Human mitochondrial cytochrome P450 27C1 is localized in skin and preferentially desaturates trans-retinol to 3,4-dehydroretinol
Human mitochondrial cytochrome P450 27C1 is localized in skin and preferentially desaturates trans-retinol to 3,4-dehydroretinol
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DOI：
10.1074/jbc.m116.773937
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发表时间：
2017-08-18
影响因子：
4.8
通讯作者：
Guengerich, F. Peter
中科院分区：
文献类型：
--
作者：
Johnson, Kevin M.;Phan, Thanh T. N.;Guengerich, F. Peter
最近，斑马鱼和人类细胞色素P450 (P450) 27C1酶被证明是类维甲酸3,4-去饱和酶。这种酶在哺乳动物p450中是不寻常的，因为主要的氧化是去饱和，而羟基化只代表一个次要的途径。我们通过蛋白质组学分析表明P450 27C1定位于人类皮肤，存在两种不同大小的蛋白质，其中一种是全长形式的裂解产物。P450 27C1以100:3:2的比例将全反式视黄醇氧化为3,4-脱氢视黄醇，4-羟基（OH）视黄醇和3-OH视黄醇。3-OH和4-OH视黄醇都不是去饱和的中间体。稳态未观察到动力爆破；底物结合速率和产物释放速率都不是限速的。在底物存在的情况下，肾上腺素还原素对铁P450 27C1的还原速度加快了3倍，比整体的还原速度快了5倍。3,3-, 4,4-和3,3,4,4,4 -氘化视黄醇的动力学同位素效应为1.5-2.3 （on k(cat)/ k -m）。由于代谢转换，C-4处的氘化使3-羟基化增加了4倍，对4-羟基化没有明显影响。C-3的氘化对3-羟基化产生强烈的动力学同位素效应，而对4-羟基化则没有。对氘化视黄醇产物的分析表明，在C-3和C-4上缺乏一个假定的烯丙基自由基的混乱。我们得出的结论是，最可能的催化机制开始于从C-4（或可能是C-3）中提取氢原子，从而启动去饱和途径，然后依次提取氢原子或质子耦合电子转移。肾上腺素还原和氢提取都有助于速率限制。
Recently, zebrafish and human cytochrome P450 (P450) 27C1 enzymes have been shown to be retinoid 3,4-desaturases. The enzyme is unusual among mammalian P450s in that the predominant oxidation is a desaturation and in that hydroxylation represents only a minor pathway. We show by proteomic analysis that P450 27C1 is localized to human skin, with two proteins of different sizes present, one being a cleavage product of the full-length form. P450 27C1 oxidized all-trans-retinol to 3,4-dehydroretinol, 4-hydroxy (OH) retinol, and 3-OH retinol in a 100:3:2 ratio. Neither 3-OH nor 4-OH retinol was an intermediate in desaturation. No kinetic burst was observed in the steady state; neither the rate of substrate binding nor product release was rate-limiting. Ferric P450 27C1 reduction by adrenodoxin was 3-fold faster in the presence of the substrate and was similar to 5-fold faster than the overall turnover. Kinetic isotope effects of 1.5-2.3 (on k(cat)/K-m) were observed with 3,3-, 4,4-, and 3,3,4,4-deuterated retinol. Deuteration at C-4 produced a 4-fold increase in 3-hydroxylation due to metabolic switching, with no observable effect on 4-hydroxylation. Deuteration at C-3 produced a strong kinetic isotope effect for 3-hydroxylation but not 4-hydroxylation. Analysis of the products of deuterated retinol showed a lack of scrambling of a putative allylic radical at C-3 and C-4. We conclude that the most likely catalytic mechanism begins with abstraction of a hydrogen atom from C-4 (or possibly C-3) initiating the desaturation pathway, followed by a sequential abstraction of a hydrogen atom or proton-coupled electron transfer. Adrenodoxin reduction and hydrogen abstraction both contribute to rate limitation.