PURIFICATION AND RECONSTITUTION OF ACTIVITY OF SACCHAROMYCES-CEREVISIAE P450-61, A STEROL DELTA(22)-DESATURASE

P450 was purified from microsomal fractions of a strain of Saccharomyc es cerevisiae which contained detectable P450 despite the disruption o f CYP51A1. The P450 had a molecular mass of 58 kDa, similar to P450 51 A1, and in a reconstituted assay with rabbit NADPH-P450 reductase and dilauryl phosphotidylcholine exhibited activity for conversion of ergo sta-5,7-dienol into ergosterol. N-Terminal amino acid sequencing of th e purified protein corresponded to the translated sequence of P450 61 which was recently identified during sequencing of chromosome XIII. Th is allowed the function of this family of P450 to be identified as ste rol Delta(22)-desaturation in the pathway of ergosterol biosynthesis.