CLONING AND FUNCTIONAL EXPRESSION IN YEAST OF A CDNA CODING FOR AN OBTUSIFOLIOL 14-ALPHA-DEMETHYLASE (CYP51) IN WHEAT

Screening of a wheat cDNA library with an heterologous CYP81B1 probe f rom Helianthus tuberosus led to the isolation of a partial cDNA coding a protein with all the characteristics of a typical P450 with high ho mology (32-39% identity) to the fungal and mammalian CYP51s. Extensive screening of several wheat cDNA libraries isolated a longer cDNA (W51 6) coding a peptide of 453 amino acids. Alignment of W516 with other P 450 sequences revealed that it was missing a segment corresponding to the N-terminal membrane anchor of the protein. The corresponding segme nt from the yeast lanosterol 14 alpha-demethylase was linked to the pa rtial wheat cDNA and the chimera expressed in Saccharomyces cerevisiae . Compared to microsomes from control yeasts, membranes of yeast expre ssing the chimera catalysed 14 alpha-demethylation of obtusifoliol wit h an increased efficiency relative to lanosterol demethylase activity. W516 is thus a plant member of the most ancient and conserved P450 fa mily, CYP51. (C) 1997 Academic Press