Generation of a Complete, Soluble, and Catalytically Active Sterol 14α-Demethylase−Reductase Complex†

Sterol 14R-demethylation is one of the key steps of sterol biosynthesis in eukaryotes and is catalyzed by cytochrome P450 sterol 14R-demethylase (other names being CYP51 and P45014DM) encoded by ERG11. This enzyme activity is supported by an associated NAPDH-dependent reductase encoded by 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 inserted just beyond the N-terminal hydrophobic segment of Candida albicans Erg11p. This modified enzyme was heterologously expressed at a level of 2.5 nmol of Erg11p/mg of protein as an integral endoplasmic reticulum protein. Following purification, treatment of the modified protein with factor Xa or thrombin resulted in sequence-specific cleavage and production of a soluble N-terminal truncated Erg11p which exhibited spectral characteristics identical to those of the purified full-length, wild-type form. Furthermore, reconstitution of the soluble enzyme with soluble yeast Ncpr1p, expressed and purified as an N-terminal deletion of 33 amino acids encompassing its membrane anchor, resulted in a fully functional and soluble eukaryotic Erg11p system. The complex was disrupted by high-salt concentration, reflecting the importance of electrostatic forces in the protein -protein interaction. The results demonstrate the membrane anchor serves to localize Erg11p to the ER where the substrate is located, but is not essential in either Ncpr1p or Erg11p activity. The possibility of cocrystallization of an active soluble eukaryotic 14R-demethylase can be envisaged. Sterol biosynthesis is ubiquitous in eukaryotes, and the membrane-bound nature of most of the enzymes involved presumably reflects the hydrophobic nature of the substrates that are dissolved in the membranes. The molecular basis of the membrane associations for these enzymes is an area of interest for determining whether this is needed for activity or protein-protein interactions between the various biosyn- thetic enzymes and their electron transport partners. Generat- ing soluble enzymes may also facilitate crystallization of the membrane-bound components of the sterol biosynthetic apparatus, leading to fundamental information and greater understanding of anti-sterol inhibitors for medicine and agriculture.