Human sterol 14 alpha-demethylase activity is enhanced by the membrane-bound state of cytochrome b(5)

Human sterol 14 alpha -demethylase (P45051; CYP51) catalyzes the oxidative removal of the C32 methyl group of dihydrolanosterol, an essential step in the cholesterol biosynthetic pathway. The reaction is dependent upon NADPH cytochrome P450 reductase (CPR) that donates the electrons for the catalyti c cycle. Here we used a recombinant yeast CPR to investigate the abilities of four different forms of cytochrome b(5) to support sterol demethylation activity of CYP51. The cytochrome b5 derivatives were genetically engineere d forms of the native rat cytochrome b(5) core-tail: the soluble globular b (5) core (core), the core linked at its N-terminus with the secretory signa l sequence of alkaline phosphatase (signal-core), and the signal sequence l inked to the native b(5) (signal-core-tail). The rat core-tail enzyme great ly stimulated sterol demethylation, whereas the signal-core-tail was only m arginally active. In contrast, the core and signal-core constructs were com pletely inactive in stimulating the demethylation reaction. Additionally, c ytochrome bs enhanced sterol demethylation by more than threefold by accept ing electrons from soluble yeast CPR and in its ability to reduce P450. We show that the nature of transient linkage between the hemoproteins and the redox partners is most likely brought about electrostatically, although pro ductive interaction between cytochrome b(5) and CYP51 is governed by the me mbrane-insertable hydrophobic region in the cytochrome b(5) which in turn d etermines the correct spatial orientation of the core. This is the first re port showing the stimulation of CYP51 by cytochrome b(5). (C) 2001 Academic Press.