Johnson Kevin M, Phan Thanh T N, Albertolle Matthew E, Guengerich F Peter
From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
J Biol Chem. 2017 Aug 18;292(33):13672-13687. doi: 10.1074/jbc.M116.773937. Epub 2017 Jul 12.
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--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 ∼5-fold faster than the overall turnover. Kinetic isotope effects of 1.5-2.3 (on / ) 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.
最近，斑马鱼和人类细胞色素P450（P450）27C1酶已被证明是类视黄醇3,4-去饱和酶。该酶在哺乳动物P450中较为独特，主要氧化反应是去饱和反应，而羟基化仅代表次要途径。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将所有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，比例为100:3:2。3-OH视黄醇和4-OH视黄醇都不是去饱和反应的中间体。在稳态下未观察到动力学爆发；底物结合速率和产物释放速率均不是限速步骤。在底物存在的情况下，肾上腺皮质铁氧化还原蛋白对铁离子P450 27C1的还原速度快3倍，比总体周转速度快约5倍。用3,3-、4,4-和3,3,4,4-氘代视黄醇观察到1.5-2.3（对/）的动力学同位素效应。C-4位的氘化由于代谢转换使3-羟基化增加了4倍，对4-羟基化没有可观察到的影响。C-3位的氘化对3-羟基化产生了强烈的动力学同位素效应，但对4-羟基化没有影响。对氘代视黄醇产物的分析表明，在C-3和C-4位没有假定的烯丙基自由基的重排。我们得出结论，最可能的催化机制始于从C-4（或可能是C-3）提取氢原子，启动去饱和途径，随后依次提取氢原子或质子耦合电子转移。肾上腺皮质铁氧化还原蛋白还原和氢原子提取都对限速有贡献。
