Yeast Cytochrome P-450 Catalyzing Lanosterol 14a-Demethylation

A form of cytochrome P-450 catalyzing lanosterol 14a-demethylation (tentatively called ‘P’45014DMn) was purified from microsomes of semi-anaerobically grown cells of Saccharomyces cerevisiae to gel electrophoreitc homogeneity. An apparent monomeric M. = 58,000 was estimated for the purified cytochrome by sodium dodecyl sulfate-polyacrylamide g l electrophoresis. Both optical and EPR spectra of oxidized P45Ol4n~ are characteristic of low spin ferric heme proteins, and its reduced CO complex showed a Soret absorption peak at 447 nm. As in the case of hepatic microsomal cytochromes P-450, the ethyl isocyanide complex of reduced P-45OIroM was in a pH-dependent equilibrium between two states having Soret peaks at 429 and 453 nm, the equilibrium being considerably shifted toward the 453-nm state. Oxidized P-45014~M was peculiar in that in its CD spectrum there was a negative shoulder at 425 nm and the 350and 414-nm troughs possessed larger and relatively smaller [e] values, respectively, than those reported for other low spin ferric cytochromes P-450. Lanosterol was the only compound which caused a Type I spectral change in oxidized P-45014DM. The lanosterol-induced low to high spin state change was, however, only slight even at saturating concentrations of the sterol, indicating that the lanosterol-P-45014D~ adduct was in a spin state equilibrium. Yeast is a primitive eukaryote which contains cytochrome P450 and, therefore, is an attractive target for comparative studies of cytochrome P-450. The occurrence of cytochrome P-450 in semi-anaerobically grown yeast was first reported by Lindenmayer and Smith (4) and by Ishidate et al. (5 ) . Early attempts to isolate cytochrome P-450 from Saccharomyces cereuisiae ( 6 ) and Candida tropicalk (7) produced preparations which were far from homogeneity. In 1977, Yoshida et al. (8) reported an improved method for purification of a form of cytochrome P-450 to near homogeneity from microsomes of semi-anaerobically grown cells of S. cereuisiae. This cytochrome P-450 was found to catalyze the 14a-demethylation of lanosterol in the presence of NADPH, molecular oxygen, and NADPH-cytochrome P-450 reductase purified from the same source (9, 10). Lanosterol 14a-demethylation is an important step in sterol biosynthesis in yeast and has been suggested to be catalyzed by a cytochrome P-450-containing system from inhibition studies (1114). Therefore, this yeast cytochrome P-450 is actually a lanosterol l4a-demethylase and a tentative name of “P45Ol4~~’’ is given to it in this and accompanying papers. This paper describes a method for purification of P-45014DM from microsomes of semi-anaerobically grown cells of S. cereuisiae and reports its spectral properties. The activity of purified P-45014DM in catalyzing lanosterol l4a-demethylation will be reported in the accompanying paper (20).