Phillips I R, Dolphin C T, Clair P, Hadley M R, Hutt A J, McCombie R R, Smith R L, Shephard E A
Department of Biochemistry, Queen Mary and Westfield College, University of London, UK.
Chem Biol Interact. 1995 Apr 28;96(1):17-32. doi: 10.1016/0009-2797(94)03580-2.
cDNA clones encoding five distinct members of the FMO family of man (FMOs 1, 2, 3, 4 and 5) were isolated by a combination of library screening and reverse transcription-polymerase chain reaction techniques. The deduced amino acid sequences of the human FMOs have 82-87% identity with their known orthologues in other mammal but only 51-57% similarity to each other. The hydropathy profiles of the proteins are very similar. From the calculated rate of evolution of FMOs (a 1% change in sequence per 6 million years) it would appear that individual members of the FMO gene family arose by duplication of a common ancestral gene some 250-300 million years ago. Each of the FMO genes was mapped by the polymerase chain reaction to the long arm of human chromosome 1. The localization of the FMO1 gene was further refined to 1q23-q25 by in situ hybridization of human metaphase chromosomes. RNase protection assays demonstrated that in man each FMO gene displays a distinct developmental and tissue-specific pattern of expression. In the adult, FMO1 is expressed in kidney but not in liver, whereas in the foetus its mRNA is abundant in both organs. FMO3 expression is essentially restricted to the liver in the adult and the mRNA is either absent, or present in low amounts, in foetal tissues. FMO4 is expressed more constitutively. Human FMO1 and FMO3 cDNAs were functionally expressed in prokaryotic and eukaryotic cells. FMO1 and FMO3, expressed in either system, displayed product stereoselectivity in their catalysis of the N-oxidation of the pro-chiral tertiary amines, N-ethyl-N-methylaniline (EMA) and pargyline. Both enzymes were stereoselective with respect to the production of the (-)-S-enantiomer of EMA N-oxide. But in the case of pargyline, the enzymes displayed opposite stereoselectivity, FMO1 producing solely the (+)-enantiomer and FMO3 predominantly the (-)-enantiomer of the N-oxide.
通过文库筛选和逆转录 - 聚合酶链反应技术相结合，分离出了编码人类FMO家族五个不同成员（FMO1、2、3、4和5）的cDNA克隆。人类FMOs的推导氨基酸序列与其在其他哺乳动物中的已知直系同源物有82 - 87%的同一性，但彼此之间只有51 - 57%的相似性。这些蛋白质的亲水性图谱非常相似。根据计算出的FMOs进化速率（每600万年序列变化1%），似乎FMO基因家族的各个成员是在约2.5亿至3亿年前通过一个共同祖先基因的复制产生的。通过聚合酶链反应将每个FMO基因定位到人类染色体1的长臂上。通过人类中期染色体的原位杂交，将FMO1基因的定位进一步精确到1q23 - q25。核糖核酸酶保护分析表明，在人类中每个FMO基因都表现出独特的发育和组织特异性表达模式。在成年人中，FMO1在肾脏中表达而不在肝脏中表达，而在胎儿中其mRNA在这两个器官中都很丰富。FMO3的表达基本上仅限于成年人的肝脏，在胎儿组织中其mRNA要么不存在，要么含量很低。FMO4的表达更具组成性。人类FMO1和FMO3的cDNA在原核细胞和真核细胞中进行了功能表达。在任一系统中表达的FMO1和FMO3在催化前手性叔胺N - 乙基 - N - 甲基苯胺（EMA）和优降宁的N - 氧化反应中表现出产物立体选择性。两种酶在生成EMA N - 氧化物的（-）-S - 对映体方面都是立体选择性的。但在优降宁的情况下，这两种酶表现出相反的立体选择性，FMO1仅产生（+）-对映体，而FMO3主要产生N - 氧化物的（-）-对映体。
