Department of Biochemistry, University at Buffalo, Buffalo, New York 14203, United States.
Biochemistry. 2022 Jan 18;61(2):57-66. doi: 10.1021/acs.biochem.1c00770. Epub 2022 Jan 3.
The improper maintenance of the bioactivated form of vitamin-D (1α,25(OH)D) 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)D 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)D 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.
维生素-D（1α,25(OH)D）的生物激活形式维护不当可能导致维生素-D 不足，从而影响膳食钙的吸收。维生素-D 代谢的一个重要调节剂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)D 侧链的碳-23（C23）或碳-24（C24）进行羟化，最终产生维生素-D 受体的拮抗剂（C23 途径）或钙三醇（C24 途径）。尽管它对人类健康很重要，但由于使用重组方法生产该酶的难度，人类同工酶（hCYP24A1）在很大程度上仍未被描述。在这项研究中，我们利用与同源还原伴侣 hAdx 的可切割融合来稳定 hCYP24A1 的生产。随后的切割和分离活性 hCYP24A1 允许对底物和类似物结合、酶活性和还原伴侣识别进行研究。我们证明涉及 hAdx 的 Leu-80 的非极性接触和 hCYP24A1 的非保守近端表面。有趣的是，缩短该残基（L80V）的长度会导致 CYP-Adx 复合物与 1α,25(OH)D 之间的结合增强，但出乎意料的是，催化作用却降低了。同样的突变对大鼠 CYP24A1（C24-羟化酶）几乎没有影响，这表明存在一种物种特异性的要求，这可能与反应的区域选择性差异相关。总之，这项工作提供了生产具有挑战性的人类 CYP 的一个例子，并提供了有关对人类维生素-D 代谢至关重要的 CYP-Adx 复合物的疏水性调节的详细信息。
