Determination of FAD-binding domain in flavin-containing monooxygenase 1 (FMO1)
Determination of FAD-binding domain in flavin-containing monooxygenase 1 (FMO1)
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DOI：
10.1006/abbi.1997.0242
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发表时间：
1997-09-15
影响因子：
3.9
通讯作者：
Kamataki, T
中科院分区：
文献类型：
--
作者：
Kubo, A;Itoh, S;Kamataki, T
含黄素单加氧酶（FMOs）是一类含黄素单加氧酶，每个单体含有1个FAD分子。为了证明FMO与FAD的相互作用，在酵母中表达了4个大鼠肝脏FMO1蛋白突变体并对其进行了表征。四种突变体在免疫化学上都与未修饰的形式相似，尽管四种突变体的FAD含量都比未修饰的形式低得多。有趣的是，将所提出的FAD结合域（GxGxxG）的第一甘氨酸改变为丙氨酸产生的突变体显示出催化活性，但比未修饰的形式低。丙咪嗪n氧化的K-m值和V-max值分别显著升高和降低。另外三个突变体（RFMOm2、RFMOm3和RFMOm4）催化活性不强。我们的研究结果表明，在FMO的催化活性中，三个甘氨酸，特别是第二个和第三个甘氨酸，是FMO表现出催化活性所必需的。以RFMOm1和未改性的形式，研究了正辛胺对FMO1活性的影响。正辛胺能提高野生型和RFMOm1酶对所有化合物的活性，正辛胺能降低和提高RFMOm1酶对丙咪嗪n-氧化的K-m和V-max值。(C) 1997学术出版社。
The flavin-containing monooxygenases (FMOs) are a family of flavoenzymes and contain one molecule of FAD per monomer, In order to demonstrate where FMO interacts with FAD, four mutants for the rat liver FMO1 protein were expressed in yeast and characterized. All four mutants were immunochemically similar to the unmodified form, although the contents of FAD in all four mutants were much lower than that in the unmodified form, Interestingly, the mutant generated by changing the first glycine of the proposed FAD-binding domain (GxGxxG) to alanine revealed catalytic activities, but was lower than those seen with the unmodified form, The conversion of the first glycine to alanine markedly increased and decreased the K-m and V-max values for imipramine N-oxidation, respectively. The other three mutants (RFMOm2, RFMOm3, and RFMOm4) were catalytically inactive. Our results suggest that three glycines, especially the second and third glycines, in the proposed FAD-binding domain were necessary for FMO to show catalytic activities. Using RFMOm1 and the unmodified form, the effects of n-octylamine on the activity of FMO1 were investigated. The activities of both wild-type and RFMOm1 enzymes for all of the compounds examined were enhanced by n-octylamine, The K-m and V-max values of both RFMOm1 and the unmodified form for imipramine N-oxidation were lowered and raised by n-octylamine, respectively. (C) 1997 Academic Press.