ANALYSIS OF GENE MUTATION IN THAI CAUSING FISH ODOR SYNDROME

我们研究了三甲胺（TMA）在人肝微粒体中的代谢。TMA N-氧化的动力学参数显示表观Km和Vmax值分别为45.9 μM和2.24 nmol/min/mg蛋白。甲巯咪唑和热处理等已知的含黄素单加氧酶（FMO）抑制剂可抑制TMA的N-氧化，而细胞色素P450抑制剂SKF-525 A则不抑制TMA的N-氧化。为了鉴定负责TMA的N-氧化的FMO形式，测量了在大肠杆菌中表达的人FMO 1、FMO 3和FMO 5的TMA N-氧化酶活性。FMO 5没有表现出TMA N-氧化酶活性。当考察TMA N-氧化的动力学参数时，FMO 3的Vmax/Km值比FMO 1高约355倍。我们分析了泰国可能患有鱼腥味综合征的患者的FMO 3基因。在2例患者的第3外显子中发现了一个新的突变，即第265位由G变为A（G265 A）。在第58位氨基酸残基（V58 I）由缬氨酸替换为异亮氨酸的突变FMO 3蛋白在沙门氏菌中表达时表现出降低的TMA N-氧化酶活性。我们的结论是，FMO 3基因与此突变产生的FMO蛋白的催化活性降低。

We examined the metabolism of trimethylamine (TMA) in human liver microsomes. The kinetic parameters of TMA N-oxidation showed the apparent Km and Vmax values of 45.9 μM and 2.24 nmol/min/mg protein, respectively. The N-oxidation of TMA was inhibited by the known flavin-containing monooxygenase (FMO) inhibitors such as methimazole and heat treatment, but not by SKF-525A, cytochrome P450 inhibitor. To identify the FMO form responsible for the N-oxidation of TMA, the TMA N-oxidase activities of human FMO1, FMO3 and FMO5 expressed in E, coli were measured. FMO5 showed no TMA N-oxidase activity. The Vmax/Km value for FMO3 was about 355 times higher than that for FMO1 when the kinetic parameters of TMA N-oxidation was examined. We analyzed the FMO3 gene of Thai possible patients with fish-odor syndrome. A novel mutation, a single base substitution from G to A at the position of 265 (G265A), was identified in exon 3 of two patients. The mutated FMO3 protein with an amino acid substitution from valine to isoleucine at residue 58 (V58I) exhibited the reduced TMA N-oxidase activity when it was expressed in Salmonella. We conclude that FMO3 gene with this mutation produce the FMO protein that has reduced catalytic activities.