Human flavin-containing monooxygenase form 3: cDNA expression of the enzymes containing amino acid substitutions observed in individuals with trimethylaminuria
Human flavin-containing monooxygenase form 3: cDNA expression of the enzymes containing amino acid substitutions observed in individuals with trimethylaminuria
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DOI：
10.1021/tx9700533
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发表时间：
1997-08-01
影响因子：
4.1
通讯作者：
Treacy, E
中科院分区：
文献类型：
--
作者：
Cashman, JR;Bi, YA;Treacy, E
三甲基氨基尿症是一种常染色体隐性遗传的人类疾病，以遗传多态性影响一小部分人群。被诊断为三甲基胺尿症的个体在他们的尿液、汗液和呼吸中排出相对大量的三甲基胺，这导致了三甲基胺特有的鱼腥味。已经提出，人含黄素单加氧酶（FMO）的活性在三甲基氨基尿症患者中是缺乏的，导致三甲基胺的代谢减少，从而导致鱼腥味。澳大利亚、美国和英国的人群都患有三甲基氨基尿症。从受影响患者的淋巴细胞中扩增人FMO 3 cDNA。我们报告的初步证据的cDNA和基因组DNA的筛选检测到的取代。通过定点诱变从野生型人FMO 3 cDNA构建变体人FMO 3 cDNA作为麦芽糖结合蛋白融合体。将5种不同的人FMO 3突变体在大肠杆菌中表达为融合蛋白，并与野生型人FMO 3麦芽糖结合蛋白（FMO 3-MBP）比较10-[（N，N-二甲基氨基）戊基]-2-（三氟甲基）酪胺和三甲胺的N-氧化作用。人Lys 158 FMO 3-MBP和人Glu 158 FMO 3-MBP在更大程度上有效地N-氧化三种胺底物。人Lys 158 Ile 66 FMOS-MBP、Glu 158 Ile 66 FMO 3-MBP、Lys 158 Leu 153 FMO 3-MBP和Glu 158 Leu 153 FMO 3-MBP均构建为突变体，经鉴定为可能导致三甲基氨基尿的FMO 3变体，并发现其作为N-加氧酶无活性。结果表明，FMO 3的密码子66和153处的突变可引起人类的三甲基氨基尿。我们观察到一个共同的多态性的FMO 3的密码子158分离几乎相等的等位基因频率在一些控制澳大利亚和北美的样本研究的赖氨酸到谷氨酸。Lys 158到Glu 158的人FMO 3多态性不降低cDNA表达酶的三甲胺N-加氧作用，因此似乎不是三甲基氨基尿症的病因。数据显示，人FMO 3的功能活性可以通过在临床诊断为三甲基氨基尿症的个体中观察到的氨基酸变化而显著改变。
Trimethylaminuria is an autosomal recessive human disorder affecting a small part of the population as an inherited polymorphism. Individuals diagnosed with trimethylaminuria excrete relatively large amounts of trimethylamine in their urine, sweat, and breath, and this results in a fishy odor characteristic of trimethylamine. Activity of the human flavin-containing monooxygenase (FMO) has been proposed to be deficient in trimethylaminuria patients causing a decrease in the metabolism of trimethylamine that results in a fishy body odor. Cohorts of Australian, American, and British individuals suffering from trimethylaminuria have been identified. The human FMO3 cDNA was amplified from lymphocytes of affected patients. We report preliminary evidence of substitutions detected by screening of the cDNA and genomic DNA. The variant human FMO3 cDNA was constructed from wild type human FMO3 cDNA by site-directed mutagenesis as maltose-binding protein fusions. Five distinct human FMO3 mutants were expressed as fusion proteins in Escherichia coli and compared with wild type human FMO3 maltose-binding proteins (FMO3-MBP) for the N-oxygenation of 10-[(N,N-dimethylamino)pentyl]-2-(trifluoromethyl) tyramine, and trimethylamine. Human Lys158 FMO3-MBP and, to a greater extent, human Glu158 FMO3-MBP efficiently N-oxygenated the three amine substrates. Human Lys158 Ile66 FMOS-MBP, Glu158 Ile66 FMO3-MBP, Lys158 Leu153 FMO3-MBP, and Glu158 Leu153 FMO3-MBP were all constructed as mutants identified as possible FMO3 variants responsible for trimethylaminuria and were found to be inactive as N-oxygenases. The results suggest that mutations at codons 66 and 153 of FMO3 can cause trimethylaminuria in humans. We observed a common polymorphism of Lys to Glu at codon 158 of FMO3 that segregated with almost equal allele frequencies in a number of control Australian and North American samples studied. The Lys158 to Glu158 human FMO3 polymorphism does not decrease trimethylamine N-oxygenation for the cDNA expressed enzyme and thus does not appear to be causative of trimethyaminuria. The data show that the functional activity of human FMO3 can be significantly altered by amino acid changes that have been observed in individuals with clinically diagnosed trimethylaminuria.