Generation of a complete, soluble, and catalytically active sterol 14 alpha-demethylase-reductase complex

Sterol 14 alpha-demethylation is one of the key steps of sterol biosynthesi s in eukaryotes and is catalyzed by cytochrome P450 sterol 14 alpha-demethy lase (other names being CYP51 and P450(14DM)) encoded by ERC11. This enzyme activity is supported by an associated NAPDH-dependent reductase encoded b y NCPR1 (NCP1), which is also associated with the endoplasmic reticulum. A diglycine linker recognition site (Gly-Gly-Ile-Glu-Gly-Arg-Gly-Gly) for the protease factor Xa, also containing a thrombin recognition site, was inser ted just beyond the N-terminal hydrophobic segment of Candida albicans Erg1 1p. This modified enzyme was heterologously expressed at a level of 2.5 nmo l of Erg11p/mg of protein as an integral endoplasmic reticulum protein. Fol lowing purification, treatment of the modified protein with factor Xa or th rombin resulted in sequence-specific cleavage and production of a soluble N -terminal truncated Erg11p which exhibited spectral characteristics identic al to those of the purified full-length, wild-type form. Furthermore, recon stitution of the soluble enzyme with soluble yeast Ncpr1p, expressed and pu rified as an N-terminal deletion of 33 amino acids encompassing its membran e anchor, resulted in a fully functional and soluble eukaryotic Erg11p syst em. The complex was disrupted by high-salt concentration, reflecting the im portance of electrostatic forces in the protein-protein interaction. The re sults demonstrate the membrane anchor serves to localize Erg11p to the ER w here the substrate is located, but is not essential in either Ncpr1p or Erg 11p activity. The possibility of cocrystallization of an active soluble euk aryotic 14 alpha-demethylase can be envisaged.