Characterization of a Cleavable Fusion of Human CYP24A1 with Adrenodoxin Reveals the Variable Role of Hydrophobics in Redox Partner Binding.
Characterization of a Cleavable Fusion of Human CYP24A1 with Adrenodoxin Reveals the Variable Role of Hydrophobics in Redox Partner Binding.
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DOI：
10.1021/acs.biochem.1c00770
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发表时间：
2022-01-18
期刊：
影响因子：
2.9
通讯作者：
Estrada DF
中科院分区：
文献类型：
--
作者：
Jay N;Duffy SR;Estrada DF
维生素d （1α,25-(OH)2D）的生物活性形式维持不当可能导致维生素d不足，从而损害膳食钙的吸收。维生素d代谢的一个重要调节因子是线粒体酶细胞色素P450 24A1 （CYP24A1）的失活功能。在人类中，CYP24A1对1α，25(OH)2D侧链的碳-23 （C23）或碳-24 （C24）进行羟基化，最终产生维生素d受体拮抗剂（C23途径）或骨化柠檬酸（C24途径）。尽管它对人类健康很重要，但人类亚型（hCYP24A1）在很大程度上仍未被表征，部分原因是利用重组手段难以生产这种酶。在这项研究中，我们利用与同源氧化还原伙伴，人类Adx （hAdx）的可切割融合，在生产过程中稳定hCYP24A1。随后裂解和分离活性hCYP24A1，研究底物和类似物结合、酶活性和氧化还原伴侣识别。我们证明了涉及hax的Leu-80和hCYP24A1的非保守近端表面的非极性接触。有趣的是，缩短该残基（L80V）的长度会导致cypa - adx复合物与1α，25(OH)2D之间的结合增强，但意外地导致催化作用下降。同样的突变对大鼠CYP24A1（一种c24 -羟化酶）的影响可以忽略不计，表明存在一种物种特异性需求，可能与反应的区域选择性差异有关。综上所述，这项工作提出了一个具有挑战性的人类CYP生产的例子，并提供了关于对人类维生素d代谢至关重要的CYP- adx复合物的疏水调节的细节。
The improper maintenance of the bioactivated form of vitamin-D (1α,25-(OH)2D) may result in vitamin-D insufficiency and therefore compromise the absorption of dietary calcium. A significant regulator of vitamin-D metabolism is the inactivating function of the mitochondrial enzyme cytochrome P450 24A1 (CYP24A1). In humans, CYP24A1 carries out hydroxylation of carbon-23 (C23) or carbon-24 (C24) of the 1α,25(OH)2D side chain, eventually resulting in production of either an antagonist of the vitamin-D receptor (C23 pathway) or calcitroic acid (C24 pathway). Despite its importance to human health, the human isoform (hCYP24A1) remains largely uncharacterized due in part to the difficulty in producing the enzyme using recombinant means. In this study, we utilize a cleavable fusion with the cognate redox partner, human Adx (hAdx), to stabilize hCYP24A1 during production. The subsequent cleavage and isolation of active hCYP24A1 allowed for an investigation of substrate and analog binding, enzymatic activity, and redox partner recognition. We demonstrate involvement of a nonpolar contact involving Leu-80 of hAdx and a nonconserved proximal surface of hCYP24A1. Interestingly, shortening the length of this residue (L80V) results in enhanced binding between the CYP-Adx complex and 1α,25(OH)2D yet unexpectedly results in decreased catalysis. The same mutation has a negligible effect on rat CYP24A1 (a C24-hydroxylase), indicating the presence of a species-specific requirement that may correlate with differences in regioselectivity of the reaction. Taken together, this work presents an example of production of a challenging human CYP as well as providing details regarding hydrophobic modulation of a CYP-Adx complex that is critical to human vitamin-D metabolism.
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DOI：
10.1073/pnas.0702093104
发表时间：
2007-07-31
影响因子：
11.1
作者：
Prosser, David E.;Kaufmann, Martin;Jones, Glenville
通讯作者：
Jones, Glenville
DOI：
10.1016/j.jbc.2021.100794
发表时间：
2021-01
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Brixius-Anderko S;Scott EE
通讯作者：
Scott EE
影响因子：
14.8
作者：
通讯作者：
--
影响因子：
4.8
作者：
Mast, Natalia;Annalora, Andrew J.;Pikuleva, Irina A.
通讯作者：
Pikuleva, Irina A.
DOI：
10.1016/j.bbrc.2004.07.040
发表时间：
2004-09-03
影响因子：
3.1
作者：
Kusudo, T;Sakaki, T;Inouye, K
通讯作者：
Inouye, K