Cholesterol 24-hydroxylase (EC 1.14.13.98), also commonly known as cholesterol 24S-hydroxylase, cholesterol 24-monooxygenase, CYP46, or CYP46A1, is an enzyme that catalyzes the conversion of cholesterol to 24S-hydroxycholesterol. It is responsible for the majority of cholesterol turnover in the human central nervous system. The systematic name of this enzyme class is cholesterol,NADPH:oxygen oxidoreductase (24-hydroxylating).

This enzyme is a member of the cytochrome P450 (CYP) superfamily of enzymes. Like many other CYP enzymes that act on cholesterol, cholesterol-24 hydroxylase is a monooxygenase that hydroxylates the side-chain of cholesterol.

Because 24S-hydroxycholesterol is more polar than cholesterol, it can more easily pass the blood–brain barrier to exit the brain and pass into the bloodstream, where it can then travel to the liver to be further degraded. This enzyme has also been found at low quantities in the retina, where it performs the same function to a lesser degree.

Genetic cloning of the encoding gene (CYP46A1) was first accomplished in 1999 and an extensive E. coli expression and purification system was later developed in 2003.

The enzymatic structure of the human cholesterol-24 hydroxylase was determined via crystallography at the Stanford Synchrotron Radiation Lightsource, and was shown to be a 57kDa (500 residue) monomeric heme-containing protein bound to the endoplasmic reticulum in neurons.

Cholesterol-24 hydroxylase is similar in structure to many other cytochrome P450s, possessing, for example, the conserved stretch of 23 hydrophobic residues in the N-terminus that make up a transmembrane-anchoring domain (residues 3-27).

Even so, the cholesterol-24 hydroxylase C-terminus has a unique proline-rich region of 5 repeated proline residues, a structural motif absent in all other related cytochrome p450 enzymes. While the exact function of these proline residues remain highly speculative, it has been shown that the deletion of this region results in a two-fold decrease in the enzyme’s catalytic efficiency.

Binding of cholesterol results in an enzymatic conformational change and a subsequent induced fit of the active site around the cholesterol molecule, anchoring the hydroxylation site (C-24, C-25) near the catalytic center of the enzyme (5.7Å from the iron core of the heme molecule to allow oxyferryl intermediates to perform the cholesterol hydroxylation). A loop region, known as the B'-C loop, has a series of 5 residues (residues 116-120) unique to cholesterol-24 hydroxylase that contribute to the positioning of the cholesterol molecule within the active site. A single cholesterol molecule takes up the entirety of the active site, with the aliphatic tail of the cholesterol held in place by interactions with the following hydrophobic residues: Phe-121, Val-126, Ile-301, Ala-302, Ala-367, Thr-475. The active site is accessed via a single entrance created by two helices (B' and F) and the β1-sheet.