Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principal cholesterol hydroxylase in the brain
Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principal cholesterol hydroxylase in the brain
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DOI：
10.1073/pnas.0803717105
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发表时间：
2008-07-15
影响因子：
11.1
通讯作者：
Pikuleva, Irina A.
中科院分区：
文献类型：
--
作者：
Mast, Natalia;Whitet, Mark Andrew;Pikuleva, Irina A.
通过将胆固醇转化为24 S-羟基胆固醇，细胞色素P450 46 A1（CYP 46 A1）启动了从大脑中清除胆固醇的主要途径。测定了CYP 46 A1的两种晶体结构。第一个是CYP 46 A1与高亲和力底物胆固醇3-硫酸酯（CH-3S）复合的1.9埃结构。第二种结构是无底物的CYP 46 A1，分辨率为2.4埃。CH-3S在生产方向上结合，并占据香蕉形疏水活性位点空腔的整个长度。独特的螺旋B '-C环插入（残基116-120）有助于定位胆固醇，以供CYP 46 A1催化氧化。与无底物结构的比较揭示了大量的底物诱导的CYP 46 A1的构象变化，并表明结构上不同的化合物可以结合在酶的活性位点。进行体外试验以表征不同治疗剂对纯化的全长重组CYP 46 A1的胆固醇羟化酶活性的影响，并鉴定了几种CYP 46 A1的强抑制剂和适度的共激活剂。结构和生化数据提供的证据表明，CYP 46 A1活性可以通过暴露于一些治疗药物和潜在的其他外源性物质而改变。
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-angstrom 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-angstrom 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.