Expression and functional characterization of cytochrome P450 26A1, a retinoic acid hydroxylase.
Expression and functional characterization of cytochrome P450 26A1, a retinoic acid hydroxylase.
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DOI：
10.1016/j.bcp.2008.10.012
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发表时间：
2009-01-15
影响因子：
5.8
通讯作者：
Isoherranen, Nina
中科院分区：
文献类型：
--
作者：
Lutz, Justin D.;Dixit, Vaishali;Yeung, Catherine K.;Dickmann, Leslie J.;Zelter, Alex;Thatcher, Jayne E.;Nelson, Wendel L.;Isoherranen, Nina
视黄酸 (RA) 是一种关键的信号分子，具有维持细胞活力所需的多种功能。它也用于治疗某些癌症。 CYP26 家族中的酶被认为负责消除 RA，而 CYP26A1 似乎在该家族中发挥着最关键的功能。尽管 CYP26A1 很重要，但它既没有被异源表达，也没有被动力学表征。我们在杆状病毒感染的昆虫细胞中表达 rCYP26A1，并将六组氨酸标签蛋白纯化至均质。通过一氧化碳差值光谱测定血红素掺入，并观察 RA 与 CYP26A1 结合的 1 型光谱。我们发现 RA 是 CYP26A1 的紧密结合配体，具有低 nM 结合亲和力。当补充 P450 氧化还原酶和 NADPH 时，CYP26A1 可有效氧化 RA（消耗 Km 9.4 ± 3.3 nM 和 Vmax 11.3 ± 4.3 pmoles/min/pmole P450），但不依赖于细胞色素 b5。 4-羟基-RA (4-OH-RA) 是 rCYP26A1 产生的主要代谢物，但也形成了其他两种主要产物。 4-OH-RA 被 CYP26A1 进一步代谢为极性更强的代谢物，并且 RA 的这种连续代谢部分发生，4-OH-RA 没有离开 CYP26A1 的活性位点。 CYP26A1 在消除 RA 及其潜在活性代谢物方面的高效性支持了该酶在调节体内 RA 清除中的主要作用。这些结果为 CYP26A1 功能提供了生化框架，并深入了解 CYP26A1 作为药物靶点以及在胎儿发育和细胞周期调节中的作用。
Retinoic acid (RA) is a critical signaling molecule that performs multiple functions required to maintain cellular viability. It is also used in the treatment of some cancers. Enzymes in the CYP26 family are thought to be responsible for the elimination of RA, and CYP26A1 appears to serve the most critical functions in this family. In spite of its importance, CYP26A1 has neither been heterologously expressed nor been characterized kinetically. We expressed the rCYP26A1 in baculovirus infected insect cells and purified the hexahistidine tagged protein to homogeneity. Heme incorporation was determined by carbon monoxide difference spectrum and a type 1 spectrum was observed with RA binding to CYP26A1. We found that RA is a tight binding ligand of CYP26A1 with low nM binding affinity. CYP26A1 oxidized RA efficiently (depletion Km 9.4 ± 3.3 nM and Vmax 11.3 ± 4.3 pmoles/min/pmole P450) when supplemented with P450 oxidoreductase and NADPH but was independent of cytochrome b5. 4-Hydroxy-RA (4-OH-RA) was the major metabolite produced by rCYP26A1 but two other primary products were also formed. 4-OH-RA was further metabolized by CYP26A1 to more polar metabolites and this sequential metabolism of RA occurred in part without 4-OH-RA leaving the active site of CYP26A1. The high efficiency of CYP26A1 in eliminating both RA and its potentially active metabolites supports the major role of this enzyme in regulating RA clearance in vivo. These results provide a biochemical framework for CYP26A1 function and offer insight into the role of CYP26A1 as a drug target as well as in fetal development and cell cycle regulation.
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影响因子：
3.9
作者：
Obach, RS;Reed-Hagen, AE
通讯作者：
Reed-Hagen, AE
影响因子：
4.8
作者：
Samokyszyn, VM;Gall, WE;Radominska-Pandya, A
通讯作者：
Radominska-Pandya, A
影响因子：
10.5
作者：
Abu-Abed, S;Dollé, P;Petkovich, M
通讯作者：
Petkovich, M
影响因子：
3.6
作者：
Marill, J;Cresteil, T;Chabot, GG
通讯作者：
Chabot, GG
影响因子：
4.8
作者：
Dong, D;Ruuska, SE;Noy, N
通讯作者：
Noy, N