MOLECULAR-CLONING AND KINETIC CHARACTERIZATION OF A FLAVIN-CONTAININGMONOOXYGENASE FROM SACCHAROMYCES-CEREVISIAE

An open reading frame from yeast coding for a homologue of flavin cont aining monooxygenase (FMO) has been cloned into several Escherichia co li expression vectors. A His(10) peptide attached to the amino terminu s produced a high yield of soluble protein when coexpressed with GroEL and GroES. The protein was purified on an affinity column and charact erized, The protein binds one mole per mole of flavin but the binding is relatively weak and 50 mu M exogenous FAD is used-to maintain full occupancy. The yeast enzyme, like mammalian enzymes, exhibits NADPH ox idase activity. The enzyme does not catalyze the oxidation of amines, but thiols, including glutathione, cysteine, and cysteamine, show subs trate activity. The fi, values for these are 7.0, 9.9, and 1.3 mM, res pectively; k(cat) values are 94, 246, and 94 per min, respectively. Th e enzyme apparently does not accept xenobiotic compounds but may be in volved in maintaining cellular reducing potential, probably through it s action on cysteamine. This activity may represent the initial role o f the FMO family of enzymes, giving rise to the multigene family of dr ug metabolizing enzymes seen in modern mammals. (C) 1996 Academic Pres s, Inc.