ABSTRACT: Cytochrome P450 27A1 (P450 27A1 or CYP27A1) is an important enzyme that participates
in different pathways of cholesterol degradation as well as in the activation of vitamin D3. Several
approaches were utilized to investigate how two physiological substrates, cholesterol and 5a-cholestane-
3R,7R,12R-triol, interact with CYP27A1. The enzyme active site was first probed spectrally by assessing
binding of the two substrates and five substrate analogues followed by computer modeling and sitedirected
mutagenesis. The computer models suggest that the spatial positions and orientations of cholesterol
and 5a-cholestane-3R,7R,12R-triol are different in the enzyme active site. As a result, some of the active
site residues interact with both substrates, although they are situated differently relative to each steroid,
and some residues bind only one substrate. Mutation of the overlapping substrate-contact residues (W100,
H103, T110, M301C, V367, I481, and V482) affected CYP27A1 binding and enzyme activity in a substratedependent
manner and allowed identification of several important side chains. T110 is proposed to interact
with the 12R-hydroxyl of 5a-cholestane-3R,7R,12R-triol, whereas V367 seems to be crucial for correct
positioning of the cholesterol C26 methyl group and for regioselective hydroxylation of this substrate.
Distinct binding of the CYP27A1 substrates may provide insight into why phenotypic manifestations of
cerebrotendinous xanthomatosis, a disease associated with CYP27A1 deficiency, are so diverse.