Dual role of the carboxyl-terminal region of pig liver l-kynurenine 3-monooxygenase: mitochondrial-targeting signal and enzymatic activity*
Dual role of the carboxyl-terminal region of pig liver l-kynurenine 3-monooxygenase: mitochondrial-targeting signal and enzymatic activity*
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DOI：
10.1093/jb/mvq099
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发表时间：
2010-12-01
影响因子：
2.7
通讯作者：
Izumi, Susumu
中科院分区：
文献类型：
--
作者：
Hirai, Kumiko;Kuroyanagi, Hidehito;Izumi, Susumu
L犬尿氨酸3-单加氧酶(KMO)是一种依赖NAD(P)H的黄素单加氧酶，催化L犬尿氨酸羟化为3-羟基犬尿氨酸，定位于线粒体外膜。在人脑中，KMO可能在3-羟基犬尿氨酸和喹啉酸两种神经毒素的形成中发挥重要作用，这两种毒素都会引发严重的神经退行性疾病。在蚊子中，它在眼睛色素的形成和一种脱毛诱导因子(黄原酸)的形成中都起着作用。在这里，我们提出了猪肝KMO的C-末端区域具有双重作用的证据。首先，它是酶活性所必需的。第二，它的功能是线粒体靶向信号，如单胺氧化酶B(MAO B)或外膜细胞色素b(5)。两个突变体(C末端标记的KMO和羧基末端截断形式KMO Delta C50)的酶活性与在COS-7细胞中表达的野生型酶的活性进行了比较，表明了第一个作用。在荧光显微镜下，通过比较野生型、三种羧基末端截短型(Delta C20、Delta C30和Delta C50)、C末端标记的野生型和一个突变体KMO的细胞内定位，证明了第二种作用，其中两个精氨酸残基Arg461-Arg462被Ser残基取代。
l-kynurenine 3-monooxygenase (KMO) is an NAD(P)H-dependent flavin monooxygenase that catalyses the hydroxylation of l-kynurenine to 3-hydroxykynurenine, and is localized as an oligomer in the mitochondrial outer membrane. In the human brain, KMO may play an important role in the formation of two neurotoxins, 3-hydroxykynurenine and quinolinic acid, both of which provoke severe neurodegenerative diseases. In mosquitos, it plays a role in the formation both of eye pigment and of an exflagellation-inducing factor (xanthurenic acid). Here, we present evidence that the C-terminal region of pig liver KMO plays a dual role. First, it is required for the enzymatic activity. Second, it functions as a mitochondrial targeting signal as seen in monoamine oxidase B (MAO B) or outer membrane cytochrome b(5). The first role was shown by the comparison of the enzymatic activity of two mutants (C-terminally FLAG-tagged KMO and carboxyl-terminal truncation form, KMO delta C50) with that of the wild-type enzyme expressed in COS-7 cells. The second role was demonstrated with fluorescence microscopy by the comparison of the intracellular localization of the wild-type, three carboxyl-terminal truncated forms (delta C20, delta C30 and delta C50), C-terminally FLAG-tagged wild-type and a mutant KMO, where two arginine residues, Arg461-Arg462, were replaced with Ser residues.