Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of RAS, P.O. Box 30, 420111, Kazan, Russia.
Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of RAS, P.O. Box 30, 420111, Kazan, Russia.
Biochim Biophys Acta Mol Cell Biol Lipids. 2019 Nov;1864(11):1580-1590. doi: 10.1016/j.bbalip.2019.07.004. Epub 2019 Jul 19.
Enzymes of CYP74 family widespread in higher plants control the metabolism of fatty acid hydroperoxides to numerous bioactive oxylipins. Hydroperoxide lyases (HPLs, synonym: hemiacetal synthases) of CYP74B subfamily belong to the most common CYP74 enzymes. HPLs isomerize the hydroperoxides to the short-lived hemiacetals, which are spontaneously decomposed to aldehydes and aldoacids. All CYP74Bs studied yet except the CYP74B16 (flax divinyl ether synthase, LuDES) possessed the 13-HPL activity. Present work reports the cloning of the expressed CYP74B33 gene of carrot (Daucus carota L.) and studies of catalytic properties of the recombinant CYP74B33 protein. In contrast to all CYP74B proteins studied yet, CYP74B33 behaved differently in few respects. Firstly, the preferred substrates of CYP74B33 are 9-hydroperoxides. Secondly and most importantly, CYP74B33 exhibits the 9-allene oxide synthase (AOS) activity. For example, the 9(S)-hydroperoxide of linoleic acid (9-HPOD) underwent the conversion to α-ketol via the short-lived allene oxide. Uncommonly, the 9-HPOD conversion affords a minority of cis-10-oxo-11-phytoenoic acid, which is also produced by CYP74C but not the CYP74A AOSs. The similar product patterns were observed upon the incubations of CYP74B33 with 9(S)-hydroperoxide of α-linolenic acid. The enzyme possessed a mixed HPL, AOS, and the epoxyalcohol synthase activity toward the 13-hydroperoxides, but the total activity was much lower than toward 9-hydroperoxides. Thus, the obtained results show that CYP74B33 is an unprecedented 9-AOS within the CYP74B subfamily.
CYP74 家族的酶广泛存在于高等植物中，控制脂肪酸氢过氧化物向众多生物活性氧代脂类的代谢。CYP74B 亚家族的过氧化物水解酶（HPLs，同义词：半缩醛合酶）属于最常见的 CYP74 酶。HPLs 将氢过氧化物异构化为短寿命的半缩醛，半缩醛自发分解为醛和aldoacids。迄今为止，除了 CYP74B16（亚麻二乙烯醚合酶，LuDES）之外，所有研究过的 CYP74B 都具有 13-HPL 活性。本工作报道了胡萝卜（Daucus carota L.）表达的 CYP74B33 基因的克隆，并研究了重组 CYP74B33 蛋白的催化特性。与迄今为止研究过的所有 CYP74B 蛋白不同，CYP74B33 在几个方面表现出不同的行为。首先，CYP74B33 的首选底物是 9-氢过氧化物。其次，也是最重要的一点，CYP74B33 表现出 9-烯氧化物合酶（AOS）活性。例如，亚油酸的 9(S)-氢过氧化物（9-HPOD）通过短寿命的烯氧化物转化为α-酮醇。不同寻常的是，9-HPOD 转化产生少量顺式-10-氧代-11-植烯酸，这也由 CYP74C 产生，但不是 CYP74A AOSs 产生。在用 9(S)-氢过氧化物-α-亚麻酸孵育 CYP74B33 时，观察到类似的产物模式。该酶对 13-氢过氧化物具有混合的 HPL、AOS 和环氧化醇合酶活性，但总活性远低于对 9-氢过氧化物的活性。因此，所得结果表明 CYP74B33 是 CYP74B 亚家族中前所未有的 9-AOS。
