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 biosynthesis in eukaryotes and is catalyzed by cytochrome P450 sterol 14 alpha-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 14 alpha-demethylase can be envisaged.
Lamb
D C
DC
Institute of Biological Sciences, University of Wales Aberystwyth, UK.
Kelly
D E
DE
Venkateswarlu
K
K
Manning
N J
NJ
Bligh
H F
HF
Schunck
W H
WH
Kelly
S L
SL
D000595:Amino Acid Sequence; D002176:Candida albicans / Q000235:genetics; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015951:Factor Xa / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors / Q000138:chemical synthesis; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009994:Osmolar Concentration; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012965:Sodium Chloride / Q000737:chemistry; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D058886:Sterol 14-Demethylase; D013917:Thrombin / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D012965:Sodium Chloride; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase; D009247:NADH, NADPH Oxidoreductases; D013917:Thrombin; D015951:Factor Xa