Suh J K, Poulsen L L, Ziegler D M, Robertus J D
Department of Chemistry and Biochemistry, University of Texas, Austin, Texas, 78712, USA.
Arch Biochem Biophys. 1996 Dec 15;336(2):268-74. doi: 10.1006/abbi.1996.0557.
An open reading frame from yeast coding for a homologue of flavin containing monooxygenase (FMO) has been cloned into several Escherichia coli expression vectors. A His10 peptide attached to the amino terminus produced a high yield of soluble protein when coexpressed with GroEL and GroES. The protein was purified on an affinity column and characterized. The protein binds one mole per mole of flavin but the binding is relatively weak and 50 microM exogenous FAD is used to maintain full occupancy. The yeast enzyme, like mammalian enzymes, exhibits NADPH oxidase activity. The enzyme does not catalyze the oxidation of amines, but thiols, including glutathione, cysteine, and cysteamine, show substrate activity. The Km values for these are 7.0, 9.9, and 1.3 mM, respectively; kcat values are 94, 246, and 94 per min, respectively. The enzyme apparently does not accept xenobiotic compounds but may be involved in maintaining cellular reducing potential, probably through its action on cysteamine. This activity may represent the initial role of the FMO family of enzymes, giving rise to the multigene family of drug metabolizing enzymes seen in modern mammals.
一个来自酵母的编码含黄素单加氧酶（FMO）同源物的开放阅读框已被克隆到几种大肠杆菌表达载体中。当与GroEL和GroES共表达时，连接在氨基末端的His10肽产生了高产率的可溶性蛋白。该蛋白在亲和柱上进行了纯化并进行了表征。该蛋白每摩尔结合一摩尔黄素，但结合相对较弱，使用50微摩尔的外源FAD来维持完全占据。酵母酶与哺乳动物酶一样，具有NADPH氧化酶活性。该酶不催化胺的氧化，但硫醇，包括谷胱甘肽、半胱氨酸和半胱胺，表现出底物活性。它们的Km值分别为7.0、9.9和1.3毫摩尔；kcat值分别为每分钟94、246和94。该酶显然不接受外源性化合物，但可能参与维持细胞的还原电位，可能是通过其对半胱胺的作用。这种活性可能代表了FMO酶家族的初始作用，产生了现代哺乳动物中所见的多基因药物代谢酶家族。
