Isoform specificity of trimethylamine N-oxygenation by human flavin containing monooxygenase (FMO) and P450 enzymes -: Selective catalysis by FMO3
Isoform specificity of trimethylamine N-oxygenation by human flavin containing monooxygenase (FMO) and P450 enzymes -: Selective catalysis by FMO3
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DOI：
10.1016/s0006-2952(98)00218-4
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发表时间：
1998-10-15
影响因子：
5.8
通讯作者：
Rettie, AE
中科院分区：
文献类型：
--
作者：
Lang, DH;Yeung, CK;Rettie, AE
在本研究中，我们在杆状病毒表达载体系统中以0.6至2.4 nmol FMO/mg膜蛋白的水平表达人黄素单加氧酶1（FMO 1）、FMO 3、FMO 4 t（截短）和FMO 5。这四种亚型，以及纯化的兔FMO 2，和11异源表达的人P450亚型的能力进行了检查，代谢三甲胺（TMA），其N-氧化物（TMAO），使用一种新的，特定的HPLC方法与放射化学检测。人FMO 3是迄今为止活性最高的同种型，在pH 7.4和0.5 mM TMA下显示出30 nmol TMAO/nmol FMO 3/min的转换数。在这些条件下，没有其它单加氧酶以大于1 nmol/nmol FMO/min的速率形成TMAO。筛选人胎肝、成人肝、肾和肠微粒体的TMA氧化，仅人成人肝微粒体提供大量TMAO形成（范围为2.9 - 9.1 nmol TMAO/mg蛋白/min，N = 5）。采用三种不同的分析方法，对重组人FMO 3形成TMAO的动力学研究，得出Km为28 +/- 1 μ M，V-max为36.3 +/- 5.7 nmol TMAO/nmol FMO 3/min。在人肝微粒体中测定的Km范围为13.0 - 54.8 μ M。因此，在生理pH下，人FMO 3是非常特异和有效的TMA N-加氧酶，并且可能负责人体内TMA的代谢清除。此外，这种特异性通过分析pH 7.4下TMA浓度不高于0.5 mM的TMAO形成，为测定人体组织中FMO 3介导的活性提供了良好的体外探针。
In the present study, we expressed human flavin-containing monooxygenase 1 (FMO1), FMO3, FMO4t (truncated), and FMO5 in the baculovirus expression vector system at levels of 0.6 to 2.4 nmol FMO/mg of membrane protein. These four isoforms, as well as purified rabbit FMO2, and eleven heterologously expressed human P450 isoforms were examined for their capacity to metabolize trimethylamine (TMA) to its N-oxide (TMAO), using a new, specific HPLC method with radiochemical detection. Human FMO3 was by far the most active isoform, exhibiting a turnover number of 30 nmol TMAO/nmol FMO3/min at pH 7.4 and 0.5 mM TMA. None of the other monooxygenases formed TMAO at rates greater than 1 nmol/nmol FMO/min under these conditions. Human fetal liver, adult liver, kidney and intestine microsomes were screened for TMA oxidation, and only human adult liver microsomes provided substantial TMAO formation (range 2.9 to 9.1 nmol TMAO/mg protein/min, N = 5). Kinetic studies of TMAO formation by recombinant human FMO3, employing three different analytical methods, resulted in a K-m of 28 +/- 1 mu M and a V-max of 36.3 +/- 5.7 nmol TMAO/nmol FMO3/min. The K-m determined in human liver microsomes ranged from 13.0 to 54.8 mu M. Therefore, at physiological pH, human FMO3 is a very specific and efficient TMA N-oxygenase, and is likely responsible for the metabolic clearance of TMA in vivo in humans. In addition, this specificity provides a good in vitro probe for the determination of FMO3-mediated activity in human tissues, by analyzing TMAO formation at pH 7.4 with TMA concentrations not higher than 0.5 mM. (C) 1998 Elsevier Science Inc.