Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principal cholesterol hydroxylase in the brain

Abstract

By converting cholesterol to 24S-hydroxycholesterol, cytochrome P450 46A1 (CYP46A1) initiates the major pathway for cholesterol removal from the brain. Two crystal structures of CYP46A1 were determined. First is the 1.9-Å structure of CYP46A1 complexed with a high-affinity substrate cholesterol 3-sulfate (CH-3S). The second structure is that of the substrate-free CYP46A1 at 2.4-Å resolution. CH-3S is bound in the productive orientation and occupies the entire length of the banana-shaped hydrophobic active-site cavity. A unique helix B -C loop insertion (residues 116 -120) contributes to positioning cholesterol for oxygenation catalyzed by CYP46A1. A comparison with the substrate-free structure reveals substantial substrate-induced conformational changes in CYP46A1 and suggests that structurally distinct compounds could bind in the enzyme active site. In vitro assays were performed to characterize the effect of different therapeutic agents on cholesterol hydroxylase activity of purified full-length recombinant CYP46A1, and several strong inhibitors and modest coactivators of CYP46A1 were identified. Structural and biochemical data provide evidence that CYP46A1 activity could be altered by exposure to some therapeutic drugs and potentially other xenobiotics.cholesterol metabolism ͉ monooxygenase ͉ drug interactions ͉ cholesterol 3-sulfate A ccumulating evidence indicates that neurodegeneration and development of neurological disorders such as Alzheimer's disease (AD) are associated with disturbances in cholesterol homeostasis in the brain (1-5). It is also becoming increasingly clear that the conversion of cholesterol to 24S-hydroxycholesterol (24OH-CH) is an important mechanism that controls cholesterol turnover in the central nervous system (6-8). Cholesterol 24-hydroxylation is carried out by cytochrome P450 46A1 (CYP46A1) and represents the first step in the major pathway for cholesterol elimination from the brain (9-11). Unlike cholesterol, 24OH-CH can cross the blood-brain barrier and be delivered to the liver for further degradation to bile acids.Studies of CYP46A1-knockout mice indicate that the continued synthesis and turnover of cerebral cholesterol via 24-hydroxylation are necessary for memory and learning (12). There appears to be a link between CYP46A1 and AD; the CYP46A1 expression pattern in the brain and levels of 24OH-CH in the serum and cerebrospinal f luid are different in healthy people and those affected by AD (13-17). The association between polymorphisms in the CYP46A1 gene and susceptibility to AD was also demonstrated in a number of studies (www.alzforum.org). However, this association has not yet been unambiguously proven and is still under investigation. Evidence has also been obtained in cultured cells and mice that supports a role for side-chain oxysterols, including 24OH-CH, as endogenous ligands for liver X receptors, that regulate the expression of genes involved in fatty acid and cholesterol metabolism (7). 24OH-CH was also shown to inhibit the formation of amyloid ␤-peptides, a ha...

Structural Diversity of Eukaryotic Membrane Cytochrome P450s

“…This is an important reaction for the clearance of excess cholesterol from the brain. Interestingly, the substratefree structure of 46A1 exhibits a much different cavity shape (34), and the 46A1 active site adapts to bind several structurally unrelated inhibitors (35,36). In contrast, structures of microsomal 2R1 (37) indicate that the secosterol moiety of vitamin D 3 and related compounds is bound between helices I and G and the helix B-C loop.…”

Structural complex of sterol 14α-demethylase (CYP51) with 14α-methylenecyclopropyl-Δ7-24, 25-dihydrolanosterol

“…The fact that CYP51s do not produce 100% high spin form upon substrate binding suggests that in CYP51s the axial water molecule might remain in close proximity to the heme after the substrate binding ( 33 ), as has been crystallographically proved for some other CYPs ( 58,59 ), therefore functioning as a shuttle for the delivery of the catalytic protons to the iron-bound oxygen during the three sequential steps of the CYP51 reaction. When MCP binds to T. cruzi CYP51, the water presumably remains within the active site ( Fig.…”

Diversity and Substrate Specificity in the Structures of Steroidogenic Cytochrome P450 Enzymes

“…However, recent efforts have succeeded in revealing the 3D structures of several mammalian CYPs with X-ray crystallography. [4][5][6][7][8][9][10][11] Information of the 3D structures of mammalian CYP enzymes will provide a great impact on the pharmaceutical studies.…”