Cloning and Functional Expression in Yeast of a cDNA Coding for an Obtusifoliol 14α-Demethylase (CYP51) in Wheat

• Published in: Biochemical and biophysical research communications Vol. 230; no. 2; pp. 381 - 385
• Main Authors: Cabello-Hurtado, Francisco, Zimmerlin, Alfred, Rahier, Alain, Taton, Maryse, DeRose, Richard, Nedelkina, Svetlana, Batard, Yannick, Durst, Francis, Pallett, Kenneth E., Werck-Reichhart, Danièle
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 13-01-1997
• Subjects: ADN; TRITICUM
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.1996.5873

Screening of a wheat cDNA library with an heterologousCYP81B1probe fromHelianthus tuberosusled to the isolation of a partial cDNA coding a protein with all the characteristics of a typical P450 with high homology (32–39% identity) to the fungal and mammalian CYP51s. Extensive screening of several wheat cDNA libraries isolated a longer cDNA (W516) coding a peptide of 453 amino acids. Alignment of W516 with other P450 sequences revealed that it was missing a segment corresponding to theN-terminal membrane anchor of the protein. The corresponding segment from the yeast lanosterol 14α-demethylase was linked to the partial wheat cDNA and the chimera expressed inSaccharomyces cerevisiae.Compared to microsomes from control yeasts, membranes of yeast expressing the chimera catalysed 14α-demethylation of obtusifoliol with an increased efficiency relative to lanosterol demethylase activity. W516 is thus a plant member of the most ancient and conserved P450 family, CYP51.