title: Human sterol 14alpha-demethylase activity is enhanced by the membrane-bound state of cytochrome b(5).
abstract: Human sterol 14alpha-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 catalytic 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 b(5) derivatives were genetically engineered 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 signal sequence of alkaline phosphatase (signal-core), and the signal sequence linked to the native b(5) (signal-core-tail). The rat core-tail enzyme greatly stimulated sterol demethylation, whereas the signal-core-tail was only marginally active. In contrast, the core and signal-core constructs were completely inactive in stimulating the demethylation reaction. Additionally, cytochrome b(5) enhanced sterol demethylation by more than threefold by accepting 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 productive interaction between cytochrome b(5) and CYP51 is governed by the membrane-insertable hydrophobic region in the cytochrome b(5) which in turn determines the correct spatial orientation of the core. This is the first report showing the stimulation of CYP51 by cytochrome b(5).
authors.affiliation: Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion, SY23 3DD, United Kingdom.
authors.forename: D C
authors.initials: DC
authors.lastname: Lamb
authors.forename: N N
authors.initials: NN
authors.lastname: Kaderbhai
authors.forename: K
authors.initials: K
authors.lastname: Venkateswarlu
authors.forename: D E
authors.initials: DE
authors.lastname: Kelly
authors.forename: S L
authors.initials: SL
authors.lastname: Kelly
authors.forename: M A
authors.initials: MA
authors.lastname: Kaderbhai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D006900:Hydroxylation / Q000187:drug effects; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D058886:Sterol 14-Demethylase; D013329:Structure-Activity Relationship
chemical_list: C548418:CYP51A1 protein, human; C548417:Cyp51 protein, rat; D011993:Recombinant Fusion Proteins; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase; D009251:NADPH-Ferrihemoprotein Reductase