CHARACTERIZATION OF 2 HUMAN FLAVIN-CONTAINING MONOOXYGENASE (FORM-3) ENZYMES EXPRESSED IN ESCHERICHIA-COLI AS MALTOSE-BINDING PROTEIN FUSIONS

To examine the possibility for drug metabolism polymorphism, adult hum an flavin-containing monooxygenases (form 3) (EC 1.14.13.8) that diffe r at one amino acid were expressed in Escherichia coli as maltose bind ing protein fusions. The cDNA that was first reported during the cloni ng of adult human flavin-containing monooxygenase was designated the w ild type lys(158) enzyme, A second cDNA has been identified as a commo n polymorphism in some human populations and was designated the glu(15 8) enzyme. The cDNA that encodes both enzymes was subcloned into a hig h yield protein fusion expression system, expressed, and the protein w as partially purified by affinity chromatography and characterized for enzyme activity with selective functional substrate probes, Nand S-ox ygenation activity of both enzymes was determined with ethylaminopenty l)-2-(trifluoromethyl)phenothiazine and methyl p-tolyl sulfide, respec tively. It was found that expression of both lys(158) and glu(158) enz ymes of the human flavin-containing monooxygenase form 3 as fusions wi th the maltose binding protein resulted in an enzyme that was soluble and greatly stabilized and had a reduced requirement for detergent dur ing enzyme purification and during the assay for activity, Expression of the fusion proteins has allowed the preparation of stable and highl y active enzyme at greater purity than was readily possible in the pas t. With the exception of the stability and solubility characteristics, the physical and chemical properties of lys(158) and glu(158) maltose binding fusion proteins of human flavin-containing monooxygenase form 3 variants resembled that of flavin-containing monooxygenase enzyme a ctivity associated with human liver microsomes and enzyme isolated fro m a previous Escherichia coli expression system that lacked the protei n fusion, Comparison of the catalytic activity of the two fusion prote ins showed that white both forms were active, there were differences i n their substrate specificities. Expression of the adult human flavin- containing monooxygenase form 3 as a maltose binding protein has allow ed considerable advances over the previously reported cDNA-expressed e nzyme systems and may provide the basis for examining the role of the flavin-containing monooxygenase in human xenobiotic or drug metabolism .