. We found that the accumulation of 7-dehydrocholesterol could be increased 16?40-fold inSpodoptera frugiperda, depending on physiological conditions, by overexpression of T. thermophila DES7.The protein was detected in the microsomal fraction, in accordance with previous reports. Althoughthe electron transfer chain for Des7p/DAF-36/Neverland Rieske-type oxygenases is presently unknown,we identified possible donors in the ciliate and insect genomes by bioinformatic analysis. In spite ofthe large evolutionary distance between S. frugiperda and T. thermophila, the results indicate that thereis significant functional conservation of the electron donors, since the ciliate?s sterol desaturase canfunction in the context of the insect electron transport system. The results achieved demonstrate thatDES7 is the first gene from a ciliate, coding for a microsomal enzyme, expressed in active form in an insectcell line.